Potential role of remimazolam in alleviating bone cancer pain in mice via modulation of translocator protein in spinal astrocytes.

Bone cancer pain (BCP) is a complex clinical challenge, with current treatments often falling short of providing adequate relief. Remimazolam, a benzodiazepine receptor agonist recognized for its anxiolytic effects, has emerged as a potential agent in managing BCP. This study explores the analgesic properties of remimazolam and its interaction with the translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, in spinal astrocytes. In the context of BCP, previous research has indicated that TSPO expression in spinal astrocytes may serve a protective regulatory function in neuropathic pain models. Building on this, the BCP mice received various doses of remimazolam on the 15th day post-inoculation, and pain behavior was assessed over time. The results showed that BCP induced an upregulation of TSPO and astrocyte activation in the spinal dorsal horn, alongside increased extracellular signal-regulated kinase (ERK) signaling and inflammatory cytokine expression. Remimazolam administration resulted in a dose-dependent reduction of pain behaviors, which corresponded with a decrease in both ERK pathway activation and inflammatory factor expression. This suggests that remimazolam's analgesic effects are mediated through its action as a TSPO agonist, leading to the attenuation of neuroinflammation and pain signaling pathways. Importantly, the analgesic effects of remimazolam were reversed by the TSPO antagonist PK11195, underscoring the pivotal role of TSPO in the drug's mechanism of action. This reversal also reinstated the heightened levels of ERK activity and inflammatory mediators, further confirming the involvement of TSPO in the modulation of these pain-related processes. These findings open new avenues for the therapeutic management of bone cancer pain, positioning remimazolam as a promising candidate for further investigation and development.

A p- adic approach to the TSPO gene.

TSPO protein is known to be involved in various cellular functions and dysregulations of TSPO expression has been found to be associated with pathologies of different human diseases, including cardiovascular disease, cancer, neuroinflammatory, neurodegenerative, neoplastic disorders. However, there are limited studies in the literature on the effects of sequence variations in the TSPO gene on the function of the protein and their relationship with human diseases. Evaluating the pathogenicity of genetic variants is crucial in terms of prioritizing the functional importance and clinical use. Therefore, various in-silico prediction tools have been developed that combine different algorithms to predict the effects of sequence variations on protein functions or gene regulation. In this study, the p-adic distance approach in modeling the genetic code, proposed and developed by Dragovich and Dragovich, was discussed in order to obtain an alternative to the existing in-silico prediction tools. Dragovichs' approach is expressed as follows: A 5-adic space of codons is constructed and 5-adic and 2-adic distances between codons are taken into account. As a result, two codons with the smallest value of 5-adic and 2-adic distances are obtained, encoded for the same amino acid and stop signal. This model describes well the degeneration of the genetic code. This study combined the data obtained from in-silico prediction tools and used a bioinformatics approach to determine the functional relevance of coding SNPs in the TSPO. Overall, we evaluate the potential utility of Dragovichs' approach by comparing it with other existing prediction tools for variant classification and prioritization.

Interplay between microbial-derived GABA and host GABA receptor signaling collectively influence the tumorigenic function of GABA in colon cancer.

Although classically recognized as a neurotransmitter, gamma aminobutyric acid (GABA) has also been identified in colonic tumors. Moreover, the gut microbiome represents another potential source of GABA. Both GABAA and GABAB receptors have been implicated in contributing to the effects of GABA in colorectal cancer, with both pro- and anti-tumorigenic functions identified. However, their subunit composition is often overlooked. Studies to date have not addressed whether the GABA-producing potential of the microbiome changes over the course of colon tumor development or whether receptor subunit expression patterns are altered in colon cancer. Therefore, we investigated the clusters of orthologous group frequencies of glutamate decarboxylase (GAD) in feces from two murine models of colon cancer and found that the frequency of microbial GAD was significantly decreased early in the tumorigenic process. We also determined that microbial-derived GABA inhibited proliferation of colon cancer cells in vitro and that this effect of GABA on SW480 cells involved both GABAA and GABAB receptors. GABA also inhibited prostaglandin E2 (PGE2)-induced proliferation and interleukin-6 (IL-6) expression in these cells. Gene expression correlations were assessed using the "Cancer Exploration" suite of the TIMER2.0 web tool and identified that GABA receptor subunits were differentially expressed in human colon cancer. Moreover, GABAA receptor subunits were predominantly positively associated with PGE2 synthase, cyclooxygenase-2 and IL-6. Collectively, these data demonstrate decreased potential of the microbiome to produce GABA during tumorigenesis, a novel anti-tumorigenic pathway for GABA, and that GABA receptor subunit expression adds a further layer of complexity to GABAergic signaling in colon cancer.

