RESUMO
The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.
Assuntos
Lectinas Tipo C , Manose , Humanos , Concanavalina A/metabolismo , Manose/química , Lectinas Tipo C/metabolismo , Oligossacarídeos/química , Sítios de Ligação , Lectinas de Ligação a Manose/químicaRESUMO
Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9 of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope.
Assuntos
Lectinas Tipo C , Manose , Humanos , Manose/química , Lectinas Tipo C/metabolismo , Oligossacarídeos/química , Açúcares , Espectroscopia de Ressonância Magnética , Epitopos , Células DendríticasRESUMO
Introduction: Lipid transfer proteins (LTPs) are allergens found in a wide range of plant-foods. Specifically, Pru p 3, the major allergen of peach, is commonly responsible for severe allergic reactions. The need for new alternatives to conventional food allergy treatments, like restrictive diets, suggests allergen immunotherapy as a promising option. It has been demonstrated that sublingual immunotherapy (SLIT) with synthetic glycodendropeptides, such as D1ManPrup3, containing mannose and Pru p 3 peptides induced tolerance in mice and that the persistence of this effect depends on treatment dose (2nM or 5nM). Moreover, it produces changes associated with differential gene expression and methylation profile of dendritic cells, as well as phenotypical changes in regulatory T cells (Treg). However, there are no works addressing the study of epigenetic changes in terms of methylation in the cell subsets that sustain tolerant responses, Treg. Therefore, in this work, DNA methylation changes in splenic-Treg from Pru p 3 anaphylactic mice were evaluated. Methods: It was performed by whole genome bisulphite sequencing comparing SLIT-D1ManPrup3 treated mice: tolerant (2nM D1ManPrup3), desensitized (5nM D1ManPrup3), and sensitized but not treated (antigen-only), with anaphylactic mice. Results: Most of the methylation changes were found in the gene promoters from both SLIT-treated groups, desensitized (1,580) and tolerant (1,576), followed by the antigen-only (1,151) group. Although tolerant and desensitized mice showed a similar number of methylation changes, only 445 genes were shared in both. Remarkably, interesting methylation changes were observed on the promoter regions of critical transcription factors for Treg function like Stat4, Stat5a, Stat5b, Foxp3, and Gata3. In fact, Foxp3 was observed exclusively as hypomethylated in tolerant group, whereas Gata3 was only hypomethylated in the desensitized mice. Discussion: In conclusion, diverse D1ManPrup3 doses induce different responses (tolerance or desensitization) in mice, which are reflected by differential methylation changes in Tregs.
Assuntos
Anafilaxia , Hipersensibilidade Alimentar , Animais , Camundongos , Linfócitos T Reguladores , Hipersensibilidade Alimentar/terapia , Anafilaxia/metabolismo , Alérgenos/metabolismo , Metilação de DNA , Fatores de Transcrição Forkhead/metabolismoRESUMO
Autoimmune diseases are life-threatening disorders that cause increasing disability over time. Systemic lupus erythematosus (SLE) and other autoimmune diseases arise when immune stimuli override mechanisms of self-tolerance. Accumulating evidence has demonstrated that protein glycosylation is substantially altered in autoimmune disease development, but the mechanisms by which glycans trigger these autoreactive immune responses are still largely unclear. In this study, we found that presence of microbial-associated mannose structures at the surface of the kidney triggers the recognition of DC-SIGN-expressing γδ T cells, inducing a pathogenic interleukin-17a (IL-17a)-mediated autoimmune response. Mice lacking Mgat5, which have a higher abundance of mannose structures in the kidney, displayed increased γδ T cell infiltration into the kidney that was associated with spontaneous development of lupus in older mice. N-acetylglucosamine supplementation, which promoted biosynthesis of tolerogenic branched N-glycans in the kidney, was found to inhibit γδ T cell infiltration and control disease development. Together, this work reveals a mannose-γδ T cell-IL-17a axis in SLE immunopathogenesis and highlights glycometabolic reprogramming as a therapeutic strategy for autoimmune disease treatment.
Assuntos
Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Animais , Camundongos , Autoimunidade , Manose , Interleucina-17/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.
