Publications by authors named "Javier Rojo"

72 Publications

Role of nanostructures in allergy: Diagnostics, treatments and safety.

Allergy 2021 Feb 9. Epub 2021 Feb 9.

Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain.

Nanotechnology is science, engineering and technology conducted at the nanoscale, which is about 1-100 nm. It has led to the development of nanomaterials, which behave very differently from materials with larger scales and can have a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition and functionalization of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers and nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalization with ligands, which provide the desired interaction with the immunological system, that is dendritic cell receptors to activate and/or modulate the response, as well as specific IgE, or effector cell receptors. However, undesired issues related to toxicity and hypersensitivity responses can also happen and would need evaluation. There are wide panels of accessible structures, and controlling their physico-chemical properties would permit obtaining safer and more efficient compounds for clinical applications goals, either in diagnosis or treatment. The application of dendrimeric antigens, nanoallergens and nanoparticles in allergy diagnosis is very promising since it can improve sensitivity by increasing specific IgE binding, mimicking carrier proteins or enhancing signal detection. Additionally, in the case of immunotherapy, glycodendrimers, liposomes, polymers and nanoparticles have shown interest, behaving as platforms of allergenic structures, adjuvants or protectors of allergen from degradation or having a depot capacity. Taken together, the application of nanotechnology to allergy shows promising facts facing important goals related to the improvement of diagnosis as well as specific immunotherapy.
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http://dx.doi.org/10.1111/all.14764DOI Listing
February 2021

Influence of the reducing-end anomeric configuration of the Man epitope on DC-SIGN recognition.

Org Biomol Chem 2020 Aug 30;18(31):6086-6094. Epub 2020 Jul 30.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Av. Américo Vespucio 49, Seville 41092, Spain.

High-mannose (ManGlcNAc) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man epitope conjugated to two terminal N-acetylglucosamines by otherwise rarely-encountered β-mannose glycosidic bond. Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to prepare high mannose. Herein, we report the synthesis of the Man epitope with both alpha and beta configurations at the reducing end, and subsequent evaluation of the impact of this configuration on binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results therefore indicate that the more synthetically-accesible Man alpha epitope may be deployed as ligand for DC-SIGN in both in vitro and in vivo biological assays.
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http://dx.doi.org/10.1039/d0ob01380cDOI Listing
August 2020

Second-Generation Dendrimers with Chondroitin Sulfate Type-E Disaccharides as Multivalent Ligands for Langerin.

Biomacromolecules 2020 07 24;21(7):2726-2734. Epub 2020 Jun 24.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

Chondroitin sulfate type-E (CS-E) is a sulfated polysaccharide that shows several interesting biological activities, such as modulation of the neuronal growth factor signaling and its interaction with langerin, a C-type lectin with a crucial role in the immunological system. However, applications of CS-E are hampered by the typical heterogeneous structure of the natural polysaccharide. Well-defined, homogeneous CS-E analogues are highly demanded. Here, we report the synthesis of monodispersed, structurally well-defined second-generation glycodendrimers displaying up to 18 CS-E disaccharide units. These complex multivalent systems have a molecular weight and a number of disaccharide repeating units comparable with those of the natural polysaccharides. In addition, surface plasmon resonance experiments revealed a calcium-independent interaction between these glycodendrimers and langerin, in the micromolar range, highlighting the utility of these compounds as CS-E mimetics.
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http://dx.doi.org/10.1021/acs.biomac.0c00476DOI Listing
July 2020

Author Correction: Glycosylated nanostructures in sublingual immunotherapy induce long-lasting tolerance in LTP allergy mouse model.

Sci Rep 2020 Apr 17;10(1):6858. Epub 2020 Apr 17.

Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-63855-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162968PMC
April 2020

Platinum nanoparticles stabilized by N-heterocyclic thiones. Synthesis and catalytic activity in mono- and di-hydroboration of alkynes.

Nanoscale 2020 Mar 17;12(12):6821-6831. Epub 2020 Mar 17.

Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, C/Américo Vespucio 49, 41092, Seville, Spain.

