Spanish Heart Transplant Registry. 31th Official Report of the Heart Failure Association of the Spanish Society of Cardiology.

INTRODUCTION AND OBJECTIVES: The present report describes the clinical characteristics and outcomes of heart transplants in Spain and updates the data to 2019. METHODS: We describe the clinical characteristics and outcomes of heart transplants performed in Spain in 2019, as well as trends in this procedure from 2010 to 2018. RESULTS: In 2019, 300 transplants were performed (8794 since 1984; 2745 between 2010 and 2019). Compared with previous years, the most notable findings were the decreasing rate of urgent transplants (38%), and the consolidation of the type of circulatory support prior to transplant, with an almost complete disappearance of counterpulsation balloon (0.7%), stabilization in the use of extracorporeal membrane oxygenation (9.6%), and an increase in the use of ventricular assist devices (29.0%). Survival from 2016 to 2018 was similar to that from 2013 to 2015 (P=.34). Survival in both these periods was better than that from 2010 to 2012 (P=.002 and P=.01, respectively). CONCLUSIONS: Heart transplant activity has remained stable during the last few years, as have outcomes (in terms of survival). There has been a trend to a lower rate of urgent transplants and to a higher use of ventricular assist devices prior to transplant.

Loading dendritic cells with gold nanoparticles (GNPs) bearing HIV-peptides and mannosides enhance HIV-specific T cell responses.

Gold nanoparticles (GNPs) decorated with glycans ameliorate dendritic cells (DC) uptake, antigen-presentation and T-cells cross-talk, which are important aspects in vaccine design. GNPs allow for high antigen loading, DC targeting, lack of toxicity and are straightforward prepared and easy to handle. The present study aimed to assess the capacity of DC to process and present HIV-1-peptides loaded onto GNPs bearing high-mannoside-type oligosaccharides (P1@HM) to autologous T-cells from HIV-1 patients. The results showed that P1@HM increased HIV-specific CD4+ and CD8+ T-cell proliferation and induced highly functional cytokine secretion compared with HIV-peptides alone. P1@HM elicits a highly efficient secretion of pro-TH1 cytokines and chemokines, a moderate production of pro-TH2 and significant higher secretion of pro-inflammatory cytokines such as TNF-α and IL-1ß. Thus, co-delivery of HIV-1 antigens and HM by GNPs is an excellent vaccine delivery system inducing HIV-specific cellular immune responses in HIV+ patients, being a promising approach to improve anti-HIV-1 vaccines.

GNP-GAPDH1-22 nanovaccines prevent neonatal listeriosis by blocking microglial apoptosis and bacterial dissemination.

Clinical cases of neonatal listeriosis are associated with brain disease and fetal loss due to complications in early or late pregnancy, which suggests that microglial function is altered. This is believed to be the first study to link microglial apoptosis with neonatal listeriosis and listeriosis-associated brain disease, and to propose a new nanovaccine formulation that reverses all effects of listeriosis and confers Listeria monocytogenes (LM)-specific immunity. We examined clinical cases of neonatal listeriosis in 2013-2015 and defined two useful prognostic immune biomarkers to design listeriosis vaccines: high anti-GAPDH1-22 titres and tumor necrosis factor (TNF)/interleukin (IL)-6 ratios. Therefore, we developed a nanovaccine with gold glyco-nanoparticles conjugated to LM peptide 1-22 of GAPDH (Lmo2459), GNP-GAPDH1-22 nanovaccinesformulated with a pro-inflammatory Toll-like receptor 2/4-targeted adjuvant. Neonates born to non-vaccinated pregnant mice with listeriosis, showed brain and vascular diseases and significant microglial dysfunction by induction of TNF-α-mediated apoptosis. This programmed TNF-mediated suicide explains LM dissemination in brains and livers and blocks production of early pro-inflammatory cytokines such as IL-1ß and interferon-α/ß. In contrast, neonates born to GNP-GAPDH1-22-vaccinated mothers before LM infection, did not develop listeriosis or brain diseases and had functional microglia. In nanovaccinated mothers, immune responses shifted towards Th1/IL-12 pro-inflammatory cytokine profiles and high production of anti-GAPDH1-22 antibodies, suggesting good induction of LM-specific memory.

Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model.

