RESULTS FROM 1 TO 20 OF 167

2021, Articolo in rivista, ENG

Robust Immune Response Induced by Schistosoma mansoni TSP-2 Antigen Coupled to Bacterial Outer Membrane Vesicles

Barbosa, Mayra M. F.; Kanno, Alex, I; Barazzone, Giovana C.; Rodriguez, Dunia; Pancakova, Violeta; Trentini, Monalisa; Faquim-Mauro, Eliana L.; Freitas, Amanda P.; Khouri, Mariana, I; Lobo-Silva, Jessica; Goncalves, Viviane M.; Schenkman, Rocilda P. F.; Tanizaki, Martha M.; Boraschi, Diana; Malley, Richard; Farias, Leonardo P.; Leite, Luciana C. C.

Purpose: The use of adjuvants can significantly strengthen a vaccine's efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around - 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-alpha, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-gamma, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.

International journal of nanomedicine (Online) 16, pp. 7153–7168

DOI: 10.2147/IJN.S315786

2021, Articolo in rivista, ENG

Direct LC-MS/MS Analysis of Extra- and Intracellular Glycerophosphoinositol in Model Cancer Cell Lines

Campos, Ana Margarida; Nuzzo, Genoveffa; Varone, Alessia; Italiani, Paola; Boraschi, Diana; Corda, Daniela; Fontana, Angelo

Glycerophosphoinositols (GPIs) are water-soluble bioactive phospholipid derivatives of increasing interest as intracellular and paracrine mediators of eukaryotic cell functions. The most representative compound of the family is glycerophosphoinositol (GroPIns), an ubiquitous component of mammalian cells that participates in cell proliferation, cell survival and cell response to stimuli. Levels and activity of this compound vary among cell types and deciphering these functions requires accurate measurements in in vitro and in vivo models. The conventional approaches for the analysis of GroPIns pose several issues in terms of sensitivity and product resolution, especially when the product is in the extracellular milieu. Here we present an UPLC-MS study for the quantitative analysis of this lipid derivative in cells and, for the first time, culture supernatants. The method is based on a solid-phase extraction that allows for fast desalting and analyte concentration. The robustness of the procedure was tested on the simultaneous measurements of intra- and extracellular levels of GroPIns in a number of human cell lines where it has been shown that the non-transformed cells are characterized by high extracellular level of GroPIns, whereas the tumor cells tended to have higher intracellular levels.

Frontiers in immunology 12

DOI: 10.3389/fimmu.2021.646681

2021, Articolo in rivista, ENG

A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses

Della Camera, Giacomo; Lipsa, Dorelia; Mehn, Dora; Italiani, Paola; Boraschi, Diana; Gioria, Sabrina

This study aims to provide guidelines to design and perform a robust and reliable physical-chemical characterization of liposome-based nanomaterials, and to support method development with a specific focus on their inflammation-inducing potential. Out of eight differently functionalized liposomes selected as "case-studies", three passed the physical-chemical characterization (in terms of size-distribution, homogeneity and stability) and the screening for bacterial contamination (sterility and apyrogenicity). Although all three were non-cytotoxic when tested in vitro, they showed a different capacity to activate human blood cells. HSPC/CHOL-coated liposomes elicited the production of several inflammation-related cytokines, while DPPC/CHOL- or DSPC/CHOL-functionalized liposomes did not. This work underlines the need for accurate characterization at multiple levels and the use of reliable in vitro methods, in order to obtain a realistic assessment of liposome-induced human inflammatory response, as a fundamental requirement of nanosafety regulations.

International journal of molecular sciences (Online) 22 (2)

DOI: 10.3390/ijms22020820

2021, Articolo in rivista, ENG

Towards bio-compatible magnetic nanoparticles Immune-related effects, in-vitro internalization, and in-vivo bio-distribution of zwitterionic ferrite nanoparticles with unexpected renal clearance

Ferretti A.M.; Usseglio S.; Mondini S.; Drago C.; La Mattina R.; Chini B.; Verderio C.; Leonzino M.; Cagnoli C.; Joshi P.; Boraschi D.; Italiani P.; Li Y.; Swartzwelter B.J.; Sironi L.; Gelosa P.; Castiglioni L.; Guerrini U.; Ponti A.

