Adenosine A2A Receptor Ligation Inhibits Osteoclast Formation

Adenosine is generated in increased concentrations at sites of injury/hypoxia and mediates a variety of physiological and pharmacological effects via G protein–coupled receptors (A1, A2A, A2B, and A3). Because all adenosine receptors are expressed on osteoclasts, we determined the role of A2A receptor in the regulation of osteoclast differentiation. Differentiation and bone resorption were studied as the macrophage colony-stimulating factor-1–receptor activator of NF-κB ligand formation of multinucleated tartrate-resistant acid phosphatase (TRAP)–positive cells from primary murine bone marrow–derived precursors. A2A receptor and osteoclast marker expression levels were studied by RT-PCR. Cytokine secretion was assayed by enzyme-linked immunosorbent assay. In vivo examination of A2A knockout (KO)/control bones was determined by TRAP staining, micro–computed tomography, and electron microscopy. The A2A receptor agonist, CGS21680, inhibited osteoclast differentiation and function (half maximal inhibitory concentration, 50 nmol/L), increased the percentage of immature osteoclast precursors, and decreased IL-1β and tumor necrosis factor-α secretion, an effect that was reversed by the A2A antagonist, ZM241385. Cathepsin K and osteopontin mRNA expression increased in control and ZM241385-pretreated osteoclasts, and this was blocked by CGS21680. Micro–computed tomography of A2AKO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. A2AKO femurs showed an increased TRAP-positive osteoclast. Electron microscopy in A2AKO femurs showed marked osteoclast membrane folding and increased bone resorption. Thus, adenosine, acting via the A2A receptor, inhibits macrophage colony-stimulating factor-1–receptor activator of NF-κB ligand–stimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated

Sobreexpresión del receptor P2Y2 tras su silenciamiento durante el proceso de cicatrización corneal

Diadenosine polyphosphates are a family of dinucleotides with relevant properties in the eye and in other tissues. Diadenosine polyphosphates can activate P2Y and P2X receptors present on the ocular surface, anterior segment and retina. In the cornea, the presence of a P2Y2, P2Y4 and P2Y6 receptor has been identified. Both diadenosine polyphosphates and other purinergic agonists modified corneal wound healing depending on the receptor that is activated by these substances. To confirm the involvement of the P2Y2 receptor in the wound healing process after the challenge with Ap4A, we have designed siRNA against P2Y2 receptor. We have observed that P2Y2 is localized in the most external layer of the corneal epithelium. The pre-treatment with siRNA produced a disappearance of the receptor at 12 and 24 hours after the wound, being the location for P2Y2 restored 36 hours after the wound. We have also observed that in half of the tested corneas, there was an increase in the P2Y2 expression after silencing compared to control and Ap4A treated corneas, being this receptor localized both in corneal epithelium and stroma.Los diadenosina polifosfatos son una familia de dinucleótidos con gran relevancia en las propiedades del ojo y de otros tejidos. Estos diadenosina polifosfatos pueden activar los receptores P2Y y P2X presentes en la superficie ocular, en el segmento anterior y en la retina. En la cornea, se ha identificado la presencia de receptores P2Y2, P2Y4 y P2Y6. Tanto los diadenosina polifosfatos como los receptores purinérgicos modifican el proceso de cicatrización corneal dependiendo del tipo de receptor activado por los distintos dinucleótidos. Para localizar el receptor P2Y2 en córneas lesionadas y tratadas con Ap4A en presencia o ausencia de un siRNA para el receptor P2Y2, hemos realizado un ensayo de inmunohistoquímica. Hemos observado que el receptor P2Y2 se localiza en el epitelio tras la lesión corneal y el consecuente tratamiento con Ap4A. El pre-tratamiento con el siRNA produce la desaparición de la señal para este receptor tanto a las 12 como a las 24 horas de la lesión corneal, siendo la localización de este receptor P2Y2 recuperada a las 36 horas de la lesión en presencia del siRNA. Además, hemos observado que en la mitad de las córneas analizadas, existía un incremento en la expresión del receptor P2Y2 tras el silenciamiento del mismo comparado con las córneas control y con las tratadas con Ap4A, localizando la presencia de este receptor tanto en el epitelio como en el estroma corneal

Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

Purpose. To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap4A and Ap3A. Methods. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap4A or Ap3A 100 μM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 μM, U0126 100 μM, Y27632 100 nM, and (−)-blebbistatin 10 μM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap4A and Ap3A (100 μM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap4A or Ap3A (100 μM) with U0126 and Y27632 (100 nM). Results. In the presence of Ap4A, U0126, Y27632, AG1478, and (−)-blebbistatin, reduced the migration rate compared to the effect of Ap4A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap4A, respectively). In the presence of Ap3A 100 μM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap3A alone, whereas AG1478 and (−)-blebbistatin (P < 0.0001 versus Ap3A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap4A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. Conclusions. The activation of the Ap4A/P2Y2 receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap3A/P2Y6 receptor signals only the MAPK pathway

Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors

Introduction This work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR). Methods After apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student's t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant. Results In vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1μM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone. Conclusions The immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature

A biodegradable antifungal-loaded sol-gel coating for the prevention and local treatment of yeast prosthetic-joint infections

Fungal prosthetic-joint infections are rare but devastating complications following arthroplasty. These infections are highly recurrent and expose the patient to the development of candidemia, which has high mortality rates. Patients with this condition are often immunocompromised and present several comorbidities, and thus pose a challenge for diagnosis and treatment. The most frequently isolated organisms in these infections are Candida albicans and Candida parapsilosis, pathogens that initiate the infection by developing a biofilm on the implant surface. In this study, a novel hybrid organo-inorganic sol-gel coating was developed from a mixture of organopolysiloxanes and organophosphite, to which different concentrations of fluconazole or anidulafungin were added. Then, the capacity of these coatings to prevent biofilm formation and treat mature biofilms produced by reference and clinical strains of C. albicans and C. Parapsilosis was evaluated. Anidulafungin-loaded sol-gel coatings were more effective in preventing C. albicans biofilm formation, while fluconazole-loaded sol-gel prevented C. parapsilosis biofilm formation more effectively. Treatment with unloaded sol-gel was sufficient to reduce C. albicans biofilms, and the sol-gels loaded with fluconazole or anidulafungin slightly enhanced this effect. In contrast, unloaded coatings stimulated C. parapsilosis biofilm formation, and loading with fluconazole reduced these biofilms by up to 99%. In conclusion, these coatings represent a novel therapeutic approach with potential clinical use to prevent and treat fungal prosthetic-joint infections.This research was funded by MUTUA MADRILEÑA FOUNDATION, grant number 04078/001 and REGIONAL GOVERNMENT OF MADRID through the ADDIMAT PROGRAM, grant number S2018/NMT-441

Adenosine deaminase as a biomarker of tenofovir mediated inflammation in naïve HIV patients

Plasma levels of adenosine deaminase (ADA), an enzyme that deaminates adenosine to inosine, are increased during inflammation. An increase in ADA activity occurs with lower human immunodeficiency virus (HIV) viral load and higher CD4+ T cell counts. We aimed to investigate the role of plasma ADA as a biomarker of inflammation in treatment-naïve HIV patients who received tenofovir or another nucleoside analog for comparison. Ninety-two treatment-naïve patients were included in the study and grouped by treatment, i.e., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or Triumeq. ADA activity was measured in plasma and cytokines were analyzed by MILLIPLEX® MAP-Luminex® Technology. Plasma concentration of monocytes and neutrophils was measured at 0, 3, and 12 months post-treatment. Treatment-naïve HIV patients had increased ADA concentrations (over 15 U/L) that decreased after treatment with TAF and Triumeq, though this did not occur in TDF-treated patients. However, all groups exhibited a pro-inflammatory systemic profile at 12 months of treatment. Plasma GM-CSF levels decreased after 12 months of treatment in the TDF group, with a concomitant decrease in blood monocyte count, and a negative correlation with ADA values was found. In conclusion, ADA levels may be modulated by antiretroviral therapy in HIV patients, possibly affecting inflammatory status.This work was supported by grants from Instituto de Salud Carlos III through the “Miguel Servet” program (CP15/00053), co-funded by Fondo Europeo de Desarrollo Regional (FEDER) and research grants from the Spanish Instituto de Salud Carlos III (PI16/00991 and PI19/00744