AIM: Nanotechnology-based fully synthetic carbohydrate vaccines are promising alternatives to classic polysaccharide/protein conjugate vaccines. We have prepared gold glyco-nanoparticles (GNP) bearing two synthetic carbohydrate antigens related to serotypes 19F and 14 of Streptococcus pneumoniae and evaluated their immunogenicity in vivo. RESULTS: A tetrasaccharide fragment of serotype 14 (Tetra-14), a trisaccharide fragment of serotype 19F (Tri-19F), a T-helper peptide and d -glucose were loaded onto GNP in different ratios. Mice immunization showed that the concomitant presence of Tri-19F and Tetra-14 on the same nanoparticle critically enhanced the titers of specific IgG antibodies toward type 14 polysaccharide compared with GNP exclusively displaying Tetra-14, while no IgG antibodies against type 19F polysaccharide were elicited. CONCLUSION: This work is a step forward toward synthetic nanosystems combining carbohydrate antigens and immunogenic peptides as potential carbohydrate-based vaccines.

Opportunities for glyconanomaterials in personalized medicine.

In this feature article we discuss the particular relevance of glycans as components or targets of functionalized nanoparticles (NPs) for potential applications in personalized medicine but we will not enter into descriptions for their preparation. For a more general view covering the preparation and applications of glyconanomaterials the reader is referred to a number of recent reviews. The combination of glyco- and nanotechnology is already providing promising new tools for more personalized solutions to diagnostics and therapy. Current applications relevant to personalized medicine include drug targeting, localized radiation therapy, imaging of glycan expression of cancer cells, point of care diagnostics, cancer vaccines, photodynamic therapy, biosensors, and glycoproteomics.

Pregnancy Vaccination with Gold Glyco-Nanoparticles Carrying Listeria monocytogenes Peptides Protects against Listeriosis and Brain- and Cutaneous-Associated Morbidities.

Listeriosis is a fatal infection for fetuses and newborns with two clinical main morbidities in the neonatal period, meningitis and diffused cutaneous lesions. In this study, we vaccinated pregnant females with two gold glyconanoparticles (GNP) loaded with two peptides, listeriolysin peptide 91-99 (LLO91-99) or glyceraldehyde-3-phosphate dehydrogenase 1-22 peptide (GAPDH1-22). Neonates born to vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice presented clear brain affections and cutaneous diminishment of melanocytes. Therefore, these nanoparticle vaccines are effective measures to offer pregnant mothers at high risk of listeriosis interesting therapies that cross the placenta.

Targeted gold-coated iron oxide nanoparticles for CD163 detection in atherosclerosis by MRI.

CD163 is a membrane receptor expressed by macrophage lineage. Studies performed in atherosclerosis have shown that CD163 expression is increased at inflammatory sites, pointing at the presence of intraplaque hemorrhagic sites or asymptomatic plaques. Hence, imaging of CD163 expressing macrophages is an interesting strategy in order to detect atherosclerotic plaques. We have prepared a targeted probe based on gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody for the specific detection of CD163 by MRI. Firstly, the specificity of the targeted probe was validated in vitro by incubation of the probe with CD163(+) or (-) macrophages. The probe was able to selectively detect CD163(+) macrophages both in human and murine cells. Subsequently, the targeted probe was injected in 16 weeks old apoE deficient mice developing atherosclerotic lesions and the pararenal abdominal aorta was imaged by MRI. The accumulation of probe in the site of interest increased over time and the signal intensity decreased significantly 48 hours after the injection. Hence, we have developed a highly sensitive targeted probe capable of detecting CD163-expressing macrophages that could provide useful information about the state of the atheromatous lesions.

Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods.

Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations.

Negatively charged glyconanoparticles modulate and stabilize the secondary structures of a gp120 V3 loop peptide: toward fully synthetic HIV vaccine candidates.

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or ß-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3ß-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3ß-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3ß-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates.

A gold glyco-nanoparticle carrying a Listeriolysin O peptide and formulated with Advax™ delta inulin adjuvant induces robust T-cell protection against listeria infection.