Assuntos
Nanopartículas Metálicas , Nanopartículas Metálicas/química , Ouro/química , Celulose/química , Química Click , Lectinas Tipo CRESUMO
Human lectins are involved in a wide variety of biological processes, both physiological and pathological, which have attracted the interest of the scientific community working in the glycoscience field. Multivalent glycosystems have been employed as useful tools to understand carbohydrate-lectin binding processes as well as for biomedical applications. The review shows the different scaffolds designed for a multivalent presentation of sugars and their corresponding binding studies to lectins and in some cases, their biological activities. We summarise this research by organizing based on lectin types to highlight the progression in this active field. The paper provides an overall picture of how these contributions have furnished relevant information on this topic to help in understanding and participate in these carbohydrate-lectin interactions.
Assuntos
Carboidratos , Lectinas , Humanos , Lectinas/metabolismoRESUMO
Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.
Assuntos
Biomimética , Lectinas de Ligação a Manose , Lectinas de Ligação a Manose/metabolismo , Antígenos CD/metabolismo , Sítios de Ligação , Lectinas Tipo C/metabolismo , Ligação ProteicaRESUMO
Food allergy induced by lipid transfer proteins (LTPs) of Rosacea fruit family, such as peach, is becoming an important health problem in the Mediterranean area. Current treatments, such as allergen specific immunotherapy (AIT) with allergenic extracts show promising, but in many cases, they need an improvement in homogeneity, availability and induction of tolerant responses. Peptide-based vaccines containing adjuvants, such as carbohydrates for C-type lectin receptors (CLRs) are presented as an alternative approach. In this work, we have prepared fucosylated glycodendropeptides (GDPs) functionalized with Pru p 3 peptides via click chemistry. These GDPs, DnFuc9Prup3, induced changes in moDC maturation and lymphocyte proliferation in food allergic patients, indicating specific recognition via DC-SIGN receptor. From these data, D4Fuc9Prup3 can be considered a promising candidate for specific immunotherapy development.
Assuntos
Antígenos de Plantas , Proteínas de Plantas , Alérgenos , Antígenos de Plantas/metabolismo , Moléculas de Adesão Celular , Células Dendríticas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superfície CelularRESUMO
The synthesis of new biocompatible antiviral materials to fight against the development of multidrug resistance is being widely explored. Due to their unique globular structure and excellent properties, [60]fullerene-based antivirals are very promising bioconjugates. In this work, fullerene derivatives with different topologies and number of glycofullerene units were synthesized by using a SPAAC copper free strategy. This procedure allowed the synthesis of compounds 1-3, containing from 20 to 40 mannose units, in a very efficient manner and in short reaction times under MW irradiation. The glycoderivatives were studied in an infection assay by a pseudotyped viral particle with Ebola virus GP1. The results obtained show that these glycofullerene oligomers are efficient inhibitors of EBOV infection with IC50s in the nanomolar range. In particular, compound 3, with four glycofullerene moieties, presents an outstanding relative inhibitory potency (RIP). We propose that this high RIP value stems from the appropriate topological features that efficiently interact with DC-SIGN.
Assuntos
Ebolavirus , Fulerenos , Doença pelo Vírus Ebola , Antivirais/uso terapêutico , Fulerenos/química , Doença pelo Vírus Ebola/tratamento farmacológico , Humanos , Manose/químicaRESUMO
In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.