N-Heterocyclic Thiones (NHT) proved to be efficient ligands for the stabilization of small platinum nanoparticles (1.3-1.7 nm), synthesized by decomposition of [Pt(dba)], under a H atmosphere, in the presence of variable sub-stoichiometric amounts of the NHT. Full characterization by means of TEM, HR-TEM, NMR, ICP, TGA and XPS have been carried out, providing information about the nature of the metal nanoparticles and the interaction of the NHT ligands to the metal surface. Importantly, DFT calculations indicate that some NHT ligands interact with the metal through the C[double bond, length as m-dash]C double bond of the imidazole fragment in addition to the sulfur atom, thus providing additional stabilization to the nanoparticles. According to XPS, TGA and ICP techniques, the surface coverage by the ligand increases by decreasing the size of the substituents on the nitrogen atom. The platinum nanoparticles have been used as catalyst in the hydroboration of alkynes. The most active system is that with a less covered surface area lacking an interaction of the ligand by means of the C[double bond, length as m-dash]C double bond. This catalyst hydroborates alkynes with excellent selectivities towards the monoborylated anti-Markovnikov product (vinyl-boronate) when one equiv. of borane is used. Very interestingly, aliphatic alkynes undergo a second hydroborylation process leading to the corresponding 1,1- and 1,2-diboroylated species with good selectivities towards the former.
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http://dx.doi.org/10.1039/d0nr00251hDOI Listing
March 2020

Phenotypic and genetic differentiation between diadromous and landlocked puyen Galaxias maculatus.

J Fish Biol 2020 Apr 27;96(4):956-967. Epub 2020 Feb 27.

Centro Austral de Investigaciones Científicas (CADIC) - CONICET, Ushuaia, Argentina.

This study reports the phenotypic and genetic differences between individuals of puyen Galaxias maculatus from two sites in the same river basin in Tierra del Fuego National Park, southern South America. Individuals from the two sampling sites presented morphometric and genetic differences. The morphometric differences indicated that individuals from Laguna Negra (LN) were short and more robust and had large eyes, whereas those from Arroyo Negro (AN) were thin and elongated and had small eyes. Genetic differences showed that AN individuals had a greater genetic structuration and an older demographic history than LN individuals. The results of this study affirmed that the individuals from the two sampling sites belong to different populations with a high degree of isolation. The demographic history could indicate that the individuals of G. maculatus which migrated to northern areas during the last glaciation settled in the Beagle Channel after its formation. The LN population could have originated after the retreat of the glaciers, migrating from AN.
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http://dx.doi.org/10.1111/jfb.14285DOI Listing
April 2020

Peptide Glycodendrimers as Potential Vaccines for Olive Pollen Allergy.

Mol Pharm 2020 03 13;17(3):827-836. Epub 2020 Feb 13.

Biochemistry and Molecular Biology, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid 28040, Spain.

Olive pollen is one of the most important causes of respiratory allergy, with Ole e 1 being the most clinically relevant sensitizing allergen. Peptide-based vaccines represent promising therapeutic approaches, but the use of adjuvants is required to strengthen the weak immunogenicity of small peptides. We propose the use of dendrimeric scaffolds conjugated to the T cell immunodominant epitope of Ole e 1 (OE) for the development of novel vaccines against olive pollen allergy. Four dendrimeric scaffolds containing an ester/ether with nine mannoses, an ester succinimidyl linker with nine -acetyl-glucosamine units or nine ethylene glycol units conjugated to OE peptide were designed, and their cytotoxicity, internalization pattern, and immunomodulatory properties were analyzed in vitro. None of the dendrimers exhibited cytotoxicity in humanized rat basophil (RBL-2H3), human bronchial epithelial Calu-3, and human mast LAD2 cell lines. Confocal images indicated that mannosylated glycodendropeptides exhibited lower colocalization with a lysosomal marker. Moreover, mannosylated glycodendropeptides showed higher transport tendency through the epithelial barrier formed by Calu-3 cells cultured at the air-liquid interface. Finally, mannosylated glycodendropeptides promoted Treg and IL10Treg proliferation and IL-10 secretion by peripheral blood mononuclear cells from allergic patients. Mannosylated dendrimers conjugated with OE peptide from Ole e 1 have been identified as suitable candidates for the development of novel vaccines of olive pollen allergy.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b01082DOI Listing
March 2020

A Mobile Solution for Rhythmic Auditory Stimulation Gait Training.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:309-312

