[18F]DPA-714 as a biomarker for positron emission tomography imaging of rheumatoid arthritis in an animal model.

International audienceINTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease, affecting 0.5-1% of adults in industrialized countries, in which systemic inflammation and synovitis drive joint destruction. [18F]DPA-714 is a specific tracer of the 18 kDa Translocator Protein (TSPO), which is overexpressed on activated macrophages, and proposed as a biomarker of neuroinflammation. Today, diagnosis of patients with early inflammatory arthritis is limited by poor sensitivity and specificity. The present study aims to investigate the potential of [18F]DPA-714 to monitor in vivo inflammatory processes at a preclinical stage via positron emission tomography (PET). METHODS: RA was induced in Dark Agouti rats by subcutaneous injection of inactivated mycobacterium tuberculosis. Development of arthritis clinical signs was investigated daily and the severity of the disease evaluated. Animals were imaged at the peak of inflammation using [18F]DPA-714 and a small-animal PET-CT tomograph. RESULTS: The first clinical signs appeared at 10 days post-injection, with a peak of inflammation at 20 days. At this time, PET-analyses showed a clear uptake of [18F]DPA-714 in swollen ankles, with mean values of 0.52 +/- 0.18%ID/cc for treated (n = 11) and 0.19 +/- 0.09 for non-treated (n = 6) rats. A good correlation between [18F]DPA-714's uptake and swelling was also found. Immunohistochemistry showed an enhanced TSPO expression in hind paws, mainly co-localized with the macrophages specific antigen CD68 expressing cells. CONCLUSION: These preliminary results demonstrates that the TSPO 18 kDa specific radioligand [18F]DPA-714 is adapted for the study and follow up of inflammation linked to RA in our experimental model, suggesting also a strong potential for clinical imaging of peripheral inflammation

Imaging Microglial/Macrophage Activation in Spinal Cords of Experimental Autoimmune Encephalomyelitis Rats by Positron Emission Tomography Using the Mitochondrial 18kDa Translocator Protein Radioligand [18F]DPA-714

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Activated microglia/macrophages play a key role in the immunopathogenesis of MS and its corresponding animal models, experimental autoimmune encephalomyelitis (EAE). Microglia activation begins at early stages of the disease and is associated with elevated expression of the 18 kDa mitochondrial translocator protein (TSPO). Thus, positron emission tomography (PET) imaging of microglial activation using TSPO-specific radioligands could be valuable for monitoring disease-associated neuroinflammatory processes. EAE was induced in rats using a fragment of myelin basic protein, yielding acute clinical disease that reflects extensive spinal cord inflammation. Enhanced TSPO expression in spinal cords of EAE rats versus those of controls was confirmed by Western blot and immunohistochemistry. Biodistribution studies in control and EAE rats were performed using the TSPO radioligand [18F]DPA-714 [N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide]. At 1 h after injection, almost fivefold higher levels of [18F]DPA-714 were measured in spinal cords of EAE rats versus controls. The specific binding of [18F]DPA-714 to TSPO in spinal cords was confirmed in competition studies, using unlabeled (R,S)-PK11195 [(R,S)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide)] or DPA-714 in excess. MicroPET studies affirm that this differential radioactivity uptake in spinal cords of EAE versus control rats could be detected and quantified. Using [18F]DPA-714, neuroinflammation in spinal cords of EAE-induced rats could be visualized by PET, offering a sensitive technique for monitoring neuroinflammatory lesions in the CNS and particularly in the spinal cord. In addition to current MRI protocols, this approach could provide molecular images of neuroinflammation for detection, monitoring, and research in MS

Longitudinal mouse-PET imaging: a reliable method for estimating binding parameters without a reference region or blood sampling

International audienceLongitudinal mouse PET imaging is becoming increasingly popular due to the large number of transgenic and disease models available but faces challenges. These challenges are related to the small size of the mouse brain and the limited spatial resolution of microPET scanners, along with the small blood volume making arterial blood sampling challenging and impossible for longitudinal studies. The ability to extract an input function directly from the image would be useful for quantification in longitudinal small animal studies where there is no true reference region available such as TSPO imaging.METHODS:Using dynamic, whole-body 18F-DPA-714 PET scans (60 min) in a mouse model of hippocampal sclerosis, we applied a factor analysis (FA) approach to extract an image-derived input function (IDIF). This mouse-specific IDIF was then used for 4D-resolution recovery and denoising (4D-RRD) that outputs a dynamic image with better spatial resolution and noise properties, and a map of the total volume of distribution (VT) was obtained using a basis function approach in a total of 9 mice with 4 longitudinal PET scans each. We also calculated percent injected dose (%ID) with and without 4D-RRD. The VT and %ID parameters were compared to quantified ex vivo autoradiography using regional correlations of the specific binding from autoradiography against VT and %ID parameters.RESULTS:The peaks of the IDIFs were strongly correlated with the injected dose (Pearson R = 0.79). The regional correlations between the %ID estimates and autoradiography were R = 0.53 without 4D-RRD and 0.72 with 4D-RRD over all mice and scans. The regional correlations between the VT estimates and autoradiography were R = 0.66 without 4D-RRD and 0.79 with application of 4D-RRD over all mice and scans.CONCLUSION:We present a FA approach for IDIF extraction which is robust, reproducible and can be used in quantification methods for resolution recovery, denoising and parameter estimation. We demonstrated that the proposed quantification method yields parameter estimates closer to ex vivo measurements than semi-quantitative methods such as %ID and is immune to tracer binding in tissue unlike reference tissue methods. This approach allows for accurate quantification in longitudinal PET studies in mice while avoiding repeated blood sampling

