Silvia Fallarini

Expression of functional GPR35 in human iNKT cells.

The aim of this study was to examine the expression of G protein-coupled receptor (GPR)35 in human invariant natural killer T (iNKT) cells and to determine the functional effects induced by selective activation of this receptor. RT-PCR analysis showed that both human iNKT cells and resting PBMC expressed GPR35; GPR35 protein resulted mostly localized in the plasma membrane, while it internalized in punctate intracellular structures following specific receptor activation (Western blot and immunofluorescence/confocal microscopy analysis). The specific activation of GPR35 by selective receptor agonists [l-kynurenic acid (KYNA)] or 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo [4,5-d]pyrimidine-7-one (zaprinast)] functionally correlated with a significant reduction in IL-4 release from alpha-galactosylceramide (alpha-GalCer)-activated human iNKT cells, and this effect resulted mediated by pertussis toxin (PTX)-sensitive Gi/o proteins. In conclusion, our results demonstrate that human iNKT cells express GPR35 functionally active in reducing IL-4 release.

Synthesis of α-Galactosyl Ceramide (KRN7000) and Analogues Thereof via a Common Precursor and Their Preliminary Biological Assessment

A new practical synthesis of alpha-GalCer and of its analogues is presented, opening the chance to easily modify the sphingosine chain. The common precursor is a disaccharide, obtained by coupling tetra-O-benzyl-D-galactose with allyl 2,3-O-isopropylidene-D-lyxofuranoside. Introduction of alkyl chains via Wittig reaction (for alpha-GalCer and OCH) or via Williamson reaction (for oxa analogues) followed by standard synthetic steps allows one to efficiently obtain such compounds. The analogues are able to activate iNKT cells when presented by CD1d expressing cells.

Synthesis of di- and tri-saccharide fragments of Salmonella typhi Vi capsular polysaccharide and their zwitterionic analogues.

Zwitterionic polysaccharides (ZPS) behave like traditional T cell-dependent antigens, suggesting the design of new classes of vaccines alternative to currently used glycoconjugates and based on the artificial introduction of a zwitterionic charge motif onto the carbohydrate structure of pathogen antigens. Here we report the new synthesis and antigenic evaluation of di-/tri-saccharide fragments of Salmonella typhi Vi polysaccharide, as well as of their corresponding zwitterionic analogues. Our strategy is based on versatile intermediates enabling chain elongation either by iterative single monomer attachment or by faster and more flexible approach using disaccharide donors. The effect of structural modifications of the synthetic compounds on antigenic properties was evaluated by competitive ELISA. All the oligosaccharides were recognized by specific anti-Vi polyclonal antibodies in a concentration-dependent manner, and the introduction of a zwitterionic motif into the synthetic molecules did not prevent the binding.

A Synthetic Disaccharide Analogue from Neisseria meningitidis A Capsular Polysaccharide Stimulates Immune Cell Responses and Induces Immunoglobulin G (IgG) Production in Mice When Protein-Conjugated

Some new phosphonoester-linked oligomers, stabilized analogues of the corresponding phosphate-bridged oligomers of Neisseria meningitidis A (MenA) capsular polysaccharide (CPS), were conjugated to human serum albumin (HSA), as a protein carrier model, and studied for immunological activities. We determined (i) in vitro, their biocompatibility (CAM test) and activity in inducing both T cell proliferation (CFSE method) and IL-2 release (ELISA), and (ii) in vivo, their ability to stimulate specific IgG antibody production (ELISA). All HSA-conjugated compounds induce T cell proliferation (40% of proliferation at 10(2) μM), whereas only the phosphonodisaccharide was effective (28% of proliferation at 10(2) μM) among the unconjugated forms. IL-2 release confirmed these results. In addition, the HSA-conjugated showed in vivo the capacity of eliciting the production of specific IgG antibodies. In conclusion, we obtained novel biocompatible, water-stable, and immunoactive MenA CPS analogues. A short disaccharide fragment showed the unusual behavior of triggering T cell proliferation in vitro.

Synthesis of a Structural Analogue of the Repeating Unit from Streptococcus pneumoniae 19F Capsular Polysaccharide Based on the Cross-Metathesis–Selenocyclization Reaction Sequence

Pseudo-oligosaccharides have attracted much interest as scaffolds for the synthesis of sugar mimics endowed with very similar biological properties but structurally and synthetically simpler than their natural counterparts. Herein, the synthesis of pseudo-oligosaccharides using the cross-metathesis reaction between distinct sugar-olefins followed by intramolecular selenocyclization of the obtained heterodimer as key steps is first investigated. This methodology has been then applied to the preparation of structural analogues of the trisaccharide repeating unit from Streptococcus pneumoniae 19F. The inhibition abilities of the synthetic molecules were evaluated by a competitive ELISA assay using a rabbit polyclonal anti-19F serum.