Recent studies showed that Parkinson's disease (PD) patients improved their gait parameters while walking with rhythmic auditory stimulation (RAS). They achieved a longer stride length, a reduced stride time variability and a higher walking speed. Combining RAS with mobile gait analysis would allow continuous monitoring of RAS effects and gait in natural environments. This paper proposes a mobile solution for home-based assessment of RAS by combining RAS gait training and a mobile system for data acquisition. Existing datasets were used to investigate the cadence of PD patients and to propose suitable frequencies for RAS gait training. The cadence calculation was implemented using a peak detection algorithm, which uses the time difference between two mid-swing events as stride time values. We validated our system as a whole using a cohort of 13 PD patients who performed RAS gait training. The algorithms were also validated against the eGaIT system, a state-of-the-art system, and achieved a mean F score for detected strides of 97.57 % ± 0.86 % and a mean absolute error for the cadence of 0.16 spm ± 0.09 spm. This study lays the ground work for further clinical studies investigating the effectiveness of mobile RAS within a home environment.
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http://dx.doi.org/10.1109/EMBC.2019.8857143DOI Listing
July 2019

Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses.

J Am Chem Soc 2019 09 11;141(38):15403-15412. Epub 2019 Sep 11.

Departamento de Química Orgánica, Facultad de Química , Universidad Complutense , 28040 Madrid , Spain.

After the last epidemic of the Zika virus (ZIKV) in Brazil that peaked in 2016, growing evidence has been demonstrated of the link between this teratogenic flavivirus and microcephaly cases. However, no vaccine or antiviral drug has been approved yet. ZIKV and Dengue viruses (DENV) entry to the host cell takes place through several receptors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), so that the blockade of this receptor through multivalent glycoconjugates supposes a promising biological target to inhibit the infection process. In order to get enhanced multivalency in biocompatible systems, tridecafullerenes appended with up to 360 1,2-mannobiosides have been synthesized using a strain-promoted cycloaddition of azides to alkynes (SPAAC) strategy. These systems have been tested against ZIKV and DENV infection, showing an outstanding activity in the picomolar range.
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http://dx.doi.org/10.1021/jacs.9b08003DOI Listing
September 2019

Pru p 3-Glycodendropeptides Based on Mannoses Promote Changes in the Immunological Properties of Dendritic and T-Cells from LTP-Allergic Patients.

Mol Nutr Food Res 2019 10 8;63(20):e1900553. Epub 2019 Aug 8.

Research Laboratory, IBIMA-Regional University Hospital of Malaga-UMA, 29009, Malaga, Spain.

Scope: Glycodendropeptides (GDPs) functionalized with mannose can enhance allergen interaction with dendritic cells (DCs) via C-type lectin receptors (CLRs), modulating the immune response. They can present multiple peptides and have potential applications for diagnosis and treatment of food allergy (FA). The immune response induced by GDPs with mannose and Pru p 3 peptides (mono/tetravalent) with ester (D ManPrup3/D ManPrup3) or ether linkers (D Man Prup3/D Man Prup3) in lipid-transfer-protein-allergic patients and tolerant controls is analyzed.

Methods And Results: The immunological response induced by GDPs is studied by assessing monocyte-derived-DC maturation, lymphocyte proliferation, cytokine production, and basophil response by flow cytometry. D ManPrup3 was recognized by DCs via CLRs inducing DC maturation in all subjects. However, CCR7 expression is significantly upregulated in allergic patients compared to tolerant controls. These changes correlate with lymphocyte proliferation and specific production of Th2/Th1 cytokines in allergic patients. Moreover, D ManPrup3 does not induce basophil activation.

Conclusion: D ManPrup3 induces changes in DC maturation and lymphocyte proliferation, indicating specific recognition via CLRs. Prup3-GDPs are recognized by immune cells, inducing a specific immune response and modulating the immunological response in FA patients. The specific geometry of D ManPrup3 in particular makes it a potential candidate for specific immunotherapy development.
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http://dx.doi.org/10.1002/mnfr.201900553DOI Listing
October 2019

Glycosylated nanostructures in sublingual immunotherapy induce long-lasting tolerance in LTP allergy mouse model.

Sci Rep 2019 03 11;9(1):4043. Epub 2019 Mar 11.

Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain.