PET imaging for the early evaluation of ocular inflammation in diabetic rats by using [18F]-DPA-714.

Ocular complications of diabetes mellitus (DM) are the leading cause of vision loss. Ocular inflammation often occurs in the early stage of DM; however, there are no proven quantitative methods to evaluate the inflammatory status of eyes in DM. The 18 kDa translocator protein (TSPO) is an evolutionarily conserved cholesterol binding protein localized in the outer mitochondrial membrane. It is a biomarker of activated microglia/macrophages; however, its role in ocular inflammation is unclear. In this study, fluorine-18-DPA-714 ([18F]-DPA-714) was evaluated as a specific TSPO probe by cell uptake, cell binding assays and micro positron emission tomography (microPET) imaging in both in vitro and in vivo models. Primary microglia/macrophages (PMs) extracted from the cornea, retina, choroid or sclera of neonatal rats with or without high glucose (50 mM) treatment were used as the in vitro model. Sprague-Dawley (SD) rats that received an intraperitoneal administration of streptozotocin (STZ, 60 mg/kg once) were used as the in vivo model. Increased cell uptake and high binding affinity of [18F]-DPA-714 were observed in primary PMs under hyperglycemic stress. These findings were consistent with cellular morphological changes, cell activation, and TSPO up-regulation. [18F]-DPA-714 PET imaging and biodistribution in the eyes of DM rats revealed that inflammation initiates in microglia/macrophages in the early stages (3 weeks and 6 weeks), corresponding with up-regulated TSPO levels. Thus, [18F]-DPA-714 microPET imaging may be an effective approach for the early evaluation of ocular inflammation in DM.

Pilot Feasibility Study: 18 F-DPA-714 PET/CT Macrophage Imaging in Triple-Negative Breast Cancers (EITHICS).

ABSTRACT: Tumor-associated macrophages are targets of interest in triple-negative breast cancer (TNBC). The translocator protein 18 kDa (TSPO) is a sensitive marker for macrophages and holds potential relevance in TNBC stratification. This pilot prospective study (EITHICS, NCT04320030) aimed to assess the potential of TSPO PET/CT imaging using 18 F-DPA-714 in primary TNBC, compared with immunohistochemistry, autoradiography, and TSPO polymorphism. PATIENTS AND METHODS: Thirteen TNBC patients were included. They underwent TSPO genotyping (HAB, MAB, LAB), 18 F-FDG PET/CT, and breast MRI. Semiquantitative PET parameters were computed. VOIs were defined on the tumor lesion, healthy breast tissue, and pectoral muscle to obtain SUV, tumor-to-background ratio (TBR), and time-activity curves (TACs). Additionally, immunohistochemistry, 3 H-DPA-714, and 3 H-PK-11195 autoradiography were conducted. RESULTS: The majority of TNBC tumors (11/13, 84%) had a preponderance of M2-polarized macrophages with a median proportion of 82% (range, 44%-94%). 18 F-DPA-714 PET/CT clearly identified TNBC tumors with an excellent TBR. Three distinct patterns of 18 F-DPA-714 TACs were identified, categorized as "above muscular," "equal to muscular," and "below muscular" with reference to the muscular background. For the "above muscular" group (2 HAB and 2 MAB), "equal muscular" group (3 HAB, 3 MAB, and 1 LAB), and "below muscular" group (1 LAB and 1 MAB), tumor TACs showed a 18 F-DPA-714 accumulation slope of 1.35, 0.62, and 0.22, respectively, and a median SUV mean of 4.02 (2.09-5.31), 1.66 (0.93-3.07), and 0.61 (0.43-1.02). CONCLUSIONS: This study successfully demonstrated TNBC tumor targeting by 18 F-DPA-714 with an excellent TBR, allowing to stratify 3 patterns of uptake potentially influenced by the TSPO polymorphism status. Further studies in larger populations should be performed to evaluate the prognostic value of this new biomarker.

