M2 Decrease Research Articles

Effect of CsCl nanocrystallization on the acousto-optic properties of GeS2-Sb2S3-CsCl chalcogenide glass-ceramics

Crystallization of chalcogenide glasses provides a new possibility to decoupling the trade-off between acousto-optic figure of merit (M2) and acoustic attenuation (α) properties of acousto-optic materials. In this work, 62.5GeS2-12.5Sb2S3-25CsCl chalcogenide glasses ceramics were prepared, and CsCl nanocrystals were precipitated and evidenced by using scanning electron microscopy and X-ray diffractometer. The precipitation of CsCl nanocrystals led to a 10 % decrease in M2 from 113.85 to 102.08 × 10−18 s3/g, but also to a substantial improvement in α, which decreased by 38 % from 3.28 to 2.03 dB/cm. The glass crystallization that together with a rearrangement of the glass network structure would result in the enhancement of network rigidity of the residual glass matrix, thus contributing to the reduction of acoustic attenuation.

235 IL-4/13 Blocking Peptide (KROS-401) Inhibits M2 Macrophage Polarization in vitro and Demonstrates Anti-tumor Activity in a Murine Model of Glioblastoma

INTRODUCTION: Glioblastoma (GBM) is the most aggressive primary brain malignancy with a median survival of 14 months. Tumor-associated macrophages (TAMs) comprise up to 40% of the GBM tumor mass and are significant enablers of an immunosuppressive tumor microenvironment (TME), limiting immunotherapeutic efficacy. TAMs are generally classified as M2 macrophages that demonstrate a pro-tumor phenotype and increased CD206 surface marker expression. Reducing the M2 macrophage population in tumors has shown benefit in preclinical studies across various cancers. Interleukins (IL)-4 and -13 both play a role in polarizing macrophages to an M2 state. We developed a peptide inhibitor (KROS-401) that targets the shared IL-4Ra subunit of the IL-4 and -13 receptor complex to inhibit macrophage polarization to an M2 phenotype and reprogram the immunosuppressive TME. METHODS: Human peripheral blood mononuclear cell (PBMC)-derived macrophages were isolated and treated with KROS-401 in the presence of IL-4 and IL-13 cytokines. M2 (CD206) surface markers were assessed by flow cytometry. The efficacy of KROS-401 peptide was examined using an intracranially implanted syngeneic murine model with luciferase-expressing GL261, a glioma cell line. Mice were inspected daily for survival and tumor growth was tracked by non-invasive in vivo bioluminescent imaging (BLI). RESULTS: Human PBMC-derived macrophages treated with KROS-401 displayed a dose-dependent decrease in M2 (CD206) cell surface markers. KROS-401 peptide inhibitor demonstrated anti-tumor activity in GL261 glioma-bearing mice with a significantly increased probability of survival compared to control (p = 0.0046) with a median survival of >40 days versus 27 days, respectively. CONCLUSIONS: KROS-401 inhibits M2 polarization of human macrophages in vitro and tumor growth in vivo. These findings highlight this IL-4/IL-13 peptide inhibitor as a potential immunomodulator of the TME for the treatment of GBM.

A Pilot Cross-Sectional Study of Immunological and Microbiome Profiling Reveals Distinct Inflammatory Profiles for Smokers, Electronic Cigarette Users, and Never-Smokers.