An effective specific immunotherapy should contain elements to generate specific recognition (T-cell peptides) and to modulate the immunological response towards a Th1/Treg pattern by enhancing dendritic cells (DCs). We propose a novel sublingual immunotherapy for peach allergy, using systems, that combine Prup3-T-cell peptides with mannose dendrons (DManPrup3 and DManPrup3). Peach anaphylactic mice were treated 1, 2 and 5 nM concentrations. Tolerance was assessed one/five weeks after finishing treatment by determining in vivo/in vitro parameters after challenge with Prup3. Only mice receiving DManPrup3 at 2 nM were protected from anaphylaxis (no temperature changes, decrease in Prup3-sIgE and -sIgG1 antibody levels, and secreting cells) compared to PBS-treated mice. Moreover, an increase of Treg-cells and regulatory cytokines (IL-10/IFN-γ) in CD4-T-cells and DCs were found. These changes were maintained at least five weeks after stopping treatment. DManPrup3 is an effective new approach of immunotherapy inducing protection from anaphylaxis which persists after finishing treatment.
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http://dx.doi.org/10.1038/s41598-019-40114-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411722PMC
March 2019

Transcriptional Profiling of Dendritic Cells in a Mouse Model of Food-Antigen-Induced Anaphylaxis Reveals the Upregulation of Multiple Immune-Related Pathways.

Mol Nutr Food Res 2019 02 17;63(3):e1800759. Epub 2018 Dec 17.

Research Laboratory, IBIMA-Regional University Hospital of Malaga, UMA, 29009, Malaga, Spain.

Scope: Much of the knowledge about gene expression during anaphylaxis comes from candidate gene studies. Despite their potential role, expression changes in dendritic cells (DCs) have not been studied in this context using high throughput methods. The molecular mechanisms underlying food-antigen-induced anaphylaxis are investigated using DCs from an animal model.

Methods And Results: RNA sequencing is used to study gene expression in lymph-node-derived DCs from anaphylactic mice sensitized intranasally with the major peach allergen Pru p 3 during the acute reaction phase, induced intraperitoneally. In total, 237 genes changed significantly, 181 showing at least twofold changes. Almost three-quarters of these increase during anaphylaxis. A subset is confirmed using RT-PCR in a second set of samples obtained from a new batch of mice. Enrichment analysis shows an overrepresentation of genes involved in key immune system and inflammatory processes, including TGF-β signaling. Comparison with a study using anaphylactic human subjects show significant overlap.

Conclusions: The findings provide a comprehensive overview of the transcriptional changes occurring in DCs during anaphylaxis and help elucidate the mechanisms involved. They add further weight to the putative role of these cells in anaphylaxis and highlight genes that may represent potential therapeutic targets.
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http://dx.doi.org/10.1002/mnfr.201800759DOI Listing
February 2019

Nanocarbon-Based Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection.

J Am Chem Soc 2018 08 30;140(31):9891-9898. Epub 2018 Jul 30.

Departamento de Química Orgánica, Facultad de Química , Universidad Complutense de Madrid , Avenida Complutense s/n , 28040 Madrid , Spain.

SWCNTs, MWCNTs, and SWCNHs have been employed as virus-mimicking nanocarbon platforms for the multivalent presentation of carbohydrates in an artificial Ebola virus infection model assay. These carbon nanoforms have been chemically modified by the covalent attachment of glycodendrons and glycofullerenes using the CuAAC "click chemistry" approach. This modification dramatically increases the water solubility of these structurally different nanocarbons. Their efficiency in blocking DC-SIGN-mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay, finding that glycoconjugates based on MWCNTs functionalized with glycofullerenes are potent inhibitors of viral infection.
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http://dx.doi.org/10.1021/jacs.8b03847DOI Listing
August 2018

Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes.

J Org Chem 2018 02 2;83(4):1727-1736. Epub 2018 Feb 2.

Departamento de Química Orgánica, Facultad de Química, Universidad Complutense de Madrid , E-28040 Madrid, Spain.

The synthesis of multivalent systems based on hexakis-adducts of [60]fullerene employing a biocompatible copper-free click chemistry strategy has been accomplished. A symmetric hexakis-adduct of fullerene bearing 12 maleimide units (3) is reported, and it has been employed to carry out the thiol-maleimide Michael addition. To achieve orthogonal click addition, an asymmetric derivative bearing one maleimide and 10 cyclooctynes has been synthesized. The sequential and one-pot transformations of the two clickable groups have been explored, finding the best results in the case of the one-pot experiment. This route has been used to obtain a biocompatible hexakis-adduct appended with two different biomolecules, carbohydrates, and amino acids.
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http://dx.doi.org/10.1021/acs.joc.7b02402DOI Listing
February 2018

Straightforward synthesis of Man, the relevant epitope of the high-mannose oligosaccharide.

Org Biomol Chem 2017 Oct;15(42):8877-8882

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Américo Vespucio 49, Sevilla, Spain.