Hypothesis: Iron oxide and other ferrite nanoparticles have not yet found widespread application in the medical field since the translation process faces several big hurdles. The incomplete knowledge of the interactions between nanoparticles and living organisms is an unfavorable factor. This complex subject should be made simpler by synthesizing magnetic nanoparticles with good physical (relaxivity) and chemical (colloidal stability, anti-fouling) properties and no biological activity (no immune-related effects, minimal internalization, fast clearance). Such an innocent scaffold is the main aim of the present paper. We systematically searched for it within the class of small-to-medium size ferrite nanoparticles coated by small (zwitter)ionic ligands. Once established, it can be functionalized to achieve targeting, drug delivery, etc. and the observed biological effects will be traced back to the functional molecules only, as the nanosized scaffold is innocent. Experiments: We synthesized nine types of magnetic nanoparticles by systematic variation of core composition, size, coating. We investigated their physico-chemical properties and interaction with serum proteins, phagocytic microglial cells, and a human model of inflammation and studied their biodistribution and clearance in healthy mice. The nanoparticles have good magnetic properties and their surface charge is determined by the preferential adsorption of anions. All nanoparticle types can be considered as immunologically safe, an indispensable pre-requisite for medical applications in humans. All but one type display low internalization by microglial BV2 cells, a process strongly affected by the nanoparticle size. Both small (3 nm) and medium size (11 nm) zwitterionic nanoparticles are in part captured by the mononuclear phagocyte system (liver and spleen) and in part rapidly (?1 h) excreted through the urinary system of mice. Findings: The latter result questions the universality of the accepted size threshold for the renal clearance of nanoparticles (5.5 nm). We suggest that it depends on the nature of the circulating particles. Renal filterability of medium-size magnetic nanoparticles is appealing because they share with small nanoparticles the decreased accumulation-related toxicity while performing better as magnetic diagnostic/therapeutic agents thanks to their larger magnetic moment. In conclusion, many of our nanoparticle types are a bio-compatible innocent scaffold with unexpectedly favorable clearance.

Journal of colloid and interface science (Print) 582, pp. 678–700

DOI: 10.1016/j.jcis.2020.08.026

2020, Articolo in rivista, ENG

The impact of nanoparticles on innate immune activation by live bacteria

Swartzwelter B.J.; Fux A.C.; Johnson L.; Swart E.; Hofer S.; Hofstatter N.; Geppert M.; Italiani P.; Boraschi D.; Duschl A.; Himly M.

The innate immune system evolved to detect and react against potential dangers such as bacteria, viruses, and environmental particles. The advent of modern technology has exposed innate immune cells, such as monocytes, macrophages, and dendritic cells, to a relatively novel type of particulate matter, i.e., engineered nanoparticles. Nanoparticles are not inherently pathogenic, and yet cases have been described in which specific nanoparticle types can either induce innate/inflammatory responses or modulate the activity of activated innate cells. Many of these studies rely upon activation by agonists of toll-like receptors, such as lipopolysaccharide or peptidoglycan, instead of the more realistic stimulation by whole live organisms. In this review we examine and discuss the effects of nanoparticles on innate immune cells activated by live bacteria. We focus in particular on how nanoparticles may interfere with bacterial processes in the context of innate activation, and confine our scope to the effects due to particles themselves, rather than to molecules adsorbed on the particle surface. Finally, we examine the long-lasting consequences of coexposure to nanoparticles and bacteria, in terms of potential microbiome alterations and innate immune memory, and address nanoparticle-based vaccine strategies against bacterial infection.

International journal of molecular sciences (Print) 21, pp. 1–22

DOI: 10.3390/ijms21249695

2020, Articolo in rivista, ENG

Induction of Innate Immune Memory by Engineered Nanoparticles in Monocytes/Macrophages: From Hypothesis to Reality

