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•Intraperitoneal injection of Stattic inhibits brain STAT3 phosphorylation.•Stattic decreases LPS-induced microglial activation in the hippocampus.•Stattic reduces brain levels of ...proinflammatory cytokines triggered by LPS administration.•Stattic reduces hippocampal BACE1 levels in LPS-treated mice.
Abnormal activation of the transcriptional factor STAT3 (signal transducer and activator of transcription 3) was recently associated with Alzheimer Disease (AD). STAT3 phosphorylation is critical for cytokine secretion linked to neuroinflammation. Moreover, STAT3 may act as a transcriptional regulator of BACE1 (β-APP cleaving enzyme-1), the key enzyme in amyloid β (Aβ) production. We have previously shown that neuroinflammation and increased brain BACE1 levels triggered by LPS-induced systemic inflammation in wild-type mice are associated with an enhanced STAT3 activation. Using this LPS model, the goal of this study was to investigate if a STAT3 inhibitor administration could be protective against neuroinflammation and abnormal BACE1 regulation. Our results show that intraperitoneal injection of Stattic, a molecule that selectively inhibits the activation of STAT3, decreases LPS-induced microglial activation in the hippocampus. In addition, STAT3 inhibition reduced brain levels of cytokines IL-6, IL-1β and TNF-α triggered by LPS systemic administration. A significant reduction of BACE1 levels was observed in the hippocampus of mice treated with LPS and Stattic compared to those exposed to LPS alone. Taking together, our results show that Stattic can protect hippocampus against two pathological hallmarks of AD, and pave the way for further explorations of the therapeutic potential of STAT3 inhibition in AD.
Traumatic brain injury (TBI) leads to a deleterious neuroinflammation, originating from microglial activation. Monitoring microglial activation is an indispensable step to develop therapeutic ...strategies for TBI. In this study, we evaluated the use of the 18-kDa translocator protein (TSPO) in positron emission tomography (PET) and cellular analysis to monitor microglial activation in a mild TBI mouse model. TBI was induced on male Swiss mice. PET imaging analysis with
FFEPPA, a TSPO radiotracer, was performed at 1, 3 and 7 days post-TBI and flow cytometry analysis on brain at 1 and 3 days post-TBI. PET analysis showed no difference in TSPO expression between non-operated, sham-operated and TBI mice. Flow cytometry analysis demonstrated an increase in TSPO expression in ipsilateral brain 3 days post-TBI, especially in microglia, macrophages, lymphocytes and neutrophils. Moreover, microglia represent only 58.3% of TSPO
cells in the brain. Our results raise the question of the use of TSPO radiotracer to monitor microglial activation after TBI. More broadly, flow cytometry results point the lack of specificity of TSPO for microglia and imply that microglia contribute to the overall increase in TSPO in the brain after TBI, but is not its only contributor.
FFEPPA is a specific ligand for the translocator protein of 18 kDa (TSPO) used as a positron emission tomography (PET) biomarker for glial activation and neuroinflammation.
FFEPPA radiosynthesis ...was optimized to assess in a mouse model the cerebral inflammation induced by an intraperitoneal injection of
serovar
lipopolysaccharides (LPS; 5 mg/kg) 24 h before PET imaging.
FFEPPA was synthesized by nucleophilic substitution (90 °C, 10 min) with tosylated precursor, followed by improved semi-preparative HPLC purification (retention time 14 min).
FFEPPA radiosynthesis were carried out in 55 min (from EOB). The non-decay corrected radiochemical yield were 34 ± 2% (
= 17), and the radiochemical purity greater than 99%, with a molar activity of 198 ± 125 GBq/µmol at the end of synthesis. Western blot analysis demonstrated a 2.2-fold increase in TSPO brain expression in the LPS treated mice compared to controls. This was consistent with the significant increase of
FFEPPA brain total volume of distribution (
) estimated with pharmacokinetic modelling. In conclusion,
FFEPPA radiosynthesis was implemented with high yields. The new purification/formulation with only class 3 solvents is more suitable for in vivo studies.
We previously reported that a novel peptide vaccine platform, based on synthetic melanin nanoaggregates, triggers strong cytotoxic immune responses and significantly suppresses tumor growth in mice. ...However, the mechanisms underlying such an efficacy remained poorly described. Herein, we investigated the role of dendritic cells (DCs) in presenting the antigen embedded in the vaccine formulation, as well as the potential stimulatory effect of melanin upon these cells, in vitro by coculture experiments and ELISA/flow cytometry analysis. The vaccine efficiency was evaluated in FLT3-L
mice constitutively deficient in DC1, DC2, and pDCs, in Zbtb46
chimera mice deficient in DC1 and DC2, and in Langerin
mice deficient in dermal DC1 and Langerhans cells. We concluded that DCs, and especially migratory conventional type 1 dendritic cells, seem crucial for mounting the immune response after melanin-based vaccination. We also assessed the protective effect of L-DOPA melanin on peptides from enzymatic digestion, as well as the biodistribution of melanin-peptide nanoaggregates, after subcutaneous injection using
FMEL050 PET imaging in mice. L-DOPA melanin proved to act as an efficient carrier for peptides by fully protecting them from enzymatic degradation. L-DOPA melanin did not display any direct stimulatory effects on dendritic cells in vitro. Using PET imaging, we detected melanin-peptide nanoaggregates up to three weeks after subcutaneous injections within the secondary lymphoid tissues, which could explain the sustained immune response observed (up to 4 months) with this vaccine technology.