The high-mannose oligosaccharide (or its corresponding Man epitope) is the most abundant structure present in pathogen envelope glycoproteins. These glycans play a key role in the pathogenesis of several pathogens and also in the communication with the immune system. Understanding the mechanism of action of these glycans requires the access to pure and chemically well-defined structures in reasonable amounts. The synthesis of these complex branched oligosaccharides is not trivial and few syntheses are reported in the literature with several synthetic and purification steps and low overall yields. In this work, we described a very efficient synthetic alternative to access this relevant Man epitope in a very straightforward manner.
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http://dx.doi.org/10.1039/c7ob02286gDOI Listing
October 2017

Topological Defects in Hyperbranched Glycopolymers Enhance Binding to Lectins.

Chemistry 2017 Nov 16;23(62):15790-15794. Epub 2017 Oct 16.

Departament de Química Analítica i Química Orgànica, Universitat Rovira i Virgili, C/ Marcel lí Domingo 1, 43007, Tarragona, Spain.

Central scaffold topology and carbohydrate density are important features in determining the binding mechanism and potency of synthetic multivalent of poly- versus monodisperse carbohydrate systems against a model plant toxin (Ricinus communis agglutinin (RCA )). Lower densities of protein receptors favour the use of heterogeneous, polydisperse glycoconjugate presentations, as determined by surface plasmon resonance and dynamic light scattering.
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http://dx.doi.org/10.1002/chem.201703432DOI Listing
November 2017

Antiviral activity of self-assembled glycodendro[60]fullerene monoadducts.

J Mater Chem B 2017 Aug 4;5(32):6566-6571. Epub 2017 Aug 4.

Departamento de Química Orgánica, Fac. C.C. Químicas, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid, Spain.

A series of amphiphilic glycodendro[60]fullerene monoadducts were efficiently synthesized using the CuAAC "click chemistry" approach. These glycodendrofullerenes can self-assemble in aqueous media, in a process favoured through π-π interactions between the [60]fullerene moieties. This aggregation process leads to big and well-defined compact micelles with a uniform size and spherical-shape. The supramolecular aggregates were characterized using electronic microscopy (SEM and TEM), light scattering methods (DLS) and X-ray methodologies (SAXS and XRD). The antiviral efficiency of these aggregates has been tested in an experimental infection assay using Ebola virus glycoprotein (EboGP) pseudotyped viral particles on Jurkat cells overexpressing DC-SIGN and an improvement in the IC value with respect to other systems endowed with a higher number of carbohydrate ligands is observed.
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http://dx.doi.org/10.1039/c7tb01379eDOI Listing
August 2017

Stabilisation of gold nanoparticles by N-heterocyclic thiones.

Dalton Trans 2017 Jul;46(26):8367-8371

Instituto de Investigaciones Químicas (IIQ), CSIC and Universidad de Sevilla, Avda. Américo Vespucio 49, 41092 Sevilla, Spain.

Gold nanoparticles (Au-NPs) have been prepared using N-heterocyclic thiones (NHTs) as ligand stabilisers. These Au-NPs have been shown to be very stable, even in air, and have been characterized by a combination of several techniques (TEM, HR-TEM, STEM-HAADF, EDX, DLS, elemental analysis and H NMR). These nanoparticles are active in the catalytic reduction of nitroarenes to anilines.
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http://dx.doi.org/10.1039/c7dt01856hDOI Listing
July 2017

Glycodendrimers as Chondroitin Sulfate Mimetics: Synthesis and Binding to Growth Factor Midkine.

Chemistry 2017 Aug 3;23(47):11338-11345. Epub 2017 Aug 3.

Instituto de Investigaciones Químicas (IIQ), CSIC- Universidad de Sevilla, Américo Vespucio 49, 41092, Seville, Spain.

Chondroitin sulfate (CS) is a member of the glycosaminoglycan (GAG) family, a class of polysaccharides implicated in relevant biological functions. The structural complexity of these carbohydrates demands the development of simple glycomimetics as useful tools to study the biological processes in which GAGs are involved. In this work we described the synthesis of the disaccharide unit of the CS-E (GlcA-GalNAc(4,6-di-OSO )), in a multivalent presentation. Using a fluorescence polarization competition assay we have demonstrated that a hexavalent dendrimer of this disaccharide interact with midkine, in the low micromolar range. This result highlights the potency of these disaccharide-displaying multivalent systems as interesting mimetics of longer and synthetically more complex GAG oligosaccharides.
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http://dx.doi.org/10.1002/chem.201701890DOI Listing
August 2017

Pru p 3-Epitope-based sublingual immunotherapy in a murine model for the treatment of peach allergy.