Translocator Protein 18 kDa Tracer 18F-FDPA PET/CTA Imaging for the Evaluation of Inflammation in Vulnerable Plaques.

Inflammation induced by activated macrophages within vulnerable atherosclerotic plaques (VAPs) constitutes a significant risk factor for plaque rupture. Translocator protein (TSPO) is highly expressed in activated macrophages. This study investigated the effectiveness of TSPO radiotracers, 18F-FDPA, in detecting VAPs and quantifying plaque inflammation in rabbits. 18 New Zealand rabbits were divided into 3 groups: sham group A, VAP model group B, and evolocumab treatment group C. 18F-FDPA PET/CTA imaging was performed at 12, 16, and 24 weeks in all groups. Optical coherence tomography (OCT) was performed on the abdominal aorta at 24 weeks. The VAP was defined through OCT images, and ex vivo aorta PET imaging was also performed at 24 weeks. The SUVmax and SUVmean of 18F-FDPA were measured on the target organ, and the target-to-background ratio (TBRmax) was calculated as SUVmax/SUVblood pool. The arterial sections of the isolated abdominal aorta were analyzed by HE staining, CD68 and TSPO immunofluorescence staining, and TSPO Western blot. The results showed that at 24 weeks, the plaque TBRmax of 18F-FDPA in group B was significantly higher than in groups A and C. Immunofluorescence staining of CD68 and TSPO, as well as Western blot, confirmed the increased expression of macrophages and TSPO in the corresponding regions of group B. HE staining revealed an increased presence of the lipid core, multiple foam cells, and inflammatory cell infiltration in the area with high 18F-FDPA uptake. This indicates a correlation between 18F-FDPA uptake, inflammation severity, and VAPs. The TSPO-targeted tracer 18F-FDPA shows specific uptake in macrophage-rich regions of atherosclerotic plaques, making it a valuable tool for assessing inflammation in VAPs.

TSPO deficiency exacerbates acute lung injury via NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in many critically ill patients. Although inflammasome activation plays an important role in the induction of acute lung injury (ALI) and ARDS, the regulatory mechanism of this process is still unclear. When cells are stimulated by inflammation, the integrity and physiological function of mitochondria play a crucial part in pyroptosis. However, the underlying mechanisms and function of mitochondrial proteins in the process of pyroptosis are largely not yet known. Here, we identified the 18-kDa translocator protein (TSPO), a mitochondrial outer membrane protein, as an important mediator regulating nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages during ALI. METHODS: TSPO gene knockout (KO) and lipopolysaccharide (LPS)-induced ALI/ARDS mouse models were employed to investigate the biological role of TSPO in the pathogenesis of ARDS. Murine macrophages were used to further characterize the effect of TSPO on the NLRP3 inflammasome pathway. Activation of NLRP3 inflammasome was preformed through LPS + adenosine triphosphate (ATP) co-stimulation, followed by detection of mitochondrial membrane potential, reactive oxygen species (ROS) production, and cell death to evaluate the potential biological function of TSPO. Comparisons between two groups were performed with a two-sided unpaired t -test. RESULTS: TSPO- KO mice exhibited more severe pulmonary inflammation in response to LPS-induced ALI. TSPO deficiency resulted in enhanced activation of the NLRP3 inflammasome pathway, promoting more proinflammatory cytokine production of macrophages in LPS-injured lung tissue, including interleukin (IL)-1ß, IL-18, and macrophage inflammatory protein (MIP)-2. Mitochondria in TSPO -KO macrophages tended to depolarize in response to cellular stress. The increased production of mitochondrial damage-associated molecular pattern led to enhanced mitochondrial membrane depolarization and pyroptosis in TSPO -KO cells. CONCLUSION: TSPO may be the key regulator of cellular pyroptosis, and it plays a vital protective role in ARDS occurrence and development.

Translocator protein expression and localization in human endometrium and endometriosis: A potential target for a noninvasive diagnosis?