Astroglial connexin 43 (Cx43) has been recognized as a crucial immunoregulating factor in the brain. Its inactivation leads to a continuous immune recruitment, cytokine expression modification and a ...specific humoral autoimmune response against the astrocytic extracellular matrix but without brain lesions or cell lysis. To assess the impact of Cx43 deletion on the brain's inflammatory response, TSPO expression was studied by positron emission tomography (PET) imaging with a specific radioligand,
FFEPPA, in basal conditions or upon Lipopolysaccharides (LPS)-induced inflammatory challenge. Astroglial Cx43-deleted mice underwent
FFEPPA PET/CT dynamic imaging with or without LPS injection (5 mg/kg) 24 h before imaging. Quantification and pharmacokinetic data modelling with a 2TCM-1K compartment model were performed. After collecting the mice brains, TSPO expression was quantified and localized by Western blot and FISH analysis. We found that astroglial Cx43 deficiency does not significantly alter TSPO expression in the basal state as observed with
FFEPPA PET imaging, FISH and Western blot analysis. However, deletion of astrocyte Cx43 abolishes the LPS-induced TSPO increase. Autoimmune encephalopathy observed in astroglial Cx43-deleted mice does not involve TSPO overexpression. Consistent with previous studies showing a unique inflammatory status in the absence of astrocyte Cx43, we show that a deficient expression of astrocytic Cx43 protects the animals from LPS-induced neuroinflammation as addressed by TSPO expression.
Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to ...implement a fully automated radiosynthesis of
FMEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of
FMEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of
FFDG.
Automated radiosynthesis of
FMEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module.
FMEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of
FMEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to
FFDG and correlated to in vivo bioluminescence imaging.
The automated radiosynthesis of
FMEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%.
FMEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with
FMEL050 and
FFDG in subcutaneous tumors and higher TBR with
FMEL050 than with
FFDG in pulmonary metastases.
We successfully implemented the radiosynthesis of
FMEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of
FMEL050 was obtained in mouse models of pigmented melanoma, where higher
FMEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to
FFDG.
Fluorine-18 (
F) is the most favorable positron emitter for radiolabeling Positron Emission Tomography (PET) probes. However, conventional
F labeling through covalent C-F bond formation is ...challenging, involving multiple steps and stringent conditions unsuitable for sensitive biomolecular probes whose integrity may be altered. Over the past decade, an elegant new approach has been developed involving the coordination of an aluminum fluoride {Al
F} species in aqueous media at a late-stage of the synthetic process. The objective of this study was to implement this method and to optimize radiolabeling efficiency using a Design of Experiments (DoE). To assess the impact of various experimental parameters on {Al
F} incorporation, a pentadentate chelating agent NODA-MP-C4 was prepared as a model compound. This model carried a thiourea function present in the final conjugates resulting from the grafting of the chelating agent onto the probe. The formation of the radioactive complex Al
F-NODA-MP-C4 was studied to achieve the highest radiochemical conversion. A complementary "cold" series study using the natural isotope
F was also conducted to guide the radiochemical operating conditions. Ultimately, Al
F-NODA-MP-C4 was obtained with a reproducible and satisfactory radiochemical conversion of 79±3.5 % (n=5).
Fluorine‐18 (18F) is the most favorable positron emitter for radiolabeling Positron Emission Tomography (PET) probes. However, conventional 18F labeling through covalent C‐F bond formation is ...challenging, involving multiple steps and stringent conditions unsuitable for sensitive biomolecular probes whose integrity may be altered. Over the past decade, an elegant new approach has been developed involving the coordination of an aluminum fluoride {Al18F} species in aqueous media at a late‐stage of the synthetic process. The objective of this study was to implement this method and to optimize radiolabeling efficiency using a Design of Experiments (DoE). To assess the impact of various experimental parameters on {Al18F} incorporation, a pentadentate chelating agent NODA‐MP‐C4 was prepared as a model compound. This model carried a thiourea function present in the final conjugates resulting from the grafting of the chelating agent onto the probe. The formation of the radioactive complex Al18F‐NODA‐MP‐C4 was studied to achieve the highest radiochemical conversion. A complementary "cold" series study using the natural isotope 19F was also conducted to guide the radiochemical operating conditions. Ultimately, Al18F‐NODA‐MP‐C4 was obtained with a reproducible and satisfactory radiochemical conversion of 79 ± 3.5% (n = 5).
Fluorine‐18 (18F) is the most favorable positron emitter for radiolabeling Positron Emission Tomography (PET) probes. However, conventional 18F labeling through covalent C−F bond formation is ...challenging, involving multiple steps and stringent conditions unsuitable for sensitive biomolecular probes whose integrity may be altered. Over the past decade, an elegant new approach has been developed involving the coordination of an aluminum fluoride {Al18F} species in aqueous media at a late‐stage of the synthetic process. The objective of this study was to implement this method and to optimize radiolabeling efficiency using a Design of Experiments (DoE). To assess the impact of various experimental parameters on {Al18F} incorporation, a pentadentate chelating agent NODA‐MP‐C4 was prepared as a model compound. This model carried a thiourea function present in the final conjugates resulting from the grafting of the chelating agent onto the probe. The formation of the radioactive complex Al18F‐NODA‐MP‐C4 was studied to achieve the highest radiochemical conversion. A complementary “cold” series study using the natural isotope 19F was also conducted to guide the radiochemical operating conditions. Ultimately, Al18F‐NODA‐MP‐C4 was obtained with a reproducible and satisfactory radiochemical conversion of 79±3.5 % (n=5).
Lesser, better, faster: Optimization of a radiolabeling method by complexation of aluminum‐18Ffluoride {Al18F} species using a Design of Experiments (DoE) approach is presented. This study demonstrates the potential of this strategy in radiochemistry for optimization of a radiochemical process.
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