In the search for an effective vaccine against the human pathogen, Listeria monocytogenes (Listeria), gold glyconanoparticles (GNP) loaded with a listeriolysin O peptide LLO91-99 (GNP-LLO) were used to immunise mice, initially using a dendritic cell (DC) vaccine approach, but subsequently using a standard parenteral immunisation approach. To enhance vaccine immunogenicity a novel polysaccharide adjuvant based on delta inulin (Advax™) was also co-formulated with the GNP vaccine. Confirming previous results, DC loaded in vitro with GNP-LLO provided better protection against listeriosis than DC loaded in vitro using free LLO peptide. The immunogenicity of GNP-LLO loaded DC vaccines was further increased by addition of Advax™ adjuvant. However, as DC vaccines are expensive and impracticable for prophylactic use, we next asked whether the same GNP-LLO antigen could be used to directly target DC in vivo. Immunisation of mice with GNP-LLO plus Advax™ adjuvant induced LLO-specific T-cell immunity and protection against Listeria challenge. Protection correlated with an increased frequency of splenic CD4(+) and CD8(+) T cells, NK cells and CD8α(+) DC, and Th1 cytokine production (IL-12, IFN-γ, TNF-α, and MCP-1), post-challenge. Enhanced T-cell epitope recruitment post-challenge was seen in the groups that received Advax™ adjuvant. Immunisation with GNP-LLO91-99 plus Advax™ adjuvant provided equally robust Listeria protection as the best DC vaccine strategy but without the complexity and cost, making this a highly promising strategy for development of a prophylactic vaccine against listeriosis.

Glycans as biofunctional ligands for gold nanorods: stability and targeting in protein-rich media.

Poly(ethylene glycol) (PEG) has become the gold standard for stabilization of plasmonic nanoparticles (NPs) in biofluids, because it prevents aggregation while minimizing unspecific interactions with proteins. Application of Au NPs in biological environments requires the use of ligands that can target selected receptors, even in the presence of protein-rich media. We demonstrate here the stabilizing effect of low-molecular-weight glycans on both spherical and rod-like plasmonic NPs under physiological conditions, as bench-marked against the well-established PEG ligands. Glycan-coated NPs are resistant to adsorption of proteins from serum-containing media and avoid phagocytosis by macrophage-like cells, but retain selectivity toward carbohydrate-binding proteins in protein-rich biological media. These results open the way toward the design of efficient therapeutic/diagnostic glycan-decorated plasmonic nanotools for specific biological applications.

Detection of mouse endogenous type B astrocytes migrating towards brain lesions.

Neuroblasts represent the predominant migrating cell type in the adult mouse brain. There are, however, increasing evidences of migration of other neural precursors. This work aims at identifying in vivo endogenous early neural precursors, different from neuroblasts, able to migrate in response to brain injuries. The monoclonal antibody Nilo1, which unequivocally identifies type B astrocytes and embryonic radial glia, was coupled to magnetic glyconanoparticles (mGNPs). Here we show that Nilo1-mGNPs in combination with magnetic resonance imaging in living mice allowed the in vivo identification of endogenous type B astrocytes at their niche, as well as their migration to the lesion site in response to glioblastoma, demyelination, cryolesion or mechanical injuries. In addition, Nilo1(+) adult radial glia-like structures were identified at the lesion site a few hours after damage. For all damage models used, type B astrocyte migration was fast and orderly. Identification of Nilo1(+) cells surrounding an induced glioblastoma was also possible after intraperitoneal injection of the antibody. This opens up the possibility of an early identification of the initial damage site(s) after brain insults, by the migration of type B astrocytes.

Assembling different antennas of the gp120 high mannose-type glycans on gold nanoparticles provides superior binding to the anti-HIV antibody 2G12 than the individual antennas.

In order to re-build Man9GlcNAc2 clusters of the HIV gp120 glycoprotein, ∼2 nm gold glyconanoparticles (GNPs) were coated with the synthetic partial structures of Man9, the tetramannoside Manα1-2Manα1-2Manα1-3Manα1- and the pentamannoside Manα1-2Manα1-3[Manα1-2Manα1-6]Manα1-. Their interactions with the anti-HIV broadly neutralizing antibody 2G12 were studied by surface plasmon resonance (SPR)-based biosensors and saturation transfer difference (STD)-NMR spectroscopy. A synergistic effect of the tetra- and pentamannosides multimerized on a same GNP was observed. The assembly of these antennas of the gp120 high-mannose type glycan on GNPs provided superior binding to the anti-HIV antibody 2G12 with respect to GNPs carrying only the individual oligomannosides. The results presented in this work provide new molecular information on the interactions between clusters of oligomannosides and 2G12 that could help in the design of a carbohydrate-based vaccine against HIV.