Mol Nutr Food Res 2017 10 6;61(10). Epub 2017 Sep 6.

Research Laboratory, IBIMA, Regional University Hospital of Malaga, UMA, Malaga, Spain.

Scope: Food-specific immunotherapy (SIT) is a promising treatment for lipid transfer protein (LTP)-syndrome. We propose a novel sublingual-SIT (SLIT) that combines a Pru p 3 T-cell peptide and an oligodeoxyribonucleotide (ODN) with CpG motifs (ODN-CpG) as adjuvants to induce a specific Th1/Treg response.

Methods And Results: LTP-peach allergic mice were treated sublingually with a combination of a CpG sequence and mono- or tetravalent systems including a Pru p 3 peptide, D (Prup3) or D (Prup3). Mice were challenged intraperitoneally with Pru p 3 one or three weeks after SLIT and tolerance was assessed. Mice treated with D (Prup3)+CpG were protected from anaphylaxis after Pru p 3 challenge. They showed no change in body temperature, lower levels of Pru p 3-specific IgE and IgG1 antibodies and higher levels of sIgG2a compared to the untreated group. They had fewer IgE and IgG1 secreting cells and more sIgG2a secreting cells. Moreover, a significantly lower number of Pru p 3-specific CD4 T cells and a higher number of Treg cells were found, alongside a Th1 cytokine pattern. These changes were maintained for three weeks after stopping treatment.

Conclusion: D Prup3+CpG represents a promising SIT for food allergy. It is easily synthesized and induces protection from anaphylaxis to Pru p 3 that is maintained for at least three weeks.
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http://dx.doi.org/10.1002/mnfr.201700110DOI Listing
October 2017

Polyvalent C-glycomimetics based on l-fucose or d-mannose as potent DC-SIGN antagonists.

Org Biomol Chem 2017 May;15(18):3995-4004

Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague, Czech Republic.

The C-type lectin DC-SIGN expressed on immature dendritic cells is a promising target for antiviral drug development. Previously, we have demonstrated that mono- and divalent C-glycosides based on d-manno and l-fuco configurations are promising DC-SIGN ligands. Here, we described the convergent synthesis of C-glycoside dendrimers decorated with 4, 6, 9, and 12 α-l-fucopyranosyl units and with 9 and 12 α-d-mannopyranosyl units. Their affinity against DC-SIGN was assessed by surface plasmon resonance (SPR) assays. For comparison, parent O-glycosidic dendrimers were synthesized and tested, as well. A clear increase of both affinity and multivalency effect was observed for C-glycomimetics of both types (mannose and fucose). However, when dodecavalent C-glycosidic dendrimers were compared, there was no difference in affinity regarding the sugar unit (l-fuco, IC 17 μM; d-manno, IC 12 μM). For the rest of glycodendrimers with l-fucose or d-mannose attached by the O- or C-glycosidic linkage, C-glycosidic dendrimers were significantly more active. These results show that in addition to the expected physiological stability, the biological activity of C-glycoside mimetics is higher in comparison to the corresponding O-glycosides and therefore these glycomimetic multivalent systems represent potentially promising candidates for targeting DC-SIGN.
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http://dx.doi.org/10.1039/c7ob00322fDOI Listing
May 2017

Multivalent Glycosylated Nanostructures To Inhibit Ebola Virus Infection.

J Am Chem Soc 2017 05 20;139(17):6018-6025. Epub 2017 Apr 20.

Departamento de Química Orgánica, Facultad de Química, Universidad Complutense , 28040 Madrid, Spain.

