Percorrer por autor "Lamghari, Meriem"
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- Bone injury and repair trigger central and peripheral NPY Neuronal PathwaysPublication . Alves, Cecília J.; Alencastre, Inês S.; Neto, Estrela; Ribas, João; Ferreira, Sofia; Vasconcelos, Daniel M.; Sousa, Daniela M.; Summavielle, Teresa; Lamghari, MeriemBone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair.
- Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometryPublication . Barreiros, Luisa; Silva, Eduarda M P; Alencastre, Inês S; Lamghari, Meriem; Segundo, Marcela ANeuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6 μm, 150 × 2.1 mm) at 30 °C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30 ng mL-1 with LOD and LOQ values as low as 0.1 and 0.3 pg in cell extracts and 16 and 48 pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.
- Fibrinogen scaffolds with immunomodulatory properties promote in vivo bone regenerationPublication . Vasconcelos, Daniel M.; Gonçalves, Raquel M.; Almeida, Catarina R.; Pereira, Inês O.; Oliveira, Marta I.; Neves, Nuno; Silva, Andreia M.; Ribeiro, António C.; Cunha, Carla; Almeida, Ana R.; Ribeiro, Cristina C.; Gil, Ana M.; Seebach, Elisabeth; Kynast, Katharina L.; Richter, Wiltrud; Lamghari, Meriem; Santos, Susana G.; Barbosa, Mário A.The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac- 1þ (CD18þ/CD11bþ) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1b and increased TGF-b1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials.
- HPLC-MS/MS method for quantification of the neuropeptide Y Y1 receptor antagonist BIBP 3226 in cell extractsPublication . Barreiros, Luisa; Silva, Eduarda M. P.; Alencastre, Inês S.; Lamghari, Meriem; Segundo, Marcela A.; Barreiros, LuisaNeuropeptide Y (NPY) is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. NPY activates different receptors in several brain regions. Recently, Y1 receptor (Y1R) has arisen as a potential regulator in the local control of bone turnover suggesting that an antireceptor strategy may be a useful therapeutic approach to prevent and/or reverse bone loss. BIBP 3226 is a potent Y1R selective antagonist that has been successfully used in in vitro studies showing a positive impact in bone turnover and thus providing good perspectives towards its application as a pharmacological tool for bone regeneration. Hence, the major aim of the present work was to implement a method based on high performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry for quantification of BIBP 3226 in cellular internalization assays. Chromatographic separation was achieved using a reversed phase Kinetex coreshell C8 column at 30 C and elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1. Total run time was 5.0 min, with retention time of 3.7 min for the target compound. The MS/MS was operated in positive ionization mode (ESI+) and data were acquired in multiple reaction monitoring (MRM) mode (m/z 474>167 for quantification and m/z 474>107 for identity confirmation). Calibration curves were linear for concentrations ranging from 0.5 to 30 ng mL-1. BIBP 3226 was quantified in cell extracts obtained from internalization assays performed with bone marrow and breast cancer cells, after solvent evaporation and resuspension in mobile phase. LOD and LOQ were 0.04 and 0.1 ng mL-1, respectively, corresponding to values as low as 0.3 and 0.8 pg per wel