Dengue induces iNOS expression and nitric oxide synthesis in platelets through IL-1R.

Introduction: Dengue is an arthropod-born disease caused by dengue virus (DENV), that may manifest as a mild illness or severe form, characterized by hemorrhagic fever and shock. Nitric oxide (NO) is a vasodilator signaling molecule and an inhibitor of platelet aggregation known to be increased in platelets from dengue patients. However, the mechanisms underlying NO synthesis by platelets during dengue are not yet elucidated. IL-1ß is a pro-inflammatory cytokine able to induce iNOS expression in leukocytes and present in dengue patients at high levels. Nevertheless, the role of IL-1ß in platelet activation, especially regarding iNOS expression, are not clear. Methods: We prospectively followed a cohort of 28 dengue-infected patients to study NO synthesis in platelets and its relationship with disease outcomes. We used in vitro infection and stimulation models to gain insights on the mechanisms. Results and Discussion: We confirmed that platelets from dengue patients express iNOS and produce higher levels of NO during the acute phase compared to healthy volunteers, returning to normal levels after recovery. Platelet NO production during acute dengue infection was associated with the presence of warning signs, hypoalbuminemia and hemorrhagic manifestations, suggesting a role in dengue pathophysiology. By investigating the mechanisms, we evidenced increased iNOS expression in platelets stimulated with dengue patients´ plasma, indicating induction by circulating inflammatory mediators. We then investigated possible factors able to induce platelet iNOS expression and observed higher levels of IL-1ß in plasma from patients with dengue, which were correlated with NO production by platelets. Since platelets can synthesize and respond to IL-1ß, we investigated whether IL-1ß induces iNOS expression and NO synthesis in platelets. We observed that recombinant human IL-1ß enhanced iNOS expression and dose-dependently increased NO synthesis by platelets. Finally, platelet infection with DENV in vitro induced iNOS expression and NO production, besides the secretion of both IL-1α and IL-1ß. Importantly, treatment with IL-1 receptor antagonist or a combination of anti-IL-1α and anti-IL-1ß antibodies prevented DENV-induced iNOS expression and NO synthesis. Our data show that DENV induces iNOS expression and NO production in platelets through mechanisms depending on IL-1 receptor signaling.

Theoretical and experimental study of inclusion complexes formed by isoniazid and modified ß-cyclodextrins: 1H NMR structural determination and antibacterial activity evaluation.

Me-ß-cyclodextrin (Me-ßCD) and HP-ß-cyclodextrin (HP-ßCD) inclusion complexes with isoniazid (INH) were prepared with the aim of modulating the physicochemical and biopharmaceutical properties of the guest molecule, a well-known antibuberculosis drug. The architectures of the complexes were initially proposed according to NMR data Job plot and ROESY followed by density functional theory (DFT) calculations of (1)H NMR spectra using the PBE1PBE functional and 6-31G(d,p) basis set, including the water solvent effect with the polarizable continuum model (PCM), for various inclusion modes, providing support for the experimental proposal. An analysis of the (1)H NMR chemical shift values for the isoniazid (H6',8' and H5',9') and cyclodextrins (H3,5) C(1)H hydrogens, which are known to be very adequately described by the DFT methodology, revealed them to be extremely useful, promptly confirming the inclusion complex formation. An included mode which describes Me-ßCD partially enclosing the hydrazide group of the INH is predicted as the most favorable supramolecular structure that can be used to explain the physicochemical properties of the encapsulated drug. Antibacterial activity was also evaluated, and the results indicated the inclusion complexes are a potential strategy for tuberculosis treatment.

DFT study of cisplatin@carbon nanohorns complexes.

This paper reports a quantum chemical investigation of the inclusion complex formation between a carbon nanohorn structure and cisplatin molecule, using the density functional theory (DFT) with the B3LYP functional and 6-31G(d,p)/LanL2DZ standard basis sets. The inclusion of the drug in host molecules such as carbon nanohorns (CNHs), aims to reduce the toxicity and enhance the effectiveness of cisplatin. In this work we carried out a search for minimum energy structures on the potential energy surface (PES) for CNH-cisplatin interaction, and then calculated the stabilization energy, charge distribution and NMR spectra, which can be of great aid for the experimental identification of the inclusion compound. Our results indicate that the CNH and cisplatin can indeed form stable inclusion complex, with the calculated (1)H NMR and (15)N NMR chemical shifts for cisplatin atoms revealing very substantial changes due to complex formation (~20ppm) that can be easily experimentally observed, which is helpful to the spectra assignment and the inclusion compound structural elucidation.

Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation.

Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1ß synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1ß in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1-dependent IL-1ß secretion. Our findings also indicate that platelet-derived IL-1ß is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1ß-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1ß.

Experimental and theoretical NMR determination of isoniazid and sodium p-sulfonatocalix[n]arenes inclusion complexes.

In this work the inclusion complex formation of isoniazid with sodium p-sulfonatocalix[n]arenes is reported aiming to improve the physicochemical and biopharmaceutical properties of isoniazid a first line antibuberculosis drug. The architectures of the complexes were proposed according to NMR data Job plot indicating details on the insertion of the isoniazid in the calix[n]arenes cavities. DFT theoretical NMR calculations were also performed for sodium p-sulfonatocalix[4]arene complex with isoniazid, with various modes of complexation being considered, to provide support for the experimental proposal. A comparison between experimental and theoretical ¹H NMR chemical shifts profiles allowed for the inclusion complex characterization confirming the isoniazid inclusion mode which is preferentially through the hydrazide moiety. The remarkable agreement between experimental and theoretical NMR profiles adds support to their use in the structural characterization of inclusion compounds. Antibacterial activity was evaluated and the results indicated the inclusion complexes as a potential strategy for tuberculosis treatment.

Supramolecular complex of fluoxetine with beta-cyclodextrin: an experimental and theoretical study.

In this work the complex formed between beta-cyclodextrin (betaCD) and fluoxetine (FLU) was investigated by experimental and computational methods. From Horizontal Attenuated Total Reflectance (HATR) was possible to verify a strong modification in the vibrational modes of betaCD and FLU, indicating interactions between them. The Nuclear Magnetic Resonance (NMR) experiments confirm these interactions through the change in chemical shifts in (1)H spectra, reduction in longitudinal relaxation times values, and the Nuclear Ouverhauser Effect confirm the inclusion of aromatic rings of FLU into the betaCD. The structures of the proposed inclusion compounds were optimized at PM3 semiempirical level of theory. In addition, single point calculations at the Density Functional Theory (DFT) level, using the Becke, Lee, Yang, and Parr functional and 6-31G(d,p) basis set, were used to determine the interaction energy for these structures. The DFT calculations identified the aromatic ring, which contains the CF(3) group as the most stable into the betaCD by an amount of, 11.7 kcal mol(-1), in the gas phase. Polarized continuum model, at the DFT level mentioned, was used to investigate the solvent effect, and the results corroborated the gas phase analysis. A high equilibrium constant (K approximately 6921+/-316) and the stoichiometry, 1:1, were obtained by Isothermal Titration Calorimetry (ITC) experiments.