The infection of humans by lethal pathogens such as Ebola and other related viruses has not been properly addressed so far. In this context, a relevant question arises: What can chemistry do in the search for new strategies and approaches to solve this emergent problem? Although initially a variety of known chemical compounds-for other purposes-proved disappointing in tests against Ebola virus (EBOV) infection, more recently, specific molecules have been prepared. In this Perspective, we present new approaches directed at the design of efficient entry inhibitors to minimize the development of resistance by viral mutations. In particular, we focus on dendrimers as well as fullerene C-with a unique symmetrical and 3D globular structure-as biocompatible carbon platforms for the multivalent presentation of carbohydrates. The antiviral activity of these compounds in an Ebola pseudotyped infection model was in the low micromolar range for fullerenes with 12 and 36 mannoses. However, new tridecafullerenes-in which the central alkyne scaffold of [60]fullerene is connected to 12 sugar-containing [60]fullerene units (total 120 mannoses)-exhibit an outstanding antiviral activity with IC in the sub-nanomolar range! The multivalent presentation of specific carbohydrates by using 3D fullerenes as controlled biocompatible carbon scaffolds represents a real advance, being currently the most efficient molecules in vitro against EBOV infection. However, additional studies are needed to determine the optimized fullerene-based leads for practical applications.
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http://dx.doi.org/10.1021/jacs.7b01683DOI Listing
May 2017

LPS promotes Th2 dependent sensitisation leading to anaphylaxis in a Pru p 3 mouse model.

Sci Rep 2017 01 13;7:40449. Epub 2017 Jan 13.

Research Laboratory, IBIMA, Regional University Hospital of Malaga, UMA, Malaga, Spain.

Pru p 3 is the major peach allergen in the Mediterranean area. It frequently elicits severe reactions, limiting its study in humans, raising the need for animal models to investigate the immunological mechanisms involved. However, no anaphylaxis model exists for Pru p 3. We aimed to develop a model of peach anaphylaxis by sensitising mice with Pru p 3 in combination with lipopolysaccharide (LPS) as an adjuvant. Four groups of mice were sensitised intranasally: untreated; treated with Pru p 3; treated with LPS; treated with Pru p 3 + LPS. After sensitisation mice were intraperitoneally challenged with Pru p 3 and in vivo and in vitro parameters were evaluated. Only mice in the Pru p 3 + LPS group showed anaphylaxis symptoms, including a decrease in temperature. Determination of in vitro parameters showed a Th2 response with an increase of Pru p 3-specific IgE and IgG1. Moreover, at the cellular level, we found increased levels of IgE and IgG1 secreting Pru p 3-specific cells and a proliferative CD4 T-cell response. These results demonstrate that Pru p 3-specific anaphylaxis can be generated after nasal sensitisation to Pru p 3 in combination with LPS. This is a promising model for evaluating food allergy immunotherapies.
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http://dx.doi.org/10.1038/srep40449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233975PMC
January 2017

Cyclooctyne [60]fullerene hexakis adducts: a globular scaffold for copper-free click chemistry.

Chem Commun (Camb) 2016 Aug;52(69):10544-6

Departamento de Química Orgánica, Fac. CC. Químicas, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid, Spain. and IMDEA-Nanoscience, Campus Cantoblanco, 28049 Madrid, Spain.

The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications.
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http://dx.doi.org/10.1039/c6cc05484fDOI Listing
August 2016

Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection.

Nat Chem 2016 Jan;8(1):50-7

The use of multivalent carbohydrate compounds to block cell-surface lectin receptors is a promising strategy to inhibit the entry of pathogens into cells and could lead to the discovery of novel antiviral agents. One of the main problems with this approach, however, is that it is difficult to make compounds of an adequate size and multivalency to mimic natural systems such as viruses. Hexakis adducts of [60]fullerene are useful building blocks in this regard because they maintain a globular shape at the same time as allowing control over the size and multivalency. Here we report water-soluble tridecafullerenes decorated with 120 peripheral carbohydrate subunits, so-called 'superballs', that can be synthesized efficiently from hexakis adducts of [60]fullerene in one step by using copper-catalysed azide–alkyne cycloaddition click chemistry. Infection assays show that these superballs are potent inhibitors of cell infection by an artificial Ebola virus with half-maximum inhibitory concentrations in the subnanomolar range.
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http://dx.doi.org/10.1038/nchem.2387DOI Listing
January 2016

Rapid and efficient synthesis of α(1-2)mannobiosides.

Org Biomol Chem 2016 Mar;14(10):2873-82

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Américo Vespucio, 49, 41092, Sevilla, Spain.

