Coumarin-poly(2-oxazoline)s as synergetic and protein-undetected nanovectors for photodynamic therapy.

Because of the difficult challenges of nanopharmaceutics, the development of a variety of nanovectors is still highly desired. Photodynamic therapy, which uses a photosensitizer to locally produce reactive oxygen species to kill the undesired cells, is a typical example for which encapsulation has been shown to be beneficial. The present work describes the use of coumarin-functionalized polymeric nanovectors based on the self-assembly of amphiphilic poly(2-oxazoline)s. Encapsulation of pheophorbide a, a known PDT photosensitizer, is shown to lead to an increased efficiency compared to the un-encapsulated version. Interestingly, the presence of coumarin both enhances the desired photocytotoxicity and enables the crosslinking of the vectors. Various nanovectors are examined, differing by their size, shape and hydrophilicity. Their behaviour in PDT protocols on HCT-116 cells monolayers is described, the influence of their crosslinking commented. Furthermore, the formation of a protein corona is assessed.

Coverage of BCG Vaccination for children aged until 7 years old and its determinants in French Guiana.

INTRODUCTION: The overall incidence of tuberculosis (TB) in France is low; thus, BCG vaccination is no longer mandatory. In French Guiana - a French overseas territory - BCG vaccination is strongly recommended because the incidence of TB is high in the context of mass immigration from endemic countries with low BCG vaccination rates. Thus, it is important to assess Bacillus Calmette-Guérin (BCG) vaccination coverage and its predictors. METHODS: We used data from the 2014 French Guiana Yellow Fever survey, which was conducted by the Observatoire Régional de la Santé de Guyane. Demographic and immunization data from eligible children and their families were collected using a questionnaire. Children who had an immunization card and who were no older than 7 years of age at the time of the survey were eligible. The Coverage for BCG and other mandatory vaccines were estimated; the delay in BCG vaccination was also computed. Univariate and multivariate analyses identified predictors associated with BCG immunization and BCG delayed immunization (after 2 months of age). RESULTS AND CONCLUSION: Overall, 469 children were eligible for this study. The total BCG coverage was 79.5 %, and the proportion of children vaccinated with delay was 50.7 %. The multivariate analysis indicated that BCVA was significantly greater among children younger than 3 years of age, whose household head was employed and whose education level was greater. None of the predictors were associated with the delay of BCG vaccination.

Synthesis and Self-Assembly of UV-Cross-Linkable Amphiphilic Polyoxazoline Block Copolymers: Importance of Multitechnique Characterization.

In the nanomedicine field, there is a need to widen the availability of nanovectors to compensate for the increasingly reported side effects of poly(ethene glycol). Nanovectors enabling cross-linking can further optimize drug delivery. Cross-linkable polyoxazolines are therefore relevant candidates to address these two points. Here we present the synthesis of coumarin-functionalized poly(2-alkyl-2-oxazoline) block copolymers, namely, poly(2-methyl-2-oxazoline)-block-poly(2-phenyl-2-oxazoline) and poly(2-methyl-2-oxazoline)-block-poly(2-butyl-2-oxazoline). The hydrophilic ratio and molecular weights were varied in order to obtain a range of possible behaviors. Their self-assembly after nanoprecipitation or film rehydration was examined. The resulting nano-objects were fully characterized by transmission electron microscopy (TEM), cryo-TEM, multiple-angle dynamic and static light scattering. In most cases, the formation of polymer micelles was observed, as well as, in some cases, aggregates, which made characterization more difficult. Cross-linking was performed under UV illumination in the presence of a coumarin-bearing cross-linker based on polymethacrylate derivatives. Addition of the photo-cross-linker and cross-linking resulted in better-defined objects with improved stability in most cases.

P2X(1) and P2X(3) purinergic receptors differentially modulate the inflammatory response in human osteoarthritic synovial fibroblasts.

BACKGROUND/AIMS: P2X receptors are membrane ion channels activated by extracellular adenosine 5'-triphosphate (ATP) which contribute to various physiological processes. The present study describes in synovial fibroblasts (SFs) obtained from osteoarthritis (OA) patients and in SW 982 cells derived from human synovial sarcoma a pharmacological characterization of P2X(1) and P2X(3) receptors implicated in the modulation of inflammatory processes in joint diseases. METHODS: mRNA, western blotting, saturation and competition binding experiments were used to characterize purinergic receptors. From a functional point of view nuclear factor kappaB (NF-kappaB) activation, tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and prostaglandin E(2) (PGE(2)) production were evaluated by means of enzyme-linked immunosorbent assays. RESULTS: P2X(1) and P2X(3) receptors were present with high affinity and density. Selected purinergic agonists and antagonists exhibited a different thermodynamic behavior. P2X(1) receptors showed an anti-inflammatory effect reducing NF-kappaB activation and TNF-alpha release whilst P2X(3) receptors mediated opposite response. No effect was mediated by P2X(1) and P2X(3) receptors on IL-6 and PGE(2) production. CONCLUSION: SFs from OA patients and SW 982 cells similarly express P2X(1) and P2X(3) receptors which are able to modulate in opposite way some functional responses closely associated with inflammation suggesting that purinergic receptors may represent a potential target in therapeutic anti-inflammatory joint interventions.