Italiani, Paola; Della Camera, Giacomo; Boraschi, Diana

The capacity of engineered nanoparticles to activate cells of the innate immune system, in particular monocytes and macrophages, is considered at the basis of their toxic/inflammatory effects. It is, however, evident that even nanoparticles that do not directly induce inflammatory activation, and are therefore considered as safe, can nevertheless induce epigenetic modifications and affect metabolic pathways in monocytes and macrophages. Since epigenetic and metabolic changes are the main mechanisms of innate memory, we had previously proposed that nanoparticles can induce/modulate innate memory, that is, have the ability of shaping the secondary response to inflammatory challenges. In light of new data, it is now possible to support the original hypothesis and show that different types of nanoparticles can both directly induce innate memory, priming macrophages for a more potent response to subsequent stimuli, and modulate bacteria-induced memory by attenuating the priming-induced enhancement. This evidence raises two important issues. First, in addition to overt toxic/inflammatory effects, we should consider evaluating the capacity to induce innate memory and the related epigenetic and metabolic changes in the immunosafety assessment of nanomaterials, since modulation of innate memory may be at the basis of long-term unwanted immunological effects. The other important consideration is that this capacity of nanomaterials could open a new avenue in immunomodulation and the possibility of using engineered nanomaterials for improving immune responses to vaccines and resistance to infections, and modulate anomalous immune/inflammatory reactions in chronic inflammatory diseases, autoimmunity, and a range of other immune-related pathologies.

Frontiers in immunology 11

DOI: 10.3389/fimmu.2020.566309

2020, Articolo in rivista, ENG

Profiling the Course of Resolving vs. Persistent Inflammation in Human Monocytes: The Role of IL-1 Family Molecules

Italiani, Paola; Mosca, Ettore; Della Camera, Giacomo; Melillo, Daniela; Migliorini, Paola; Milanesi, Luciano; Boraschi, Diana

Monocytes and macrophages have a central role in all phases of an inflammatory reaction. To understanding the regulation of monocyte activation during a physiological or pathological inflammation, we propose twoin vitromodels that recapitulate the different phases of the reaction (recruitment, initiation, development, and resolution vs. persistence of inflammation), based on human primary blood monocytes exposed to sequential modifications of microenvironmental conditions. These models exclusively describe the functional development of blood-derived monocytes that first enter an inflammatory site. All reaction phases were profiled by RNA-Seq, and the two models were validated by studying the modulation of IL-1 family members. Genes were differentially modulated, and distinct clusters were identified during the various phases of inflammation. Pathway analysis revealed that both models were enriched in pathways involved in innate immune activation. We observe that monocytes acquire an M1-like profile during early inflammation, and switch to a deactivated M2-like profile during both the resolving and persistent phases. However, during persistent inflammation they partially maintain an M1 profile, although they lose the ability to produce inflammatory cytokines compared to M1 cells. The production of IL-1 family molecules by ELISA reflected the transcriptomic profiles in the distinct phases of the two inflammatory reactions. Based on the results, we hypothesize that persistence of inflammatory stimuli cannot maintain the M1 activated phenotype of incoming monocytes for long, suggesting that the persistent presence of M1 cells and effects in a chronically inflamed tissue is mainly due to activation of newly incoming cells. Moreover, being IL-1 family molecules mainly expressed and secreted by monocytes during the early stages of the inflammatory response (within 4-14 h), and the rate of their production decreasing during the late phase of both resolving and persistent inflammation, we suppose that IL-1 factors are key regulators of the acute defensive innate inflammatory reaction that precedes establishment of longer-term adaptive immunity, and are mainly related to the presence of recently recruited blood monocytes. The well-described role of IL-1 family cytokines and receptors in chronic inflammation is therefore most likely dependent on the continuous influx of blood monocytes into a chronically inflamed site.

Frontiers in immunology 11

DOI: 10.3389/fimmu.2020.01426

2020, Articolo in rivista, ENG

The IL-1 family cytokines and receptors in autoimmune diseases

Migliorini, Paola; Italiani, Paola; Pratesi, Federico; Puxeddu, Ilaria; Boraschi, Diana

The role of the cytokines and receptors of the IL-1 family in inflammation is well known. Several cytokines of the family have a powerful inflammatory activity, with IL-1 beta being the best-characterized factor. The inflammatory activity of IL-1 cytokines is regulated by other factors of the family, including receptor antagonists, soluble receptors and anti-inflammatory cytokines.