Smokers (SM) have increased lung immune cell counts and inflammatory gene expression compared to electronic cigarette (EC) users and never-smokers (NS). The objective of this study is to further assess associations for SM and EC lung microbiomes with immune cell subtypes and inflammatory gene expression in samples obtained by bronchoscopy and bronchoalveolar lavage (n = 28). RNASeq with the CIBERSORT computational algorithm were used to determine immune cell subtypes, along with inflammatory gene expression and microbiome metatranscriptomics. Macrophage subtypes revealed a two-fold increase in M0 (undifferentiated) macrophages for SM and EC users relative to NS, with a concordant decrease in M2 (anti-inflammatory) macrophages. There were 68, 19, and 1 significantly differentially expressed inflammatory genes (DEG) between SM/NS, SM/EC users, and EC users/NS, respectively. CSF-1 and GATA3 expression correlated positively and inversely with M0 and M2 macrophages, respectively. Correlation profiling for DEG showed distinct lung profiles for each participant group. There were three bacteria genera-DEG correlations and three bacteria genera-macrophage subtype correlations. In this pilot study, SM and EC use were associated with an increase in undifferentiated M0 macrophages, but SM differed from EC users and NS for inflammatory gene expression. The data support the hypothesis that SM and EC have toxic lung effects influencing inflammatory responses, but this may not be via changes in the microbiome.

Symmetry Analysis of the Complex Polytypism of Layered Rare-Earth Tellurites and Related Selenites: The Case of Introducing Transition Metals

Our systematic explorations of the complex rare earth tellurite halide family have added several new [Ln12(TeO3)12][M6X24] (M = Cd, Mn, Co) representatives containing strongly deficient and disordered metal-halide layers based on transition metal cations. The degree of disorder increases sharply with decrease of M2+ radius and the size disagreements between the cationic [Ln12(TeO3)12]+12 and anionic [M6Cl24]−12 layers. From the crystal chemical viewpoint, this indicates that the families of both rare-earth selenites and tellurites can be further extended; one can expect formation of some more complex structure types, particularly among selenites. Analysis of the polytypism of compounds have been performed using the approach of OD (“order–disorder”) theory.

Abstract 16: Altered Testicular Macrophage Polarization Is Associated With Reproductive Dysfunction In Hypertensive Mice

Elevated circulating proinflammatory (M1) and decreased anti-inflammatory (M2) macrophages contribute to hypertension (HTN) and end-organ damage. HTN is associated with reproductive dysfunction in men. However, the impact of HTN on testicular macrophages and inflammation is unknown. We hypothesized that HTN increases M1 and decreases M2 testicular macrophages, which is associated with inflammation and reproductive dysfunction. Male mice were made hypertensive by either providing L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks [L-NAME-induced HTN (LHTN)] or L-NAME water for 2 weeks, followed by a 2-week washout period and a subsequent 3-week 4% high salt diet [salt-sensitive hypertension (SSHTN)]. Control (C) mice received tap water and normal diet. Flow cytometry analysis revealed a significant increase in both M1 (C: 15%±1, LHTN: 22%±2; p<0.05) and M2 (C: 10%±1, LHTN: 21%±2; p<0.05) macrophages in testes from LHTN mice. Similarly, testes from SSHTN mice had a significant increase in M1 (C: 17%±1, SSHTN: 28%±2; p<0.05) but had a significant decrease in M2 (C: 14%±1, SSHTN: 7%±1; p<0.05) macrophages. Testes from both hypertension models had a significant increase in gene expression of the proinflammatory cytokines TNFa, IFNg, IL-1b, IL-6, and IL-17. Sperm concentration (C: 8.5±0.7, LHTN: 6.5±0.2, SSHTN: 4.7±0.5; both p<0.05) and the percentage of sperm mitochondrial activity (C: 88%±3, LHTN: 71±5, SSHTN: 64%±3; both p<0.05) were decreased significantly in both hypertension groups. Hypertensive mice presented a significantly increased percentage of sperm with abnormal morphology (C: 5%±1, LHTN: 8%±1, SSHTN: 13%±2; both p<0.05) and damaged acrosome (C: 1.4%±0.2, LHTN: 2.8%±0.2, SSHTN: 4%±0.5; both p<0.05). There was a significant decrease in gene expression of the hormone receptors AR, ERa, and LHR, and the steroidogenic enzymes StAR, 3bHSD, 17bHSD, and CYP17a1 in the testes of LHTN and SSHTN mice. These data demonstrate that HTN alters testicular macrophage polarization which is associated with inflammation and impaired reproductive health. Therapeutic strategies may be developed to improve reproductive health in male hypertensive patients by targeting testicular macrophage imbalance.