RESUMO
To investigate food allergy-tolerance mechanisms induced through allergen-specific immunotherapy we used RNA-Sequencing to measure gene expression in lymph-node-derived dendritic cells from Pru p 3-anaphylactic mice after immunotherapy with glycodendropeptides at 2 nM and 5 nM, leading to permanent tolerance and short-term desensitization, respectively. Gene expression was also measured in mice receiving no immunotherapy (anaphylaxis); and in which anaphylaxis could never occur (antigen-only). Compared to anaphylaxis, the antigen-only group showed the greatest number of expression-changes (411), followed by tolerant (186) and desensitized (119). Only 29 genes changed in all groups, including Il12b, Cebpb and Ifngr1. The desensitized group showed enrichment for genes related to chronic inflammatory response, secretory granule, and regulation of interleukin-12 production; the tolerant group showed genes related to cytokine receptor activity and glucocorticoid receptor binding, suggesting distinct pathways for similar outcomes. We identified genes and processes potentially involved in the restoration of long-term tolerance via allergen-specific immunotherapy, representing potential prognostic biomarkers.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Dessensibilização Imunológica , Tolerância Imunológica/genética , Subunidade p40 da Interleucina-12/genética , Receptores de Interferon/genética , Alérgenos/imunologia , Alérgenos/farmacologia , Anafilaxia/genética , Anafilaxia/imunologia , Animais , Antígenos de Plantas/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicopeptídeos/farmacologia , Humanos , Interleucina-12/genética , Linfonodos/imunologia , Camundongos , Proteínas de Plantas/farmacologia , RNA-Seq , Receptor de Interferon gamaRESUMO
Introduction: Allergen-specific immunotherapy (AIT) is applied as treatment to rise tolerance in patients with food allergies. Although AIT is thoroughly used, the underlying epigenetic events related to tolerant induction are still unknown. Thus, we aim to investigate epigenetic changes that could be related to tolerance in dendritic cells (DCs) from anaphylactic mice to lipid transfer proteins, Pru p 3, in the context of a sublingual immunotherapy (SLIT) with a glycodendropeptide (D1ManPrup3) that has demonstrated tolerant or desensitization responses depending on the treatment dose. Methods: Changes in DNA methylation in CpG context were determined comparing Sensitized (Antigen-only) animals and two groups receiving SLIT with the D1ManPrup3 nanostructure (D1ManPrup3-SLIT): Tolerant (2nM D1ManPrup3) and Desensitized (5nM D1ManPrup3), against anaphylactic animals. DNA from lymph nodes-DCs were isolated and then, Whole Genome Bisulphite Sequencing was performed to analyze methylation. Results: Most differentially methylated regions were found on the area of influence of gene promoters (DMPRs). Compared to the Anaphylactic group, the highest value was found in Desensitized mice (n = 7,713 DMPRs), followed by Tolerant (n = 4,091 DMPRs) and Sensitized (n = 3,931 DMPRs) mice. Moreover, many of these epigenetic changes were found in genes involved in immune and tolerance responses (Il1b, Il12b, Il1a, Ifng, and Tnf) as shown by functional enrichment (DCs regulation, B cell-mediated immunity, and effector mechanisms). Discussion: In conclusion, different doses of D1ManPrup3-SLIT induce different DNA methylation changes, which are reflected in the induction of distinct responses, tolerance, or desensitization.
Assuntos
Anafilaxia , Hipersensibilidade Alimentar , Imunoterapia Sublingual , Animais , Camundongos , Metilação , Alérgenos , Dessensibilização ImunológicaRESUMO
Investigators traditionally use randomized designs and corresponding analysis procedures to make causal inferences about the effects of interventions, assuming independence between an individual's outcome and treatment assignment and the outcomes of other individuals in the study. Often, such independence may not hold. We provide examples of interdependency in model organism studies and human trials and group effects in aging research and then discuss methodologic issues and solutions. We group methodologic issues as they pertain to (1) single-stage individually randomized trials; (2) cluster-randomized controlled trials; (3) pseudo-cluster-randomized trials; (4) individually randomized group treatment; and (5) two-stage randomized designs. Although we present possible strategies for design and analysis to improve the rigor, accuracy and reproducibility of the science, we also acknowledge real-world constraints. Consequences of nonadherence, differential attrition or missing data, unintended exposure to multiple treatments and other practical realities can be reduced with careful planning, proper study designs and best practices.
Assuntos
Gerociência , Humanos , Animais , Camundongos , Reprodutibilidade dos Testes , Distribuição Aleatória , CausalidadeRESUMO
Langerin is a C-type Lectin expressed at the surface of Langerhans cells, which play a pivotal role protecting organisms against pathogen infections. To address this aim, Langerin presents at least two recognition sites, one Ca2+-dependent and another one independent, which are capable to recognize a variety of carbohydrate ligands. In contrast to other lectins, Langerin recognizes sulfated glycosaminoglycans (GAGs), a family of complex and heterogeneous polysaccharides present in the cell membrane and the extracellular matrix, at the interphase generated in the trimeric form of Langerin but absent in the monomeric form. The complexity of these oligosaccharides has impeded the development of welldefined monodisperse structures to study these interaction processes. However, in the last few decades, an improvement of synthetic developments to achieve the preparation of carbohydrate multivalent systems mimicking the GAGs has been described. Despite all these contributions, very few examples are reported where the GAG multivalent structures are used to evaluate the interaction with Langerin. These molecules should pave the way to explore these GAG-Langerin interactions.