Increased frequency of circulating invariant natural killer T cells in malignant pleural mesothelioma patients

Invariant natural killer T (iNKT) cells are a distinct subset of human T cells, which expresses an invariant T cell receptor Vα24 Jα18 and recognizes glycolipid antigens in the context of CD1d molecules. iNKT cells exert pivotal regulatory roles in many immune responses, including antitumor immune responses. Alterations in iNKT cell frequency, phenotype, and activation state have been reported in cancer patients. No data are available on the iNKT cells in malignant pleural mesothelioma (MPM), a rare, but very aggressive, malignancy of the pleura with a very poor prognosis. Here, we studied the frequency, phenotype, and cytokine profile of circulating iNKT cells in MPM patients, and correlated results with tumor histological types (epithelioid, sarcomatoid, biphasic) and clinical stages (I, II, III). We found that the iNKT cell frequency was significantly increased in MPM patients with epithelioid and sarcomatoid types in comparison with healthy volunteers (HV); only three biphasic mesotheliomas were available in this study, thus no conclusions can be drawn for this MPM type. The increased frequency significantly correlates with the clinical stage of tumor with the highest value at the stage III in both epithelioid and sarcomatoid subtypes. According to the histological types, we measured changes in the frequencies of CD4+CD8+ (DP) and CD4-CD8- (DN), but not in the cytokine profiles (IFN-γ/IL-4 expression). These results demonstrate that the frequency of iNKT cells is increased in MPM patients and that this increase correlates with MPM type and stage.

“In vitro” studies on galectin-3 in human natural killer cells

Galectin-3 (Gal-3) is a β-galactoside binding protein able to modulate both innate and adaptive immune responses. First identified in macrophages, Gal-3 has been studied widely in many mammalian immune cells, but scarcely in natural killer (NK) cells. The aim of this study was to analyze Gal-3 in human NK cells, isolated from peripheral blood mononuclear cells. Both PCR and RT-PCR analysis showed that resting human NK cells express Gal-3 mRNA, which can be modulated upon cytokine stimulation (100 U/ml IL-2 + 20 ng/ml IL-15) for different period of time (1-24 h). Western blot, cytofluorimetry, and confocal microscopy analysis clearly demonstrated that the Gal-3 gene can translate into the corresponding protein. From our results, resting NK cells, isolated from different healthy donors, can express high or low basal levels of Gal-3. In NK cells, Gal-3 was always intracellularly detected at both cytoplasm and nucleus levels, while never at the membrane surface, and its localization resulted independent from the cellular activation status. In addition, the intracellular Gal-3 can co-localize with perforin in exocytic vesicles. Cell treatment with a thiodigalactoside-based Gal-3 inhibitor (1-30 μM) slightly increased the number of degranulating NK cells, while it significantly increased the percentage of cells releasing high amounts of cytotoxic granules (+ 36 ± 3% vs. inhibitor-untreated cells at 30 μM Gal-3). In conclusion, our results demonstrate that human resting NK cells express Gal-3 at both gene and protein levels and that the Gal-3 expression can be modulated upon cytokine stimulation. In the same cells, Gal-3 always localizes intracellularly and functionally correlates with the degree of NK cell degranulation.

Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells

The ability of a rigid α-Tn mimetic (compound 1) to activate murine invariant natural killer T (iNKT) and human natural killer (NK) cells, two subsets of lymphocytes involved in cancer immunesurveillance, was investigated. For this purpose, the mimetic 1 was properly conjugated to a stearic acid containing glycerol-based phospholipid (compound 5) to be presented, in the context of the conserved non polymorphic major histocompatibility complex class I-like molecules (CD1d), to iNKT cells. On the contrary, the mimetic 1 was conjugated to a multivalent peptide-based scaffold (compound 6) to induce NK cell activation.

Synthetic isoforms of endogenous sulfatides differently modulate indoleamine 2,3-dioxygenase in antigen presenting cells.

AIMS To investigate whether sulfatides modulate indoleamine 2,3-dioxygenase (IDO)1, a fine-tuned enzymatic mechanism for controlling immune responses, gene expression/function in antigen presenting cells (APC). The relationship between structure and activity (SAR) of newly synthesized sulfatide isoforms (C16:0, C18:0, C22:0, C24:1) was also evaluated. MAIN METHODS CD1d-transfected THP-1 human cells were used as APC and treated with increasing concentrations (0.01-10μΜ) of each compound for an appropriate period of time. The gene expression and the enzymatic activity of IDO1 were examined using reverse transcription-polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC). Compound-untreated cells were taken as negative, while 1000U/ml interferon (IFN)-γ-treated cells as positive controls. KEY FINDINGS Not all sulfatides induced the same effect: the basal IDO1 expression was significantly reduced (-48 ± 3% at 0.01μΜ) by C16:0 sulfatide, while it was increased by C18:0 or C24:1 sulfatide (+87 ± 7% and +50 ± 5% at 1μΜ, respectively) over negative controls; C22:0 sulfatide resulted ineffective at all concentrations tested. These effects functionally correlated with changes in IDO1 activity: l-kynurenine contents in the culture media were significantly reduced by C16:0 sulfatide (-29 ± 4% at 0.01μM), while it was increased by C18:0 or C24:1 sulfatide (+61 ± 8% and +48 ± 4% at 1μM, respectively) over negative controls. C22:0 sulfatide resulted ineffective at all concentration tested. SIGNIFICANCE The overall data demonstrate that specific sulfatide isoforms differently modulate IDO1 in APC. The sulfatide-induced effects are structurally dependent on the length/saturation of their fatty acid chain.

Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.