Abstract 1730: Up-regulation of C/EBPα inhibits suppressive activity of myeloid cells and potentiates antitumor response in mice and cancer patients

Abstract Tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are widely implicated in the suppression of immune responses in cancer and are associated with negative clinical outcomes. We report that in mouse tumor models, therapeutic up-regulation of the transcriptional factor C/EBPα with small activating RNA (MTL-CEBPA) blocks the suppressive activity of monocytic (M) MDSC and TAM without affecting polymorphonuclear (PMN) MDSC. MTL-CEBPA treatment demonstrated antitumor activity that was dependent on T cells. Combination of MTL-CEBPA and anti-PD1 antibody or with PMN-MDSC targeted therapy resulted in marked antitumor effect. A phase I trial was conducted in 36 patients with advanced hepatocellular carcinoma (HCC). Marked decrease in M-MDSC and the expression of genes and proteins involved in immune suppressive activity of these cells were observed. Combined treatment with MTL-CEBPA and sorafenib resulted in a 27% objective radiological response and three complete responses in patients with virally associated HCC. Tissue biopsies from these patients demonstrated decrease in M2 polarized macrophages within the tumors. Thus, therapeutic up-regulation of C/EBPα inhibited suppressive myeloid cells that resulted in potent antitumor effect in mice and with encouraging clinical response in cancer patients. We are currently recruiting to a multi-centre study of MTL-CEBPA in combination with a PD-1 inhibitor (pembrolizumab) in adult patients with advanced solid tumors (TIMEPOINT - ClinicalTrials.gov Identifier: NCT04105335). Citation Format: Ayumi Hashimoto, Debashis Sarker, Vikash Reebye, Sheba Jarvis, Mikael H. Sodergren, Andrew Kossenkov, Nina Raulf, Jenni Vasara, Pinelopi Andrikakou, Tim Meyer, Kai-Wen Huang, Ruth Plummer, Cheng Ean Chee, Duncan Spalding, Madhava Pai, David J. Pinato, Rohini Sharma, Bristi Basu, Daniel Palmer, Yuk-Ting Ma, Robert Habib, Anna Martirosyan, Naouel Elasri, Adeline Reynaud, John Rossi, Mark Cobbold, Nagy Habib, Dmitry Gabrilovich. Up-regulation of C/EBPα inhibits suppressive activity of myeloid cells and potentiates antitumor response in mice and cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1730.

Pro-inflammatory macrophage polarization enhances the anti-cancer efficacy of self-assembled galactomannan nanoparticles entrapped with hydrazinocurcumin.

Galactomannan (GM), a natural polymer, is recognized to specifically target macrophage mannose receptors (CD206). Interestingly, some reports indicate that GM has an ability to induce pro-inflammatory (M1-like, tumericidal) polarization in macrophages, suggesting its potential use as an anti-cancer agent. Hydrazinocurcumin (HC), a pyrazole derivative of curcumin, is reported to possess increased anti-cancer efficacy over curcumin. Moreover, HC-encapsulated nanoparticles (NPs) have been reported to re-polarize tumor-associated macrophages (TAMs) from anti-inflammatory (M2-like, tumor-promoting) to pro-inflammatory phenotype. To club the therapeutic properties of both GM and HC, we synthesized self-assembled amphiphilic PEGylated GM NPs loaded with HC (PSGM-HCNPs) and evaluated their potential to re-polarize TAMs towards M1-like phenotype. PSGM-HCNPs re-polarized IL-4 polarized RAW 264.7 cells via a phenotypic switch from M2- to M1-like by elevating ROS level, decreasing CD206 and arginase-1 expressions and increasing pro-inflammatory cytokines' secretion. Conditioned medium (CM) taken from re-polarized RAW 264.7 cells containing residual PSGM-HCNPs elevated ROS, arrested cell cycle, and induced apoptosis in 4T1, breast cancer cells, and Ehrlich's ascites carcinoma (EAC) cells. Decreased levels of MMP-2, MMP-9, and Bcl-2 with increased levels of Bax in both 4T1 and EAC cells indicated anti-metastatic and apoptosis-inducing potential of the CM. Treatment of PSGM-HCNPs in EAC-bearing mice reduced tumor burden, increased their survival time, decreased CD206+F4/80+ cells, and increased TNF-α+F4/80+ cells signifying decrease in M2- and increase in M1-like skewness among ascitic TAMs.