Autoimmunity reviews 19 (9)

DOI: 10.1016/j.autrev.2020.102617

2020, Articolo in rivista, ENG

Probing the immune responses to nanoparticles across environmental species. A perspective of the EU Horizon 2020 project PANDORA

Annalisa Pinsino; Neus G. Bastús; Martí Busquets-Fité; Laura Canesi; Paola Cesaroni; Damjana Drobne; Albert Duschl; Marie-Ann Ewart; Ignasi Gispert; Jutta Horejs-Hoeck; Paola Italiani; Birgit Kemmerling; Peter Kille; Petra Procházková; Victor F. Puntes; David J. Spurgeon; Claus Svendsen; Colin J. Wilde; Diana Boraschi

Understanding how engineered nanomaterials affect immune responses of living organisms requires a strong collaborative effort between immunologists, toxicologists, ecologists, physiologists, inorganic chemists, nanomaterial scientists and experts in law and risk management. This perspective aims to provide a new viewpoint on the interaction between engineered nanomaterials and the immune defensive systems across living species, gained within the EU Horizon 2020 project PANDORA. We consider the effects of nanoparticle exposure on immune functions in plants, marine and terrestrial invertebrates and their relation to the current state of knowledge for vertebrates (in particular humans). These studies can shed light on the broader perspective of defensive and homeostatic mechanisms (immunity, inflammation, stress responses, microbiota, stem cell differentiation) suggesting ways to: i) perform a comparative analysis of the nanoparticle impact on immunity across model organisms; ii) inspire best practices in experimental methodologies for nanosafety/nanotoxicity studies; iii) regroup and harmonise fragmented research activities; iv) improve knowledge transfer strategies and nano-security; v) propose innovative tools and realistic solutions, thereby helping in identifying future research needs and tackling their challenges.

Environmental science. Nano (Online)

DOI: 10.1039/D0EN00732C

2020, Articolo in rivista, ENG

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species

Diana Boraschi; Andi Alijagic; Manon Auguste; Francesco Barbero; Eleonora Ferrari; Szabolcs Hernadi; Craig Mayall; Sara Michelini; Pacheco Natividad I. Navarro; Alessandra Prinelli; Elmer Swart; Benjamin J Swartzwelter; Neus G Bastus; Laura Canesi; Damjana Drobne; Albert Duschl; Marie-Ann Ewart; Jutta Horejs-Hoeck; Paola Italiani; Birgit Kemmerling; Peter Kille; Petra Prochazkova; Victor F Puntes; David J Spurgeon; Claus Svendsen; Colin J Wilde; Annalisa Pinsino

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

Small (Weinh., Print) 16 (21)

DOI: 10.1002/smll.202000598

2019, Articolo in rivista, ENG

The association between frailty and MRI features of cerebral small vessel disease

Kant, Ilse M. J.; Mutsaerts, Henri J. M. M.; van Montfort, Simone J. T.; Jaarsma-Coes, Myriam G.; Witkamp, Theodoor D.; Winterer, Georg; Spies, Claudia D.; Hendrikse, Jeroen; Slooter, Arjen J. C.; de Bresser, Jeroen; Armbruster, Franz Paul; Boecher, Axel; Boraschi, Diana; Borchers, Friedrich; Della Camera, Giacomo; van Delien, Edwin; Diehl, Ina; Dschietzig, Thomas Bernd; Feinkohl, Insa; Filmer, Ariane; Gallinat, Juergen; Hafen, Bettina; Hartmann, Katarina; Heidtke, Karsten; Helmschrode, Anja; Italiani, Paola; Ittermann, Bernd; Krause, Roland; Kronabel, Marion; Kuehn, Simone; Lachmann, Gunnar; Melillo, Daniela; Menon, David K.; Moreno-Lopez, Laura; Moergeli, Rudolf; Nuernberg, Peter; Ofosu, Kwaku; Olbert, Maria; Pietzsch, Malte; Pischon, Tobias; Preller, Jacobus; Ruppert, Jana; Schneider, Reinhard; Stamatakis, Emmanuel A.; Weber, Simon; Weyer, Marius; Winzeck, Stefan; Wolf, Alissa; Yeurek, Fatima; Zacharias, Norman