Assuntos
Antígenos CD , Lectinas de Ligação a Manose , Antígenos CD/química , Células de Langerhans/metabolismo , Lectinas Tipo C/química , Ligantes , Lectinas de Ligação a Manose/químicaRESUMO
Covalent conjugation of allergens to toll-like receptor (TLR) agonists appears to be a powerful strategy for the development of safety compounds for allergen-specific immunomodulatory response toward tolerance in allergy. In this work, we have synthesized two family of ligands, an 8-oxoadenine derivative as a ligand for TLR7 and a pyrimido[5,4-b]indole as a ligand for TLR4, both conjugated with a T-cell peptide of Pru p 3 allergen, the lipid transfer protein (LTP) responsible for LTP-dependent food allergy. These conjugates interact with dendritic cells, inducing their specific maturation, T-cell proliferation, and cytokine production in peach allergic patients. Moreover, they increased the Treg-cell frequencies in these patients and could induce the IL-10 production. These outcomes were remarkable in the case of the TLR7 ligand conjugated with Pru p 3, opening the door for the potential application of these allergen-adjuvant systems in food allergy immunotherapy.
Assuntos
Hipersensibilidade Alimentar/metabolismo , Imunomodulação , Peptídeos/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Alérgenos/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células , Citocinas/biossíntese , Hipersensibilidade Alimentar/imunologia , Humanos , Ligantes , Linfócitos T/citologia , Linfócitos T/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 7 Toll-Like/agonistasRESUMO
Glycodendron microarrays with defined valency have been constructed by on-chip synthesis on hydrophobic indium tin oxide (ITO) coated glass slides and employed in lectin-carbohydrate binding studies with several plant and human lectins. Glycodendrons presenting sugar epitopes at different valencies were prepared by spotwise strain-promoted azide-alkyne cycloaddition (SPAAC) between immobilised cyclooctyne dendrons and azide functionalised glycans. The non-covalent immobilisation of dendrons on the ITO surface by hydrophobic interaction allowed us to study dendron surface density and SPAAC conversion rate by in situ MALDI-TOF MS analysis. By diluting the dendron surface density we could study how the carbohydrate-lectin interactions became exclusively dependant on the valency of the immobilised glycodendron.
Assuntos
LectinasRESUMO
DC-SIGN, a lectin discovered two decades ago, plays a relevant role in innate immunity. Since its discovery, it has turned out to be a target for developing antiviral drugs based on carbohydrates due to its participation in the infection process of several pathogens. A plethora of carbohydrate multivalent systems using different scaffolds have been described to achieve this goal. Our group has made significant contributions to this field, which are revised herein.
Assuntos
Antracenos/química , Moléculas de Adesão Celular/química , Glicosídeos/química , Lectinas Tipo C/química , Receptores de Superfície Celular/química , Antracenos/farmacologia , Antivirais/química , Antivirais/farmacologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Vírus da Dengue/efeitos dos fármacos , Fulerenos/química , Lectinas Tipo C/metabolismo , Nanopartículas Metálicas/química , Conformação Molecular , Receptores de Superfície Celular/metabolismo , Zika virus/efeitos dos fármacosRESUMO
Suitably engineered molecular systems exhibiting triplet excited states with very long lifetimes are important for high-end applications in nonlinear optics, photocatalysis, or biomedicine. We report the finding of an ultra-long-lived triplet state with a mean lifetime of 93â ms in an aqueous phase at room temperature, measured for a globular tridecafullerene with a highly compact glycodendrimeric structure. A series of three tridecafullerenes bearing different glycodendrons and spacers to the C60 units have been synthesized and characterized. UV/Vis spectra and DLS experiments confirm their aggregation in water. Steady-state and time-resolved fluorescence experiments suggest a different degree of inner solvation of the multifullerenes depending on their molecular design. Efficient quenching of the triplet states by O2 but not by waterborne azide anions has been observed. Molecular modelling reveals dissimilar access of the aqueous phase to the internal structure of the tridecafullerenes, differently shielded by the glycodendrimeric shell.
RESUMO
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.