Impacts of Interleukin-17 Neutralization on the Inflammatory Response in a Healing Ligament.

In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.

Compensatory mechanisms of balance to the scaling of arm-swing frequency

The present study investigated the contribution of the Hof (2007) mechanism 1 (M1-moving the center of pressure (COP) with respect to the vertical projection of the center of mass (COMTotal)); and mechanism 2 (M2-rotating the trunk and upper limbs around the COMTotal) to postural control and the stability of COP–COMTotal cophase as a function of lateral arm-swing frequency. Young adults were instructed to stand still on a force platform while alternating their arm swinging from above the head to the side of their thigh to create perturbations to postural control. Scaling the frequency of arm-swing (random step changes of 0.2Hz within a bandwidth of 0.2 to 1.6Hz) increased the SD of COP but decreased the SD of COMTotal. Increments in arm-swing frequency induced a progressive increase in M1 and decrease in M2 in terms of their relative contribution to postural stability. The cophase between COP and COMTotal became more tightly in-phase over increments of arm-swing frequency. These findings show an adaptive compensatory role of M1 and M2 within the stability of COP–COMTotal coupling in the regulation of human balance control.

Microwave hydrothermal synthesis of MSnO3 (M2+ = Ca2+, Sr2+, Ba2+): effect of M2+ on crystal structure and photocatalytic properties

A series of alkaline earth metal stannates, MSnO3 (M2+ = Ca2+, Sr2+, Ba2+), has been synthesized by a microwave hydrothermal method and subsequent heat treatment process for the first time. The obtained samples are characterized by X-ray diffraction, Brunauer–Emmett–Teller (BET) surface area analysis, UV–Vis diffuse reflectance spectroscopy, scanning electron microscopy, and a photoluminescence technique with terephthalic acid. It is found that the variation of M2+ radius in MSnO3 leads to a change in the crystal structure and the photocatalytic degradation activity for methyl orange. With the increase of M2+ radius, dipole moments are generated in octahedral units because of the octahedral tilting distortion, which may facilitate the mobility of charge carriers and improve the activities of MSnO3. The wider band gap and larger BET surface area caused by the decrease of M2+ radius also contribute to photocatalytic performances. In addition, compared with the conventional hydrothermal method, higher activity is achieved on the samples prepared by means of the microwave method. Our findings may provide insights in fabrication of highly efficient photocatalysts by adjusting the counter-cation radius.

The partitioning of trace elements between clinopyroxene and trachybasaltic melt during rapid cooling and crystal growth