A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in Parkinson's disease.

The antagonistic interaction between adenosine and dopamine receptors could have important pathophysiological and therapeutic implications in Parkinson's disease (PD). The primary aim of this study was to investigate the expression, affinity, and density of A(1), A(2A), A(2B), and A(3) adenosine receptors (ARs) and D(2) dopamine receptors (D(2)Rs) in PD. An increase in A(2A)AR density in putamen was found. The presence and functionality of ARs in human lymphocyte and neutrophil membranes from patients with PD revealed a specific A(2A)AR alteration compared with healthy subjects. A statistically significant linear correlation among the A(2A)AR density, functionality, or tumor necrosis factor-alpha (TNF-alpha) levels and Unified Parkinson's Disease Rating Scale (UPDRS) motor score was reported. Adenosine concentration and TNF-alpha levels were increased in plasma of patients with PD. In rat adrenal pheochromocytoma (PC12) cells, a widely useful model, adenosine antagonists decreased dopamine uptake, and an opposite effect was mediated by A(2A) agonists. This is the first report showing the presence of an A(2A)AR alteration in putamen in PD that mirrors a similar up-regulation in human peripheral blood cells. Moreover, the correlation found between A(2A)AR density or A(2A) agonist potency and UPDRS motor score highlights the central role of A(2A)ARs in the pharmacological treatment of PD.

Oxidative/nitrosative stress selectively altered A(2B) adenosine receptors in chronic obstructive pulmonary disease.

The primary aim of this study was to investigate adenosine receptors (ARs) in bronchoalveolar lavage (BAL) macrophages from patients with chronic obstructive pulmonary disease (COPD) and age-matched healthy smokers. A(2B)ARs were significantly decreased in BAL macrophages from patients with COPD when compared with healthy smokers. The effect of proinflammatory cytokines and oxidative/nitrosative stress on AR expression and function in U937 cells before and after PMA treatment was evaluated. IL-1beta and TNF-alpha treatment up-regulated A(2A)- and A(3)ARs but not A(1)- or A(2B)ARs, whereas IL-6 did not modify AR expression. In contrast, oxidative/nitrosative stress selectively decreased A(2B)AR expression, which was associated with a reduction in the potency of the adenosine agonist 5'-N-ethylcarboxamideadenosine (NECA) to induce cAMP. Further, the ability of NECA to enhance cell proliferation was increased after oxidative/nitrosative stress. The specific involvement of A(2B)ARs was investigated by using potent and selective A(2B)AR antagonist and by A(2B)AR knockdown using siRNA and demonstrated responses similar to those obtained with oxidative/nitrosative stress. N-acetylcysteine (NAC), an antioxidant agent, counteracted the decrease in A(2B)AR expression, as well as the altered NECA effects on cAMP and cell proliferation. These findings highlight the central role of A(2B)ARs in alveolar macrophages, suggesting that their modulation could represent an innovative pharmacological strategy to manage COPD.-Varani, K., Caramori, G., Vincenzi, F., Tosi, A., Barczyk, A., Contoli, M., Casolari, P., Triggiani, M., Hansel, T., Leung, E., MacLennan, S., Barnes, P. J., Fan Chung, K., Adcock, I., Papi, A., Borea, P. A. Oxidative/nitrosative stress selectively altered A(2B) adenosine receptors in chronic obstructive pulmonary disease.

Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti-tumor necrosis factor alpha but not methotrexate.

OBJECTIVE: To investigate A(1), A(2A), A(2B), and A(3) adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNFalpha), as compared with those in age-matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNFalpha release and NF-kappaB activation. METHODS: Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A(2A) and A(3) agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti-TNFalpha. In a separate cohort of RA patients, TNFalpha release and NF-kappaB activation were evaluated in plasma and nuclear extracts, respectively. RESULTS: In ERA patients, we found a high density and altered functionality of A(2A) and A(3) receptors. The binding and functional parameters of A(2A) and A(3) receptors normalized after anti-TNFalpha, but not MTX, treatment. TNFalpha release was increased in ERA patients and in MTX-treated RA patients, whereas in anti-TNFalpha-treated RA patients, release was comparable to that in the controls. NF-kappaB activation was elevated in ERA patients and in MTX-treated RA patients. Anti-TNFalpha treatment mediated decreased levels of NF-kappaB activation. CONCLUSION: A(2A) and A(3) receptor up-regulation in ERA patients and in MTX-treated RA patients was associated with high levels of TNFalpha and NF-kappaB activation. Treatment with anti-TNFalpha normalized A(2A) and A(3) receptor expression and functionality. This new evidence of A(2A) and A(3) receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component.

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors.

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.