Frailty is a common syndrome in older individuals that is associated with poor cognitive outcome. The underlying brain correlates of frailty are unclear. The aim of this study was to investigate the association between frailty and MRI features of cerebral small vessel disease in a group of non-demented older individuals. We included 170 participants who were classified as frail (n = 30), pre-frail (n = 85) or non-frail (n = 55). The association of frailty and white matter hyperintensity volume and shape features, lacunar infarcts and cerebral perfusion was investigated by regression analyses adjusted for age and sex. Frail and pre-frail participants were older, more often female and showed higher white matter hyperintensity volume (0.69 [95%-CI 0.08 to 1.31], p = 0.03 respectively 0.43 [95%-CI: 0.04 to 0.82], p = 0.03) compared to non-frail participants. Frail participants showed a non-significant trend, and pre-frail participants showed a more complex shape of white matter hyperintensities (concavity index: 0.04 [95%-CI: 0.03 to 0.08], p = 0.03; fractal dimensions: 0.07 [95%-CI: 0.00 to 0.15], p = 0.05) compared to non-frail participants. No between group differences were found in gray matter perfusion or in the presence of lacunar infarcts. In conclusion, increased white matter hyperintensity volume and a more complex white matter hyperintensity shape may be structural brain correlates of the frailty phenotype.

Scientific reports (Nature Publishing Group) 9

DOI: 10.1038/s41598-019-47731-2

2019, Articolo in rivista, ENG

Assessing Immunological Memory in the Solitary Ascidian Ciona robusta

Melillo, Daniela; Marino, Rita; Della Camera, Giacomo; Italiani, Paola; Boraschi, Diana

The immune defensive mechanisms active in the solitary ascidian Ciona robusta include phagocytic and encapsulating activity, largely brought about by phagocytic cells within the haemocyte population, the presence of complement components, which have been molecularly and functionally identified, and expression of a number of immune-related genes and pathways, identified by genome-based homology with vertebrate counterparts. Since C. robusta only displays highly conserved innate immune mechanisms, being devoid of an adaptive immune system, this organism is an excellent model for studying the features of innate memory, i.e., the capacity of the innate immune system to re-programming its responsiveness to potentially dangerous agents upon repeated exposure. In this study, we have developed an in vivo model for assessing the establishment and molecular/functional features of innate memory, by sequentially exposing C. robusta to a priming stimulus (microbial molecules), followed by a period of resting to return to basal conditions, and a challenge with microbial agents in homologous or cross-stimulation. The endpoints of immune activation were a functional activity (phagocytosis) and the molecular profiles of immune-related gene expression. The results show that exposure of C. robusta to microbial agents induces a reaction that primes animals for developing a different (expectedly more protective) response to subsequent challenges, showing the effective establishment of an immune memory. This immune memory relies on the modulation of a number of different mechanisms, some of which are priming-specific, others that are challenge-specific, and others that are non-specific, i.e., are common to all priming/challenge combinations (e.g., up-regulation of the Tnf and Lbp genes). Memory-dependent expression of the humoral immunity-related gene C3ar inversely correlates with memory-dependent variations of phagocytic rate, suggesting that complement activation and phagocytosis are alternative defensive mechanisms in C. robusta. Conversely, memory-dependent expression of the cellular immunity-related gene Cd36 directly correlates with variations of phagocytic rate, suggesting a direct involvement of this gene in the functional regulation of phagocytosis.

Frontiers in immunology 10

DOI: 10.3389/fimmu.2019.01977

2019, Articolo in rivista, ENG

Bovine colon organoids: From 3D bioprinting to cryopreserved multi-well screening platforms

Topfer, Elfi; Pasotti, Anna; Telopoulou, Aikaterini; Italiani, Paola; Boraschi, Diana; Ewart, Marie-Ann; Wilde, Colin