We present the variation in trace element partition coefficients measured at the interface between rapidly cooled clinopyroxene crystals and co-existing melts. Results indicate that, as the cooling rate is increased, clinopyroxene crystals are progressively depleted in Si, Ca and Mg counterbalanced by enrichments in Al (mainly tetrahedral Aliv), Na and Ti. Partition coefficients (Ds) for rare earth elements (REE), high field strength elements (HFSE) and transition elements (TE) increase with increasing cooling rate, in response to clinopyroxene compositional variations. The entry of REE into the M2 site is facilitated by a coupled substitution where either Na substitutes for Ca on the M2 site or Aliv substitutes for Si in the tetrahedral site. The latter substitution reflects an increased ease of locally balancing the excess charge at M2 as the number of surrounding Aliv atoms increases. Due to the lower concentration of Ca in rapidly cooled clinopyroxenes, divalent large ion lithophile elements (LILE) on M2 decrease at the expense of monovalent cations. Conversely, higher concentrations of HFSE and TE on the M1 site are facilitated as the average charge on this site increases with the replacement of divalent-charged cations by Alvi. Although crystallization kinetics modify clinopyroxene composition, deviations from equilibrium partitioning are insufficient to change the tendency of a trace element to be compatible or incompatible. Consequently, there are regular relationships between ionic radius, valence of the trace element and D. At both equilibrium and cooling rate conditions, Ds for isovalent cations define parabola-like curves when plotted against ionic radius, consistent with the lattice strain model, demonstrating that the partitioning of trace elements is driven by charge balance mechanisms; cation substitution reactions can be treated in terms of the energetics of the various charge-imbalanced configurations.

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy

Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation. We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68(+)CCR2(+)) and M2 (CX3CR1(+)CD206(+)/CD163(+)) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals. We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals. We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

Identification and quantification of potential metabolites of Gd-based contrast agents by electrochemistry/separations/mass spectrometry

Oxidative and potentially metabolic pathways of the five most frequently used contrast agents for magnetic resonance imaging (MRI) based on gadolinium (Gd) are examined. The oxidation of gadopentetate (Gd-DTPA) was studied with a focus on electrochemical oxidation coupled to analytical separation methods and mass spectrometric detection. Mass voltammograms generated with online electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) gave a first overview of oxidation products. Two potential metabolites could be detected, with the major metabolite originating from an N-dealkylation (M1). Four other Gd complexes used as MRI contrast agents showed similar reactions in the EC/ESI-MS set-up. To obtain more information about the properties and the quantity of the generated products, a wide range of separation and detection techniques was applied in further experiments. Gd-DTPA and its N-dealkylation product were successfully separated by capillary electrophoresis (CE) and detected by ESI-MS and inductively coupled plasma (ICP)-MS, respectively. CE experiments indicated that the second oxidation product (M2) detected in the mass voltammogram is unstable and decomposes to M1. Employing EC/CE/ICP-MS, the quantification of the metabolites could be achieved. Under the employed conditions, 8.8% of Gd-DTPA was oxidized. Online experiments with high performance liquid chromatography (HPLC) coupled to ESI-MS confirmed the decomposition of M2. Time-resolved measurements showed a decrease of M2 and a simultaneous increase in M1 within only a few minutes, confirming the conclusion that M2 degrades to M1, while EC/LC/ICP-MS measurements provided quantitative evidence as well. The EC/MS simulation shows that a metabolic transformation should not be disregarded in further research regarding the trigger of nephrogenic systemic fibrosis (NSF), a disease exclusively observed for several hundred dialysis patients after delivery of Gd-based MRI contrast agents with linear structure. Furthermore, the used methods may allow the prediction of options for the oxidative removal of these contrast agents from wastewaters.

Rapid change in semi-diurnal tides in the North Atlantic since 1980

[1] An anomalously large trend of M2 and S2 amplitudes and phases, beginning in the early 1980s, is observed in the North Atlantic. Harmonic analysis of long-term sea-level records from tide-gauge stations along the North American Atlantic coast and European Atlantic coast shows a large decrease in M2 and S2 amplitudes and an increase in phase lag. The characteristics of the evolution of M2 and S2 tides are similar, but not fully conclusive, which may suggesting different causes. In general, the trends may be due to global warming, which significantly increased in the early 1980s in the North Atlantic region. Several climate-related driving mechanisms for the observed changes in tides are discussed.