α(1,2)mannobiosides with different substituents at the reducing end have been synthesized by a common strategy using benzoyls as the permanent protecting groups and an acetyl as the orthogonal protecting group at position C2 of the glycosyl acceptor. The new synthetic strategy has been performed remarkably reducing the number of purification steps, the time of synthesis (less than 72 hours) and improving the overall yield at least three times with respect to the best procedure described in the literature at the moment. Additionally, this protecting group strategy is compatible with the presence of azido groups and the use of Cu catalyzed azide alkyne cycloaddition (CuAAC) also called "click chemistry" for conjugating the α(1-2)mannobiosides to different scaffolds for the preparation of mannosyl multivalent systems.
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http://dx.doi.org/10.1039/c6ob00083eDOI Listing
March 2016

Multivalent Glycosylation of Fluorescent Gold Nanoclusters Promotes Increased Human Dendritic Cell Targeting via Multiple Endocytic Pathways.

ACS Appl Mater Interfaces 2015 Sep 8;7(37):20945-56. Epub 2015 Sep 8.

Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), CSIC-University of Seville , 41092 Seville, Spain.

We report the synthesis and characterization of gold nanoclusters (Au NCs) stabilized by a mixture of zwitterionic and multivalent mannose ligands. Characterization of this carbohydrated nanosystem confirms its small size (∼2 nm), intense red-NIR fluorescence, relatively high affinity to lectin (ConA), and stability in physiological media. Cell studies performed using human-monocyte-derived dendritic cells (DCs) show that Au NC uptake efficiency is greatly enhanced by the presence of surface carbohydrate (>250% compared to noncarbohydrated Au NCs), allowing their detection in cells by fluorescence following incubation with concentrations as low as 1 μg mL(-1). Investigation using electron microscopy and pharmacological inhibitors indicates that Au NC uptake is mediated by multiple endocytic pathways involving the engulfment of Au NCs into endosomes and partial transport to lysosomes. Results show that clathrin- and F-actin-dependent pathways play major roles in Au NC uptake by DCs, regardless of whether or not they are coated with carbohydrates. In contrast, a specific C-lectin inhibitor induces a 60% decrease in DC particle uptake only for the carbohydrate-coated Au NCs. This study demonstrates that the combination of ultrasmall gold NCs and functionalization with multivalent mannose ligands results in greatly enhanced human DC targeting, presumably due to increased diffusion and target cell binding, respectively.
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http://dx.doi.org/10.1021/acsami.5b06541DOI Listing
September 2015

Investigation of glycofullerene dynamics by NMR spectroscopy.

Org Biomol Chem 2015 Aug 17;13(32):8750-5. Epub 2015 Jul 17.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

Glycofullerenes, in which carbohydrate molecules are attached via a linker to a [60]fullerene core, facilitate spherical presentation of glyco-based epitopes. We herein investigate the dynamics of two glycofullerenes, having 12 and 36 mannose residues at their periphery, by NMR translational diffusion and quantitative (13)C relaxation studies employing a model-free approach for their interpretation. The sugar residues are shown to be highly flexible entities with S(2) < 0.2 in both compounds. Notably, the larger glycofullerene with longer linkers shows faster internal dynamics and higher flexibility than its smaller counterpart. The dynamics and flexibility as well as the slower translational diffusion of the larger glycofullerene, thereby favoring rebinding to a receptor, may together with its spatial extension explain why it is better than the smaller one at blocking the DC-SIGN receptor and inhibiting the infection by pseudotyped Ebola virus particles.
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http://dx.doi.org/10.1039/c5ob00929dDOI Listing
August 2015

Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.

J Am Chem Soc 2015 Apr 23;137(12):4100-10. Epub 2015 Mar 23.

†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.
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http://dx.doi.org/10.1021/ja511529xDOI Listing
April 2015

Human dendritic cell activation induced by a permannosylated dendron containing an antigenic GM3-lactone mimetic.

Beilstein J Org Chem 2014 10;10:1317-1324. Epub 2014 Jun 10.

Department of Chemistry, University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino (FI), Italy.

Vaccination strategies based on dendritic cells (DCs) armed with specific tumor antigens have been widely exploited due the properties of these immune cells in coordinating an innate and adaptive response. Here, we describe the convergent synthesis of the bifunctional multivalent glycodendron 5, which contains nine residues of mannose for DC targeting and one residue of an immunogenic mimetic of a carbohydrate melanoma associated antigen. The immunological assays demonstrated that the glycodendron 5 is able to induce human immature DC activation in terms of a phenotype expression of co-stimulatory molecules expression and MHCII. Furthermore, DCs activated by the glycodendron 5 stimulate T lymphocytes to proliferate in a mixed lymphocytes reaction (MLR).
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http://dx.doi.org/10.3762/bjoc.10.133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077398PMC
July 2014