Three-dimensional (3D) colon organoids, termed "colonoids", derived from adult stem cells represent a powerful tool in in vitro pharmaceutical and toxicological research. Murine and human colonoid models exist. Here we describe the establishment of bovine colonoids for agri-biotechnological applications, and extend the repertoire of colonoid culture options through proof-of-principle for bioprinting and novel in-plate cryopreservation technology. As a first step, we differentiated established long-term bovine colonoid cultures into mature colonoids. Tissue-specific differentiation was demonstrated by gene expression. Second, we investigated cryopreservation of colonoids in situ within an extracellular matrix in multi-well plates. Upon controlled thawing, cryopreserved 3D cultures grew at similar rates to unfrozen colonoids. Cytotoxic sensitivity to staurosporine was not significantly different between in situ freeze-thawed and unfrozen control cultures. Third, scalability of colonoid culture assembly by extrusion bioprinting into multi-well plates using GelMA bioink was assessed. With optimised bioprinting and crosslinking parameters, colonoids in GelMA were printed into 96 well culture plates and remained viable and proliferative post-print. For tissue-relevant in vitro studies we furthermore established differentiated colonoid-derived monolayer cultures on permeable membranes. Taken together, we outline novel in vitro approaches to study the ruminant colonic epithelium and introduce in-plate cryopreservation as convenient alternative to conventional in-vial cryopreservation.

Toxicology in vitro 61

DOI: 10.1016/j.tiv.2019.104606

2019, Articolo in rivista, ENG

ANTIBODY THERAPY One hits (almost) all

Boraschi, Diana; Apte, Ron N.; Martin, Michael U.

Therapeutic efficacy in inflammatory diseases is hampered by disease complexity. A monoclonal antibody that neutralizes IL-1R3, the common co-receptor of most inflammatory IL-1 cytokines, opens up new perspectives in effective disease management.

Nature immunology (Print) 20 (9), pp. 1095–1097

DOI: 10.1038/s41590-019-0463-5

2019, Articolo in rivista, ENG

In vitro Evidence of Human Immune Responsiveness Shows the Improved Potential of a Recombinant BCG Strain for Bladder Cancer Treatment

Rodriguez, Dunia; Goulart, Cibelly; Pagliarone, Ana C.; Silva, Eliane P.; Cunegundes, Priscila S.; Nascimento, Ivan P.; Borra, Ricardo C.; Dias, Waldely O.; Tagliabue, Aldo; Boraschi, Diana; Leite, Luciana C. C.

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1 beta, IL-2, IFN-gamma, and TNF-alpha. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4(+) T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

Frontiers in immunology 10

DOI: 10.3389/fimmu.2019.01460

2019, Articolo in rivista, ENG

Cerebral microbleeds are not associated with postoperative delirium and postoperative cognitive dysfunction in older individuals

Lachmann, Gunnar; Kant, Ilse; Lammers, Florian; Windmann, Victoria; Spies, Claudia; Speidel, Saya; Borchers, Friedrich; Hadzidiakos, Daniel; Hendrikse, Jeroen; Winterer, Georg; de Bresser, Jeroen; Wolf, Alissa; Spies, Claudia; Yuerek, Fatima; Borchers, Friedrich; Lachmann, Gunnar; Feinkohl, Insa; Ofosu, Kwaku; Olbert, Maria; Zacharias, Norman; Moergeli, Rudolf; Pischon, Tobias; Gallinat, Juergen; Kuehn, Simone; Slooter, Arjen; van Dellen, Edwin; Kant, Ilse; de Bresser, Jeroen; Hendrikse, Jeroen; van Montfort, Simone; Menon, David; Stamatakis, Emmanuel; Preller, Jacobus; Moreno-Lopez, Laura; Winzeck, Stefan; Melillo, Daniela; Boraschi, Diana; Della Camera, Giacomo; Italiani, Paola; Schneider, Reinhard; Krause, Roland; Heidtke, Karsten; Nuernberg, Peter; Helms-Chrodt, Anja; Boecher, Axel; Hafen, Bettina; Armbruster, Franz Paul; Diehl, Ina; Ruppert, Jana; Hartmann, Katarina; Kronabel, Marion; Weyer, Marius; Dschietzig, Thomas Bernd; Pietzsch, Malte; Weber, Simon; Ittermann, Bernd; Fillmer, Ariane

Background

PloS one 14 (6)

DOI: 10.1371/journal.pone.0218411

2019, Articolo in rivista, ENG

Surface Exposure of PEG and Amines on Biodegradable Nanoparticles as a Strategy to Tune Their Interaction with Protein-Rich Biological Media

Claudia Conte, Giovanni Dal Poggetto , Benjamin J. Swartzwelter , Diletta Esposito, Francesca Ungaro, Paola Laurienzo, Diana Boraschi and Fabiana Quaglia

Nanoparticles (NPs) based on amphiphilic block copolymers of polyethylene glycol (PEG) and biodegradable polyesters are of particular current interest in drug nanodelivery due to their easily manipulated properties. The interaction of these NPs with biological environments is highly influenced by shell features, which drive biological identity after administration. To widen the strategies available for tuning particle surface chemistry, here we developed a panel of amine-bearing PEGylated NPs with a poly("-caprolactone) (PCL) core for the delivery of lipophilic drugs, and investigated the impact of NP modifications on their interaction with abundant circulating proteins (human serum albumin--HSA--and mucin), as well as their transport through biological barriers (artificial mucus--AM, extracellular matrix--ECM).We prepared NPs based on a diamino-terminated PCL (amine-NPs) and its mixture with PEG-PCL copolymers (amine/PEG-NPs) at dierent PEG molecular weights by nanoprecipitation, as well as corresponding NPs of PEG-PCL (PEG-NPs). The presence of an amine-bearing polymer resulted in NPs with a net positive charge and a zeta potential dependent on the length of PEG in the copolymer. Amine/PEG-NPs had a larger fixed aqueous layer thickness as compared to PEG-NPs, suggesting that PEG conformation is aected by the presence of positive charges. In general, amine-bearing NPs promptly interacted with the dysopsonic protein HSA, due to electrostatic interactions, and lose stability, thereby undergoing time-related aggregation. On the other hand, amine/PEG-NPs interaction with mucin induced switching to a negative surface charge but did not alter the quality of the dispersion. The transport kinetics of NPs through a layer of artificial mucus and tumor extracellular matrix was studied by means of fluorescent NPs based upon FRET. Amine/PEG-NPs did not cross the ECM, but they were promptly transported through the AM, with swifter transport noted at increasing MWs of PEG in the copolymer. Finally, we demonstrated that all the dierent NP types developed in this study are internalized by human monocytes and, despite the positive charge, they did not induce a measurable inflammatory eect. In conclusion, we showed that the concurrent presence of both PEG and amine groups on NP surface is a promising strategy for directing their interaction with body compartments. While PEG-NPs are confirmed for their capacity to cross ECM-like compartments, amine/PEG-NPs are revealed as a powerful platform to widen the arsenal of nanotools available for overcoming mucus-covered epithelia.

Nanomaterials (Basel) 9

DOI: 10.3390/nano9101354

2018, Articolo in rivista, ENG

Cytokines and soluble receptors of the IL-1 family in Schnitzler syndrome.

Migliorini, Paola, Italiani, Paola, Puxeddu, Ilaria, and Boraschi Diana

Objectives: Schnitzler syndrome (SchS) is an autoinflammatory disorder characterized by chronic urticaria, fever, monoclonal gammopathy. The success of IL-1 blocking therapies suggests a crucial role of IL-1 in disease induction. The aim of this study is to perform a comprehensive analysis of IL-1 family cytokines and soluble receptors in a group of SchS patients. Methods: Three patients fulfilling the criteria for the diagnosis of SchS were recruited; 86 blood donors formed the control group. IL-1 family cytokines (IL-1?, IL-1?, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, sIL-1R4) and antagonists (IL-1Ra, IL-18 binding protein -IL-18BP) were measured by a multiarray ELISA assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Cytokine levels were compared by Mann Whitney test. Results: IL-18 and free IL-18 were increased in patients vs. controls (p=0.005 and p=0.0082, respectively), while IL-18BP levels were not different. IL-1?, IL-1? and IL-33 were undetectable in both patients and controls. The soluble receptors sIL-1R1, sIL-1R2 and ST2/sIL-1R4, and the IL-1 antagonist IL-1Ra were all within the normal range; sIL-1R3 levels were significantly lower in patients vs. controls (p= 0.039). Conclusions: The data indicate that SchS is characterized by increased circulating levels of free IL-18, possibly leading to a higher activation of innate/inflammatory effector cells. At variance with other inflammatory diseases, the lack of increase in sIL-1R1 and sIL-1R2 and the decreased levels of sIL-R3 imply a failure in the counterbalancing mechanism aimed at inhibiting excessive IL-1? in the tissues.

