| 1. |
- Eftekhari, Sajedeh, et al.
(author)
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Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.
- 2015
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In: Brain Research. - : Elsevier BV. - 1872-6240 .- 0006-8993. ; 1600:Nov 20, s. 93-109
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Journal article (peer-reviewed)abstract
- Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.
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| 2. |
- Eftekhari, Sajedeh, et al.
(author)
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Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence and autoradiography.
- 2016
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In: Journal of Comparative Neurology. - : Wiley. - 1096-9861 .- 0021-9967. ; 524:1, s. 90-118
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Journal article (peer-reviewed)abstract
- Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene-related peptide (CGRP) is associated with activation of the trigeminovascular system, transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP it is critical to identify the regions within the brainstem that processes CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3) H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus and spinal trigeminal nucleus and the spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, motor trigeminal nucleus; where protein co-expression of CLR and RAMP1 was observed via immunofluorescence. [(3) H]MK-3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that CGRP receptor antagonists may not need to be CNS-penetrant to antagonize receptors in these brain regions. This article is protected by copyright. All rights reserved.
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| 3. |
- Eftekhari, Sajedeh, et al.
(author)
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Localization of CGRP Receptor Components, CGRP, and Receptor Binding Sites in Human and Rhesus Cerebellar Cortex.
- 2013
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In: Cerebellum. - : Springer Science and Business Media LLC. - 1473-4230. ; 12:6, s. 937-949
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Journal article (peer-reviewed)abstract
- The cerebellum is classically considered to be mainly involved in motor processing, but studies have suggested several other functions, including pain processing. Calcitonin-gene-related peptide (CGRP) is a neuropeptide involved in migraine pathology, where there is elevated release of CGRP during migraine attacks and CGRP receptor antagonists have antimigraine efficacy. In the present study, we examined CGRP and CGRP receptor binding sites and protein expression in primate cerebellar cortex. Additionally, mRNA expression of the CGRP receptor components, calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), was examined. In addition, expression of procalcitonin was studied. We observed high [(3)H]MK-3207 (CGRP receptor antagonist) binding densities in the molecular layer of rhesus cerebellar cortex; however, due to the limit of resolution of the autoradiographic image the exact cellular localization could not be determined. Similarly, [(125)I]CGRP binding was observed in the molecular layer and Purkinje cell layer of human cerebellum. CLR and RAMP1 mRNA was expressed within the Purkinje cell layer and some expression was found in the molecular layer. Immunofluorescence revealed expression of CGRP, CLR, and RAMP1 in the Purkinje cells and in cells in the molecular layer. Procalcitonin was found in the same localization. Recent research in the biology of cerebellum indicates that it may have a role in nociception. For the first time we have identified CGRP and CGRP receptor binding sites together with CGRP receptor expression through protein and mRNA localization in primate cerebellar cortex. These results point toward a functional role of CGRP in cerebellum. Further efforts are needed to evaluate this.
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| 4. |
- Trotter, Dinko E. Gonzalez, et al.
(author)
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In Vivo Imaging of the Programmed Death Ligand 1 by F-18 PET
- 2017
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In: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 58:11, s. 1852-1857
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Journal article (peer-reviewed)abstract
- Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-Z(PD-L1_1) with F-18 and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1-binding Affibody ligand Z(PD-L1_1) was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of F-18-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by gamma-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-Z(PD-L1_1) was labeled, with a radiochemical yield of 15.1% +/- 5.6%, radiochemical purity of 96.7% +/- 2.0%, and specific activity of 14.6 +/- 6.5 GBq/mu mol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 +/- 0.33) and -negative (% ID/g = 0.32 +/- 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.
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