Arabic gum plus colistin coated moxifloxacin-loaded nanoparticles for the treatment of bone infection caused by Escherichia coli

Osteomyelitis is an inflammatory process of bone and bone marrow that may even lead to patient death. Even though this disease is mainly caused by Gram-positive organisms, the proportion of bone infections caused by Gram-negative bacteria, such as Escherichia coli, has significantly increased in recent years. In this work, mesoporous silica nanoparticles have been employed as platform to engineer a nanomedicine able to eradicate E. coli- related bone infections. For that purpose, the nanoparticles have been loaded with moxifloxacin and further functionalized with Arabic gum and colistin (AG+CO-coated MX-loaded MSNs). The nanosystem demonstrated high affinity toward E. coli biofilm matrix, thanks to AG coating, and marked antibacterial effect because of the bactericidal effect of moxifloxacin and the disaggregating effect of colistin. AG+CO-coated MX-loaded MSNs were able to eradicate the infection developed on a trabecular bone in vitro and showed pronounced antibacterial efficacy in vivo against an osteomyelitis provoked by E. coli. Furthermore, AG+CO-coated MX-loaded MSNs were shown to be essentially non-cytotoxic with only slight effect on cell proliferation and mild hepatotoxicity, which might be attributed to the nature of both antibiotics. In view of these results, these nanoparticles may be considered as a promising treatment for bone infections caused by enterobacteria, such as E. coli, and introduce a general strategy against bone infections based on the implementation of antibiotics with different but complementary activity into a single nanocarrier. Statement of significance: In this work, we propose a methodology to address E.coli bone infections by using moxifloxacin-loaded mesoporous silica nanoparticles coated with Arabic gum containing colistin (AG+CO-coated MX-loaded MSNs). The in vitro evaluation of this nanosystem demonstrated high affinity toward E. coli biofilm matrix thanks to the Arabic gum coating, a disaggregating and antibacterial effect of colistin, and a remarkable antibiofilm action because of the bactericidal ability of moxifloxacin and colistin. This anti-E. coli capacity of AG+CO-coated MX-loaded MSNs was brought out in an in vivo rabbit model of osteomyelitis where the nanosystem was able to eradicate more than 90% of the bacterial load within the infected boneFinancial support from the Eu-ropean Research Council through ERC-2015-AdG-694160 (VERDI) grant. AM is funded by grants from Instituto de Salud Carlos III through the “Miguel Servet” program (CP15/00053

Effect of gold nanostars plus amikacin against cabapenem-resistant Klebsiella pneumoniae biofilm.

(1) Background: Carbapenem-resistant Klesiella pneumoniae (CR-KP) infection rates depict an almost pre-antibiotic scenario since the pipeline for effective antibiotics against this pathogen has been almost entirely depleted. This study aims to evaluate the antibacterial effect of gold nanostars (GNS) alone or associated with some of the most widely used antibiotics for the treatment of CR-KP strains, i.e., meropenem or amikacin, on both planktonic and sessile forms. Additionally, we measured the effect of GNS on cell proliferation and biocompatibility in invertebrate in vivo models. (2) Materials and methods: GNS were made from gold seeds grown using a seeded-growth surfactant-free method assisted by silver ions and functionalized with mercapto-poly(ethylene glycol)amino by ligand exchange. The antimicrobial capacity, effect on cell proliferation, and biocompatibility of the most effective combination was evaluated in a Galleria mellonella model. (3) Results:The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 80 and 160 µM of GNS for all strains, respectively. The minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were >320 µM of GNS for both. A synergy was found between GNS and amikacin. Larvae administered GNS plus amikacin were found to tolerate the treatment well, which prevented infection. (4) Conclusions: GNS are a promising anti-CR-KP nanomaterial