Lesions of the Cerebellar Nodulus and Uvula Impair Downward Pursuit

We studied sinusoidal (SIN) and step-ramp (SR) pursuit in two rhesus monkeys, before and after surgical lesions of the cerebellar nodulus and uvula (Nod/Uv). Eye movements were recorded using the magnetic field scleral search coil method. Pursuit targets were generated by an LCD projector and back-projected onto a tangent screen in an otherwise dark room. After the Nod/Uv lesions, both monkeys showed a reduced eye velocity during downward pursuit (SIN: 42% decrease in M1, 91% decrease in M2; SR: 37% decrease in M1, 85% decrease in M2). For SR, the decrease was seen only for the closed-loop response; initial eye acceleration did not change (P>0.05). Upward pursuit gains increased for SIN (M1: 9%, M2: 11%); they decreased for SR (M1: 27%, M2: 18%), but to a lesser degree than for downward pursuit. Horizontal pursuit was little changed in M1 but was reduced in one direction in M2, the animal with the larger lesion. The deficit in downward tracking was limited to foveal pursuit; ocular following of random-dot stimuli was retained, even when the target subtended only several degrees. Our findings support a critical role for the Nod/Uv in vertical pursuit, particularly for sustained downward pursuit. Finally, in both monkeys, the lesion increased spontaneous upward ocular drift in the dark (mean prelesion, 1.43 degrees/s; postlesion, 5.92 degrees/s), suggesting a role for the Nod/Uv in holding the eyes still and in the genesis of downbeat nystagmus.

Manual planimetric measurement of carotid plaque volume using three‐dimensional ultrasound imaging

We investigated the utility of three manual planimetric methods to quantify carotid plaque volume. A single observer measured 15 individual plaques from 15 three-dimensional (3D) ultrasound (3D US) images of patients ten times each using three different planimetric approaches. Individual plaque volumes were measured (range: 32.6-597.1 mm3) using a standard planimetric approach (M1) whereby a plaque end was identified and sequential contours were measured. The same plaques were measured using a second approach (M2), whereby plaque ends were first identified and the 3D US image of the plaque was then subdivided into equal intervals. A third method (M3) was used to measure total plaque burden (range: 165.1-1080.0 mm3) in a region (+/- 1.5 cm) relative to the carotid bifurcation. M1 systematically underestimated individual plaque volume compared to M2 (V2 = V1 + 14.0 mm3, r = 0.99, p = 0.006) due to a difference in the mean plaque length measured. Coefficients of variance (CV) for M1 and M2 decrease with increasing plaque volume, with M2 results less than M1. Root mean square difference between experimental and theoretical CV for M2 was 3.2%. The standard deviation in the identification of the transverse location of the carotid bifurcation was 0.56 mm. CVs for plaque burden measured using M3 ranged from 1.2% to 7.6% and were less than CVs determined for individual plaque volumes of the same volume. The utility of M3 was demonstrated by measuring carotid plaque burden and volume change over a period of 3 months in three patients. In conclusion, M2 was determined to be a more superior measurement technique than M1 to measure individual plaque volume. Furthermore, we demonstrated the utility of M3 to quantify regional plaque burden and to quantify change in plaque volume.

Evaluation of [18F]fluoroxanomeline {5-{4-[(6-[18F]fluorohexyl)oxy]-1,2,5-thiadiazol-3-yl}-1-methyl-1,2,3,6-tetrahydropyridine} in muscarinic knockout mice