The limitations of current imaging methods to detect small or superficial endometriotic lesions prompt the search for new molecular targets. TSPO is an 18 KDa protein located in the outer mitochondrial membrane, which can be traced by positron emission tomography (PET) using specific ligands. TSPO is located mostly in neurons and inflammatory sites outside the brain. We hypothesized that it might also be expressed in the human endometrium and endometrial-like tissue, being a target for molecular imaging of endometriosis. This prospective cross-sectional study included 28 women with endometriosis and 11 endometriosis-free controls. Endometriotic lesions (n = 49) and normal peritoneum (n = 13) from endometriosis patients were obtained during laparoscopy, while samples of eutopic endometrium from patients with endometriosis (n = 28) and from control women (n = 11) were collected in the operating room using a flexible device. TSPO mRNA expression was evaluated by quantitative reverse-transcription real-time PCR while protein expression was evaluated by immunohistochemistry with a monoclonal antibody antihuman TSPO. TSPO mRNA expression was detected in an invariable fashion in all tissue types evaluated; however, TSPO protein was found to be more abundant in the glandular epithelium than in the stroma, both in the endometrium and in the endometriotic lesions. Interestingly, hormone therapies did not alter the expression of TSPO, and its presence was mostly negative in tissues adjacent to endometriotic implants. As a proof of concept, the protein expression pattern of TSPO in endometriotic tissue and along the adjacent areas suggests that TSPO-based molecular imaging might be used for noninvasive endometriosis detection.

Regulation of astrocyte metabolism by mitochondrial translocator protein 18 kDa.

The mitochondrial translocator protein 18 kDa (TSPO) has been linked to functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in Leydig cells and microglia indicate that TSPO function may vary between cells depending on their specialized roles. Astrocytes are critical for providing trophic and metabolic support in the brain. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. Relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/-) mouse primary astrocytes in vitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed extracellular metabolic flux analyses. We found that TSPO deficiency reduced basal cellular respiration and attenuated the bioenergetic response to glucopenia. Fatty acid oxidation was increased, and lactate production was reduced in TSPO-/- MPAs and U373 cells. Co-immunoprecipitation studies revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a in U373 and MPAs, presenting a mechanism wherein TSPO may regulate FAO in these cells. Compared to TSPO+/+ cells, in TSPO-/- MPAs we observed attenuated tumor necrosis factor release following 3 h lipopolysaccharide (LPS) stimulation, which was enhanced at 24 h post-LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility, TSPO deficiency does not appear to modulate the metabolic response of MPAs to inflammation, at least in response to the model used in this study.

Enhancing predictability of IDH mutation status in glioma patients at initial diagnosis: a comparative analysis of radiomics from MRI, [18F]FET PET, and TSPO PET.

PURPOSE: According to the World Health Organization classification for tumors of the central nervous system, mutation status of the isocitrate dehydrogenase (IDH) genes has become a major diagnostic discriminator for gliomas. Therefore, imaging-based prediction of IDH mutation status is of high interest for individual patient management. We compared and evaluated the diagnostic value of radiomics derived from dual positron emission tomography (PET) and magnetic resonance imaging (MRI) data to predict the IDH mutation status non-invasively. METHODS: Eighty-seven glioma patients at initial diagnosis who underwent PET targeting the translocator protein (TSPO) using [18F]GE-180, dynamic amino acid PET using [18F]FET, and T1-/T2-weighted MRI scans were examined. In addition to calculating tumor-to-background ratio (TBR) images for all modalities, parametric images quantifying dynamic [18F]FET PET information were generated. Radiomic features were extracted from TBR and parametric images. The area under the receiver operating characteristic curve (AUC) was employed to assess the performance of logistic regression (LR) classifiers. To report robust estimates, nested cross-validation with five folds and 50 repeats was applied. RESULTS: TBRGE-180 features extracted from TSPO-positive volumes had the highest predictive power among TBR images (AUC 0.88, with age as co-factor 0.94). Dynamic [18F]FET PET reached a similarly high performance (0.94, with age 0.96). The highest LR coefficients in multimodal analyses included TBRGE-180 features, parameters from kinetic and early static [18F]FET PET images, age, and the features from TBRT2 images such as the kurtosis (0.97). CONCLUSION: The findings suggest that incorporating TBRGE-180 features along with kinetic information from dynamic [18F]FET PET, kurtosis from TBRT2, and age can yield very high predictability of IDH mutation status, thus potentially improving early patient management.

A first-in-man study of [18F] FEDAC: a novel PET tracer for the 18-kDa translocator protein.