Effective targeting of DC-SIGN by α-fucosylamide functionalized gold nanoparticles.

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-ß-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.

Glycosystems in nanotechnology: Gold glyconanoparticles as carrier for anti-HIV prodrugs.

The therapeutic approach for the treatment of HIV infection is based on the highly active antiretroviral therapy (HAART), a cocktail of antiretroviral drugs. Notwithstanding HAART has shown different drawbacks like toxic side effects and the emergence of viral multidrug resistance. Nanotechnology offers new tools to improve HIV drug treatment and prevention. In this scenario, gold nanoparticles are an interesting chemical tool to design and prepare smart and efficient drug-delivery systems. Here we describe the preparation and antiviral activity of carbohydrate-coated gold nanoparticles loaded with anti-HIV prodrug candidates. The nucleoside reverse transcriptase inhibitors abacavir and lamivudine have been converted to the corresponding thiol-ending ester derivatives and then conjugated to ~3 nm glucose-coated gold nanoparticles by means of "thiol-for-thiol" ligand place exchange reactions. The drugs-containing glyconanoparticles were characterized and the pH-mediated release of the drug from the nanoparticle has been determined. The antiviral activity was tested by evaluating the replication of NL4-3 HIV in TZM-bl infected cells. The proof-of-principle presented in this work aims to introduce gold glyconanoparticles as a new multifunctional drug-delivery system in the therapy against HIV.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

A quantitative study of the intracellular dynamics of fluorescently labelled glyco-gold nanoparticles via fluorescence correlation spectroscopy.

The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc-Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc-Au NPs fluorescently-labelled with HiLyte Fluor647 (Glc-Au-Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a "prebleaching" methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc-Au-Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media.

68Ga-labeled gold glyconanoparticles for exploring blood-brain barrier permeability: preparation, biodistribution studies, and improved brain uptake via neuropeptide conjugation.

New tools and techniques to improve brain visualization and assess drug permeability across the blood-brain barrier (BBB) are critically needed. Positron emission tomography (PET) is a highly sensitive, noninvasive technique that allows the evaluation of the BBB permeability under normal and disease-state conditions. In this work, we have developed the synthesis of novel water-soluble and biocompatible glucose-coated gold nanoparticles (GNPs) carrying BBB-permeable neuropeptides and a chelator of the positron emitter (68)Ga as a PET reporter for in vivo tracking biodistribution. The small GNPs (2 nm) are stabilized and solubilized by a glucose conjugate. A NOTA ligand is the chelating agent for the (68)Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The radioactive labeling of the GNPs is completed in 30 min at 70 °C followed by purification via centrifugal filtration. As a proof of principle, a biodistribution study in rats is performed for the different (68)Ga-GNPs. The accumulation of radioactivity in different organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected organs. The biodistribution of the (68)Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show different distribution profiles. One of the targeted (68)Ga-GNPs improves BBB crossing near 3-fold (0.020 ± 0.0050% ID/g) compared to nontargeted GNPs (0.0073 ± 0.0024% ID/g) as measured by dissection and tissue counting.

Galactofuranose-coated gold nanoparticles elicit a pro-inflammatory response in human monocyte-derived dendritic cells and are recognized by DC-SIGN.

Galactofuranose (Galf) is the five-membered ring form of galactose exclusively found in nonmammalian species, among which several are pathogens. To determine the putative role of this carbohydrate in host-pathogen interactions, we synthesized multivalent gold nanoparticles carrying Galf (Galf-GNPs) and show that they are recognized by the EB-A2 antibody, which is widely used to detect Galf-containing galactomannan in the serum of Aspergillosis patients. We demonstrated that human monocyte-derived dendritic cells bound Galf-GNPs via interaction with the lectin DC-SIGN. Moreover, interaction of dendritic cells with Galf-GNPs resulted in increased expression of several maturation markers on these cells and induced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. These data indicate that Galf is able to modulate the innate immune response via dendritic cells. In conclusion, Galf-GNPs are a versatile tool that can be applied in multiple functional studies to gain a better understanding of the role of Galf in host-pathogen interaction.

High sensitive detection of carbohydrate binding proteins in an ELISA-solid phase assay based on multivalent glyconanoparticles.

Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.