We set out to develop a muscarinic M1-selective agonist (based on the structure of the functionally M1-selective xanomeline) that could be radiolabeled with fluorine-18 for use as an imaging agent for positron emission tomography. The radiochemical synthesis was achieved, employing the arts of organic and radiochemical syntheses. Binding selectivity studies employed biodistribution studies, using autoradiography and/or tissue dissection, in wild-type or muscarinic receptor knockout mice. [(18)F]Fluoroxanomeline shows rather uniform uptake in all mouse brain regions and high specific binding, with a brain-to-blood ratio of 32 at 60 min postinjection. In addition, the specific binding is demonstrated by a 58% to 75% decrease in brain uptake upon coinjection with 5 nmol of unlabeled fluoroxanomeline or xanomeline. Brain uptake studies with [(3)H]xanomeline in muscarinic knockout mice show decreased uptake in M1 (17-34%) and M2 (2-20%) knockout mice compared with control. However, statistical significance was observed in only a few regions. Comparison of [(18)F]fluoroxanomeline in knockout mice showed no difference in M1 or M4 knockout mice but a general decrease in M2 (2-24%) knockout mice. The decrease of [(18)F]fluoroxanomeline uptake in M2 knockout mice reached statistical significance in brain stem, cerebellum, frontal cortex, hippocampus, inferior colliculus and superior colliculus. Although xanomeline displays highly selective M1 agonist activity in functional assays, little selectivity for muscarinic subtype binding was observed for xanomeline or its fluorine-containing analogue, fluoroxanomeline. This emphasizes the lack of correlation between functional selectivity and binding selectivity.

Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells.

We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.

Chronic Effects of Traumatic Brain Injury on Hippocampal Vesicular Acetylcholine Transporter and M2Muscarinic Receptor Protein in Rats

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may be due to presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and M2receptors can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2muscarinic subtype receptor protein levels at four time points: 1 day, 1 week, 2 weeks, and 4 weeks following injury. Rats were anesthetized and surgically prepared for controlled cortical impact injury (4 m/s, 2.5- to 2.9-mm depth) and sham surgery. Animals were sacrificed and coronal sections (35 μm thick) were cut through the dorsal hippocampus for VAChT and M2immunohistochemistry. Semiquantitative measurements of VAChT and M2protein in hippocampal homogenates from injured and sham rats were assessed using Western blot analysis. Immunohistochemistry showed no obvious changes in VAChT and M2immunoreactivity at 1 day and 1 week postinjury. At 2 and 4 weeks postinjury, an increase in hippocampal VAChT protein and a corresponding loss of hippocampal M2protein was observed compared to sham controls. Consistent with these results, Western blot analyses at 4 weeks postinjury demonstrated a 40–50% increase in VAChT and a 25–30% decrease in M2. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI, by upregulating the storage capacity and subsequent release of ACh and downregulating presynaptic inhibitory receptors.

Studies on the molecular motion of [M(daco)2]2+ Ions in bis‐daco complexes of MX2 (M = Cu(II) and Ni(II) and X = ClO4 or NO3; daco = 1,5‐diazacyclooctane)

AbstractThe temperature dependence of the second moment (M2) of 1H NMR absorption of the partially deuterated (deuterated amine protons) complexes [M (daco‐d2)2] X2 · n D2O (M = Cu(II) and Ni(II); daco = 1,5‐diazacyclooctane; X = ClO4 or NO3; n = 0 for X = NO3 and n = 2 for X = ClO4) has been measured. Among them, [Cu(daco‐d2)2](NO3)2 showed a distinct drop in M2 at the same temperature region (ca. 363 K) where the complex is reported to undergo the thermochromic phase transition. The complexes [Cu(daco‐d2)2](ClO4)2·2D2O and [Ni(daco‐d2)2](ClO4)2·2D2O showed a gradual decrease of M2 around 220 and 280 k, respectively, without any colour changes and phase transitions, whilst M2 of [Ni(daco‐d2)2](NO3)2 shows no marked decrease with increase of temperature up to 400 K. M2 of rigid and motional lattices of [Cu(daco‐d2)2](NO3)2 is theoretically calculated using the reported X‐ray crystal structure data. The molecular motions of [Cu(daco‐d2)2](NO3)2, [Cu(daco‐d2)2](ClO4)2·2D2O and [Ni(daco‐d2)2](ClO4)2·2D2O are explained in terms of dynamic disorder of metal diamine chelate rings.