PURPOSE: N-benzyl-N-methyl-2-[7, 8-dihydro-7-(2-[18F] fluoroethyl) -8-oxo-2-phenyl-9H-purin-9-yl] acetamide ([18F] FEDAC) is a novel positron emission tomography (PET) tracer that targets the translocator protein (TSPO; 18 kDa) in the mitochondrial outer membrane, which is known to be upregulated in various diseases such as malignant tumors, neurodegenerative diseases, and neuroinflammation. This study presents the first attempt to use [18F]FEDAC PET/CT and evaluate its biodistribution as well as the systemic radiation exposure to the radiotracer in humans. MATERIALS AND METHODS: Seventeen whole-body [18F]FEDAC PET/CT (injected dose, 209.1 ± 6.2 MBq) scans with a dynamic scan of the upper abdomen were performed in seven participants. Volumes of interest were assigned to each organ, and a time-activity curve was created to evaluate the biodistribution of the radiotracer. The effective dose was calculated using IDAC-Dose 2.1. RESULTS: Immediately after the intravenous injection, the radiotracer accumulated significantly in the liver and was subsequently excreted into the gastrointestinal tract through the biliary tract. It also showed high levels of accumulation in the kidneys, but showed minimal migration to the urinary bladder. Thus, the liver was the principal organ that eliminated [18F] FEDAC. Accumulation in the normal brain tissue was minimal. The effective dose estimated from biodistribution in humans was 19.47 ± 1.08 µSv/MBq, and was 3.60 mSV for 185 MBq dose. CONCLUSION: [18F]FEDAC PET/CT provided adequate image quality at an acceptable effective dose with no adverse effects. Therefore, [18F]FEDAC may be useful in human TSPO-PET imaging.

Ginger (Zingiber Officinale Roscoe) ameliorates ethanol-induced cognitive impairment by modulating NMDA and GABA-A receptors in rat hippocampus.

Brain damage caused by ethanol abuse may lead to permanent damage, including severe dementia. The aim of this study was to investigate the effects of ginger powder on ethanol-induced cognitive disorders by examining oxidative damage and inflammation status, and the gene expression of N-methyl-D-aspartate (NMDA) and γ-Aminobutyric acid (GABA)-A receptors in the hippocampus of male rats. 24 adult male Sprague-Dawley rats were allocated randomly to four groups as follows control, ethanol (4g/kg/day, by gavage), ginger (1g/kg/day, by gavage), and ginger-ethanol. At the end of the study, memory and learning were evaluated by the shuttle box test. Moreover, to explore mechanisms involved in ethanol-induced cognitive impairment and the protective effect of ginger, the expression of Nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), NMDA receptor, and GABA-A receptor was measured along with inflammatory and oxidative biomarkers in the hippocampus tissue. The results showed that ethanol could induce cognitive impairment in the ethanol group, while pretreatment with ginger could reverse it. The gene expression of the NF-κB/ Tumor necrosis factor (TNF)-α/Interleukin (IL)-1ß pathway and NMDA and GABA-A receptors significantly increased in the ethanol group compared to the control group. While pretreatment with ginger could significantly improve ethanol-induced cognitive impairment through these pathways in the ginger-ethanol group compared to the ethanol group (P < 0.05). It can be concluded that ginger powder could ameliorate ethanol-induced cognitive impairment by modulating the expression of NMDA and GABA-A receptors and inhibiting oxidative damage and the NF-κB/TNF-α/IL-1ß pathway in the rat hippocampus.

Alternative Splicing and Nonsense-Mediated Decay of a Zebrafish GABA Receptor Subunit Transcript.

The superfamily of Cys-loop ionotropic neurotransmitter receptors includes those that detect GABA, glutamate, glycine, and acetylcholine. There is ample evidence that many Cys-loop receptor subunit genes include alternatively spliced exons. In this study, we report a novel example of alternative splicing (AS): we show that the 68-bp exon 3 in the zebrafish gabrr2b gene-which codes for the ρ2b GABAAR subunit-is an alternative cassette exon. Skipping of gabrr2b exon 3 results in a downstream frame shift and a premature termination codon (PTC). We provide evidence in larval zebrafish that transcripts containing the PTC are subject to degradation through nonsense-mediated decay. We also compile reports of AS of homologous exons in other Cys-loop receptor genes in multiple species. Our data add to a large body of research demonstrating that exon 3 in Cys-loop receptor genes is a conserved site for AS, the effects of which can vary from novel splice-isoform generation to downregulation of gene expression through transcript degradation.

Epiberberine induced p53/p21-dependent G2/M cell cycle arrest and cell apoptosis in gastric cancer cells by activating γ-aminobutyric acid receptor- ß3.

BACKGROUND AND PURPOSE: Epiberberine (EPI) is one of the most important bioalkaloid found in the rhizome of Coptis chinensis, which has been observed to exhibit pharmaceutical effects against gastric cancer (GC). Nevertheless, the potential mechanism of EPI against GC cells still remains unclear. This study aimed to identify the core receptor on GC cells through which EPI inhibited the growth of GC cells and to explore the underlying inhibitory mechanisms. METHODS: To identify hub receptor targets that respond to EPI treatment, RNA sequencing (RNA-Seq) data from a tumor-bearing mouse model were analyzed using bioinformatics method and molecular docking. The binding interaction between EPI and GABRB3 was validated through western blotting based-cellular thermal shift assay (WB-CETSA). To further verify the binding region between EPI and GABRB3 through circular dichroism (CD) chromatography, fragments of the extracellular and transmembrane domains of the GABRB3 protein were expressed and purified in vitro. Stable cell lines with the overexpression or knockdown of GABRB3 were established using the recombinant lentivirus system. MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)) assay, colony formation assay, invasion and migration experiments, and flow cytometry were conducted to validate the inhibitory effect of EPI on the GC cells via GABRB3. Additionally, western blotting was utilized to explore the potential inhibitory mechanisms. RESULTS: Through the combination of multiple bioinformatics methods and molecular docking, we found that the γ-aminobutyric acid type A receptor subunit -ß3 (GABRB3) might be the critical receptor target in response to EPI treatment. The results of WB-CETSA analysis indicated that EPI significantly promoted the thermostability of the GABRB3 protein. Importantly, EPI could directly bind to GABRB3 and alter the secondary structure of GABRB3 fragments similar to the natural agonist, γ-aminobutyric acid (GABA). The EPI-induced suppression of the malignant phenotype of GC cells was dependent on the presence of GABRB3. GABRB3 expression was positively correlated with TP53 in patients with GC. The binding of EPI to GABRB3 stimulated p53 accumulation in GC cells. This activated the p21/CDK1/cyclinB1 pathway, resulting in G2/M cell cycle arrest, and induced the Bcl-2/BAX/Caspase axis-dependent cell apoptosis. CONCLUSION: This study revealed the target receptor for EPI in GC cells and provided new insights into its anticancer mechanisms.

TOM40 mediates the effect of TSPO on postpartum depression partially through regulating calcium homeostasis in microglia.

AIMS: To assess the effect of the translocator protein 18 kDa (TSPO) on postpartum depression and explore its mechanism. METHODS: Postpartum depression (PPD) mouse model was established, and flow cytometry, immunofluorescence, Western blot analysis, real-time quantitative PCR, adeno-associated virus (AAV), co-immunoprecipitation-mass spectrometry and immunofluorescence co-staining were used to detect the effect of TSPO ligand ZBD-2 on PPD mice. RESULTS: ZBD-2 inhibits the overactivation of microglia in the hippocampus and amygdala of PPD model mice. ZBD-2 not only inhibited the inflammation but also repressed the burst of reactive oxygen species (ROS) and mitochondrial ROS (mtROS). Meanwhile, ZBD-2 protects mitochondria from LPS-induced damages through inhibiting the influx of calcium. ZBD-2 modulated the calcium influx by increasing the level of translocase of the outer mitochondrial membrane 40 (TOM40) and reducing the interaction of TSPO and TOM40. In addition, the effect of ZBD-2 was partially dependent on anti-oxidative process. Knockdown of TOM40 by adeno-associated virus (AAV) in the hippocampus or amygdala dramatically reduced the effect of ZBD-2 on PPD, indicating that TOM40 mediates the effect of ZBD-2 on PPD. CONCLUSIONS: TOM40 is required for the effect of ZBD-2 on treating anxiety and depression in PPD mice. This study reveals the role of microglia TSPO in PPD development and provides the new therapeutic strategy for PPD.

Multiplex immunohistochemistry defines two cholesterol metabolism patterns predicting immunotherapeutic outcomes in gastric cancer.

BACKGROUND: The role of cholesterol metabolism in gastric cancer (GC) and its implications for tumor characteristics and immunotherapy response remain poorly understood. In this study, our aim was to investigate this role, identify associated metabolic subtypes, and assess their clinical implications in GC. METHODS: We conducted a comprehensive analysis of cholesterol metabolism genes (CMGs) using transcriptomic data from TCGA and GEO. Based on 23 representative CMGs, we classified GC into metabolic subtypes. We evaluated clinical features and immune cell infiltration between these subtypes. Additionally, we identified a CMG signature and assessed its clinical relevance in GC. We retrospectively enrolled thirty-five GC patients receiving chemotherapy plus a PD-1 inhibitor to assess the CMG signature using multiplex immunohistochemistry. RESULTS: Our analysis revealed two cholesterol metabolism subtypes in GC: Cholesterol Metabolism Type 1 (CMT1) and Cholesterol Metabolism Type 2 (CMT2). These subtypes exhibited distinct patterns: CMT1 indicated heightened cholesterol biosynthesis, while CMT2 showed abnormal cholesterol transport. CMT2 was associated with unfavorable clinical features, enriched malignant pathways, and a pro-tumor immune microenvironment. Furthermore, we developed a five-CMG prognostic signature (ABCA1, NR1H3, TSPO, NCEH1, and HMGCR) that effectively predicted the prognosis of patients with GC and their response to chemotherapy plus a PD-1 inhibitor. This signature was validated in a clinical cohort using multiplex immunohistochemistry. CONCLUSION: Our results highlight the effectiveness of cholesterol metabolism patterns as biomarkers for predicting the prognosis and immunotherapy response in GC. The expression of cholesterol metabolism genes and the assessment of cholesterol metabolism patterns have the potential to predict the outcome of immunotherapy and guide treatment strategies.

Radiotracers for Imaging of Inflammatory Biomarkers TSPO and COX-2 in the Brain and in the Periphery.

Inflammation involves the activation of innate immune cells and is believed to play an important role in the development and progression of both infectious and non-infectious diseases such as neurodegeneration, autoimmune diseases, pulmonary and cancer. Inflammation in the brain is marked by the upregulation of translocator protein (TSPO) in microglia. High TSPO levels are also found, for example, in macrophages in cases of rheumatoid arthritis and in malignant tumor cells compared to their relatively low physiological expression. The same applies for cyclooxgenase-2 (COX-2), which is constitutively expressed in the kidney, brain, thymus and gastrointestinal tract, but induced in microglia, macrophages and synoviocytes during inflammation. This puts TSPO and COX-2 in the spotlight as important targets for the diagnosis of inflammation. Imaging modalities, such as positron emission tomography and single-photon emission tomography, can be used to localize inflammatory processes and to track their progression over time. They could also enable the monitoring of the efficacy of therapy and predict its outcome. This review focuses on the current development of PET and SPECT tracers, not only for the detection of neuroinflammation, but also for emerging diagnostic measures in infectious and other non-infectious diseases such as rheumatic arthritis, cancer, cardiac inflammation and in lung diseases.

Translocator protein (TSPO) expression in neoplastic cells and tumor-associated macrophages in meningiomas.

Meningiomas are the most common primary intracranial tumors and show extensive infiltration of macrophages. The mitochondrial membrane protein translocator protein (TSPO) has been used as an in vivo marker of microglia and macrophage activation to visualize neuroinflammation. However, it is unknown which cell types express TSPO in meningiomas. Immunohistochemistry of 38 WHO grade 1-3 meningiomas was subjected to segmentation and deep learning classification of TSPO expression to either Iba1-positive tumor-associated macrophages (TAMs) or all other (mainly neoplastic) cells. A possible association between clinical data and TSPO expression intensities was also investigated. TAMs accounted for 15.9%-26% of all cells in the meningioma tissue. Mean fluorescence intensity of TSPO was significantly higher in TAMs (p < 0.0001), but the mass of neoplastic cells in the tumors exceeded that of TAMs. Thus, the summed fluorescence intensity of TSPO in meningioma cells was 64.1% higher than in TAMs (p = 0.0003). We observed no correlation between TSPO expression intensity and WHO grade. These results indicate that both macrophage-lineage and neoplastic cells in meningiomas express TSPO and that the SPECT-TSPO signal in meningiomas mainly reflects the latter; TSPO is expressed equally in parenchymal activated and resting macrophage/microglia lineage cells.

Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution.

Various cellular sources hamper interpretation of positron emission tomography (PET) biomarkers in the tumor microenvironment (TME). We developed an approach of immunomagnetic cell sorting after in vivo radiotracer injection (scRadiotracing) with three-dimensional (3D) histology to dissect the cellular allocation of PET signals in the TME. In mice with implanted glioblastoma, translocator protein (TSPO) radiotracer uptake per tumor cell was higher compared to tumor-associated microglia/macrophages (TAMs), validated by protein levels. Translation of in vitro scRadiotracing to patients with glioma immediately after tumor resection confirmed higher single-cell TSPO tracer uptake of tumor cells compared to immune cells. Across species, cellular radiotracer uptake explained the heterogeneity of individual TSPO-PET signals. In consideration of cellular tracer uptake and cell type abundance, tumor cells were the main contributor to TSPO enrichment in glioblastoma; however, proteomics identified potential PET targets highly specific for TAMs. Combining cellular tracer uptake measures with 3D histology facilitates precise allocation of PET signals and serves to validate emerging novel TAM-specific radioligands.

Single-Cell Meta-Analysis of Neutrophil Activation in Kawasaki Disease and Multisystem Inflammatory Syndrome in Children Reveals Potential Shared Immunological Drivers.

BACKGROUND: Kawasaki disease (KD) and multisystem inflammatory syndrome in children (MIS-C) share similar clinical manifestations, including cardiovascular complications, suggesting similar underlying immunopathogenic processes. Aberrant neutrophil activation may play a crucial role in the shared pathologies of KD and MIS-C; however, the associated pathogenic mechanisms and molecular drivers remain unknown. METHODS: We performed a single-cell meta-analysis of neutrophil activation with 103 pediatric single-cell transcriptomic peripheral blood mononuclear cell data across 9 cohorts, including healthy controls, KD, MIS-C, compared with dengue virus infection, juvenile idiopathic arthritis, and pediatric celiac disease. We used a series of computational analyses to investigate the shared neutrophil transcriptional programs of KD and MIS-C that are linked to systemic damage and cardiac pathologies, and suggested Food and Drug Administration-approved drugs to consider as KD and MIS-C treatment. RESULTS: We meta-analyzed 521 950 high-quality cells. We found that blood signatures associated with risks of cardiovascular events are enriched in neutrophils of KD and MIS-C. We revealed the expansion of CD177+ neutrophils harboring hyperactivated effector functions in both KD and MIS-C, but not in healthy controls or in other viral-, inflammatory-, or immune-related pediatric diseases. KD and MIS-C CD177+ neutrophils had highly similar transcriptomes, marked by conserved signatures and pathways related to molecular damage. We found the induction of a shared neutrophil expression program, potentially regulated by SPI1 (Spi-1 proto-oncogene), which confers enhanced effector functions, especially neutrophil degranulation. CD177 and shared neutrophil expression program expressions were associated with acute stages and attenuated during KD intravenous immunoglobulin treatment and MIS-C recovery. Network analysis identified hub genes that correlated with the high activation of CD177+ neutrophils. Disease-gene association analysis revealed that the KD and MIS-C CD177+ neutrophils' shared expression program was associated with the development of coronary and myocardial disorders. Last, we identified and validated TSPO (translocator protein) and S100A12 (S100 calcium-binding protein A12) as main molecular targets, for which the Food and Drug Administration-approved drugs methotrexate, zaleplon, metronidazole, lorazepam, clonazepam, temazepam, and zolpidem, among others, are primary candidates for drug repurposing. CONCLUSIONS: Our findings indicate that CD177+ neutrophils may exert systemic pathological damage contributing to the shared morbidities in KD and MIS-C. We uncovered potential regulatory drivers of CD177+ neutrophil hyperactivation and pathogenicity that may be targeted as a single therapeutic strategy for either KD or MIS-C.