Scandinavian journal of rheumatology (Online) 22, pp. 1–4

DOI: 10.1080/030009742.2018.1550210

2018, Articolo in rivista, ENG

Circulating levels of IL-1 family cytokines and receptors in Alzheimer's disease: new markers of disease progression?

Italiani, Paola; Puxeddu, Ilaria; Napoletano, Sabrina; Scala, Emanuele; Melillo, Daniela; Manocchio, Simone; Angiolillo, Antonella, Migliorini, Paola; Boraschi, Diana; Vitale, Emilia, and Di Costanzo, Alfonso

Background. Although the mechanisms underlying AD neurodegeneration are not fully understood, it is now recognised that inflammation could play a crucial role in the initiation and progression of AD neurodegeneration. A neuro-inflammatory network, based on the anomalous activation of microglial cells, includes the production of a number of inflammatory cytokines both locally and systemically. These may serve as diagnostic markers or therapeutic targets for AD neurodegeneration. Methods. We have measured the levels of the inflammation-related cytokines and receptors of the IL-1 family in serum of subjects with AD, compared to mild cognitive impairment (MCI), subjective memory complaints (SMC), and normal healthy subjects (NHS). Using a custom-made multiplex ELISA array, we examined ten factors of the IL-1 family, the inflammation-related cytokines IL-1, IL-1, IL-18, and IL-33, the natural inhibitors IL-1Ra and IL-18BP, and the soluble receptors sIL-1R1, sIL-1R2, sIL-1R3, and sIL-1R4. Results. The inflammatory cytokines IL-1 and IL-1, their antagonist IL-1Ra, and their soluble receptor sIL-1R1 were increased in AD. The decoy IL-1 receptor sIL-1R2 was only increased in MCI. IL-33 and its soluble receptor sIL-1R4 were also significantly higher in AD. The soluble form of the accessory receptor for both IL-1 and IL-33 receptor complexes, sIL-1R3, was increased in SMC and even more in AD. Total IL-18 levels were unchanged, whereas the inhibitor IL-18BP was significantly reduced in MCI and SMC, and highly increased in AD. The levels of free IL-18 were significantly higher in MCI. Conclusions. AD is characterised by a significant alteration in the circulating levels of the cytokines and receptors of the IL-1 family. The elevation of sIL-1R4 in AD is in agreement with findings in other diseases and can be considered a marker of ongoing inflammation. Increased levels of IL-1Ra, sIL-1R1, sIL-1R4, and IL-18BP distinguished AD from MCI and SMC, and from other inflammatory diseases. Importantly, sIL-1R1, sIL-1R3, sIL-1R4, and IL-18BP negatively correlated with cognitive impairment. A significant elevation of circulating sIL-1R2 and free IL-18, not present in SMC, is characteristic of MCI and disappears in AD, making them additional interesting markers for evaluating progression from MCI to AD.

Journal of neuroinflammation 15, pp. 342

DOI: 10.1186/s12974-018-1376-1

2018, Articolo in rivista, ENG

Innate Immune Memory in Invertebrate Metazoans: A Critical Appraisal

Melillo, Daniela; Marino, Rita; Italiani, Paola; Boraschi, Diana

The ability of developing immunological memory, a characteristic feature of adaptive immunity, is clearly present also in innate immune responses. In fact, it is well known that plants and invertebrate metazoans, which only have an innate immune system, can mount a faster and more effective response upon re-exposure to a stimulus. Evidence of immune memory in invertebrates comes from studies in infection immunity, natural transplantation immunity, individual, and transgenerational immune priming. These studies strongly suggest that environment and lifestyle take part in the development of immunological memory. However, in several instances the formal correlation between the phenomenon of immune memory and molecular and functional immune parameters is stillmissing. In this review, we have critically examined the cellular and humoral aspects of the invertebrate immune memory responses. In particular, we have focused our analysis on studies that have addressed immune memory in the most restrictive meaning of the term, i.e., the response to a challenge of a quiescent immune system that has been primed in the past. These studies highlight the central role of an increase in the number of immune cells and of their epigenetic re-programming in the establishment of sensu stricto immune memory in invertebrates.

Frontiers in immunology 9

DOI: 10.3389/fimmu.2018.01915

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