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis

Background & Aims Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene ( REG3A ) alters the fecal microbiota and affects development of colitis in mice. Methods We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2′,7′-dichlorofluorescein diacetate and flow cytometry. Results The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A- TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A -TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A- TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria ( Faecalibacterium prausnitzii and Roseburia intestinalis ). Conclusions Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A -TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation

Legislative Documents

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ROLE DES PHOSPHOLIPASES D ET A 2 DANS LE TRANSPORT INTRA CELLULAIRE ET LA SECRETION DES PROTEINES PAR LA CELLULE EPITHELIALE MAMMAIRE

PENDANT LA LACTATION, LES CELLULES EPITHELIALES MAMMAIRES (MEC) SYNTHETISENT ET SECRETENT DE GRANDES QUANTITES DE PROTEINES. LES PRINCIPALES PROTEINES DU LAIT DE LAPIN SONT LES CASEINES ET LA PROTEINE ACIDE DE PETIT LAIT (WAP : WHEY ACIDIC PROTEIN). POUR OBTENIR DAVANTAGE D'INFORMATION SUR LES MECANISMES DE TRANSPORT DANS LA VOIE DE SECRETION ET PARTICULIEREMENT SUR LA FORMATION DES VESICULES A PARTIR DU RESEAU TRANS GOLGIEN (TGN), NOUS AVONS ETUDIE LE ROLE DES PHOSPHOLIPASES D ET A 2. LE FAIT QUE LES PROTEINES DU LAIT SOIENT SOUMISES A UN CERTAIN NOMBRE DE MODIFICATIONS POST-TRADUCTIONNELLES AU NIVEAU DE L'APPAREIL DE GOLGI A ETE UTILISES POUR ETUDIER L'IMPLICATION EVENTUELLE DE CES PHOSPHOLIPASES DANS LES MECANISMES DE TRANSPORT DU RETICULUM VERS L'APPAREIL DE GOLGI. LE TRAITEMENT DES EXPLANTS MAMMAIRES PAR DU BUTANOL, INHIBE LA FORMATION D'ACIDE PHOSPHATIDIQUE, DIMINUE LA SECRETION DES CASEINES ET D'UNE FACON MOINS IMPORTANTE CELLE DE LA WAP. LE BUTANOL AFFECT LE TRANSPORT DES CASEINES DU RETICULUM ENDOPLASMIQUE VERS LE COMPLEXE GOLGIEN AINSI QUE LA FORMATION DES VESICULES A PARTIR DU TGN. EN REVANCHE, DANS CES CONDITIONS, LE TRANSPORT DE LA WAP VERS L'APPAREIL DE GOLGI EST MOINS AFFECTE. L'ACTIVATION DE LA PROTEINE KINASE C (PKC) AUGMENTE LE NIVEAU DE SECRETION DES DEUX PROTEINES ET MAIS SEULEMENT CELUI DE LA WAP EN PRESENCE DE BUTANOL. LE DOSAGES DES PRODUITS DE TRANSPHOSPHATIDYLATION ONT MONTRE L'EXISTENCE D'UNE ACTIVITE CONSTITUTIVE DE LA PHOSPHOLIPASE D QUI EST STIMULEE PAR ACTIVATION DE LA PKC. EN CE QUI CONCERNE L'IMPLICATION DES PLA 2S DANS LA SECRETION ET LE TRANSPORT DES PROTEINES DU LAIT, NOS RESULTATS MONTRENT QUE LA CPCA 2 N'EST PAS IMPLIQUEE DANS LES MECANISMES DE SECRETION DE BASE DE CES PROTEINES MAIS SEMBLE PARTICIPER A LA STIMULATION DE SECRETION INDUITE PAR PROLACTINE. AU CONTRAIRE, LA IPLA 2 NE SEMBLE PAS IMPLIQUEE DANS CET EFFET MAIS PARTI$ CLAIREMENT AUX MECANISMES DE TRANSPORT DES PROTEINES DU LAIT DANS LA VOIE DE SECRETION.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF