Structurally Related Kappa Opioid Receptor Agonists with Substantial Differential Signaling Bias: Neuroendocrine and Behavioral Effects in C57BL6 Mice.

Background: The kappa opioid receptor system has been revealed as a potential pharmacotherapeutic target for the treatment of addictions to substances of abuse. Kappa opioid receptor agonists have been shown to block the rewarding and dopamine-releasing effects of psychostimulants. Recent investigations have profiled the in vivo effects of compounds biased towards G-protein-mediated signaling, with less potent arrestin-mediated signaling. The compounds studied here derive from a series of trialkylamines: N-substituted-N- phenylethyl-N-3-hydroxyphenylethyl-amine, with N-substituents including n-butyl (BPHA), methylcyclobutyl (MCBPHA), and methylcyclopentyl (MCPPHA). Methods: BPHA, MCBPHA, and MCPPHA were characterized in vitro in a kappa opioid receptor-expressing cell line in binding assays and functional assays. We also tested the compounds in C57BL6 mice, assaying incoordination with rotarod, as well as circulating levels of the neuroendocrine kappa opioid receptor biomarker, prolactin. Results: BPHA, MCBPHA, and MCPPHA showed full kappa opioid receptor agonism for G-protein coupling compared with the reference compound U69,593. BPHA showed no measurable ß-arrestin-2 recruitment, indicating that it is extremely G-protein biased. MCBPHA and MCPPHA, however, showed submaximal efficacy for recruiting ß-arrestin-2. Studies in C57BL6 mice reveal that all compounds stimulate release of prolactin, consistent with dependence on G-protein signaling. MCBPHA and MCPPHA result in rotarod incoordination, whereas BPHA does not, consistent with the reported requirement of intact kappa opioid receptor/ß-arrestin-2 mediated coupling for kappa opioid receptor agonist-induced rotarod incoordination. Conclusions: BPHA, MCBPHA, and MCPPHA are thus novel differentially G-protein-biased kappa opioid receptor agonists. They can be used to investigate how signaling pathways mediate kappa opioid receptor effects in vitro and in vivo and to explore the effects of candidate kappa opioid receptor-targeted pharmacotherapeutics.

Improving Transcriptome Fidelity Following Synovial Tissue Disaggregation.

Objective: To improve the fidelity of the cellular transcriptome of disaggregated synovial tissue for applications such as single-cell RNA sequencing (scRNAseq) by modifying the disaggregation technique. Methods: Osteoarthritis (OA) and rheumatoid arthritis (RA) synovia were collected at arthroplasty. RNA was extracted from intact or disaggregated replicate pools of tissue fragments. Disaggregation was performed with either a proprietary protease, Liberase TL (Lib) as a reference method, Liberase TL with an RNA polymerase inhibitor flavopyridol (Flavo), or a cold digestion with subtilisin A (SubA). qPCR on selected markers and RNAseq were used to compare disaggregation methods using the original intact tissue as reference. Results: Disaggregated cell yield and viability were similar for all three methods with some viability improved (SubA). Candidate gene analysis showed that Lib alone dramatically increased expression of several genes involved in inflammation and immunity compared with intact tissue and was unable to differentiate RA from OA. Both alternative methods reduced the disaggregation induced changes. Unbiased analysis using bulk RNAseq and the 3 protocols confirmed the candidate gene studies and showed that disaggregation-induced changes were largely prevented. The resultant data improved the ability to distinguish RA from OA synovial transcriptomes. Conclusions: Disaggregation of connective tissues such as synovia has complex and selective effects on the transcriptome. We found that disaggregation with an RNA polymerase inhibitor or using a cold enzyme tended to limit induction of some relevant transcripts during tissue processing. The resultant data in the disaggregated transcriptome better represented the in situ transcriptome. The specific method chosen can be tailored to the genes of interest and the hypotheses being tested in order to optimize the fidelity of technique for applications based on cell suspensions such as sorted populations or scRNAseq.

Lipopolysaccharide Exposure Differentially Alters Plasma and Brain Inflammatory Markers in Adult Male and Female Rats.

Humans and rodents have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. Therefore, we examined whether inflammatory response to LPS and the associated BBB disruption differed in male and female adult rats. Rats were treated with saline or two injections of 1 mg/kg LPS and studied 24 h after the second LPS injection. Plasma isolated from trunk blood and brain tissue homogenates of the prefrontal cortex (PFC), dorsal striatum (DS), hippocampus, and cerebellum were analyzed for cytokines and chemokines using a 9-plex panel from Meso Scale Discovery. BBB disruption was analyzed with tight junction proteins claudin-5 and VE-cadherin via Western blotting and VEGF by ELISA. This allowed us to compare sex differences in the levels of individual cytokines as well as associations among cytokines and expression of tight junction proteins between the plasma and specific brain regions. Higher levels of interferon-γ, interleukin-10 (IL-10), IL-13, IL-4, CXCL-1, and VEGF in the plasma were revealed compared to the brain homogenates, and higher levels of TNFα, IL-1ß, IL-6, and IL-5 in the PFC were seen compared with plasma and other brain regions in males. Females showed higher levels of plasma CXCL1 and VEGF compared to males, and males showed higher levels of PFC TNFα, IL-6, IL-4, and VEGF compared to females. LPS induced significant increases in plasma cytokines and VEGF in both sexes. LPS did not significantly alter cytokines in brain tissue homogenates, however, it increased chemokines in the PFC, DS, and hippocampus. In the PFC, LPS produced BBB disruption, which is evident as reduced expression of claudin-5 in males and reduced expression of VE-cadherin in both sexes. Taken together, our results reveal significant sex differences in pro-inflammatory cytokine and chemokine levels in plasma and brain that were associated with BBB disruption after LPS, and validate the use of multiplex assay for plasma and brain tissue samples.

Parachute Mitral Valve: A Case of Isolated Accessory Mitral Valve Tissue.

A 69-year-old male presented to the emergency room with dyspnea on exertion lasting more than 2 weeks. Echocardiography showed an ill-defined subaortic structure. Subsequent transesophageal echocardiography revealed a parachute-like structure prolapsing into the left ventricular outflow tract causing subvalvular aortic obstruction. Surgical excision confirmed this structure as an accessory anterior mitral leaflet. (Level of Difficulty: Intermediate.).

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Validation of gene expression biomarker analysis for biopsy-based clinical trials in Crohn's disease.

BACKGROUND: The ability to measure the expression of proinflammatory cytokines from intestinal biopsies in patients with Crohn's disease in an accurate and reproducible way is critical for proof-of-concept and mechanism-of-action trials; however, the number of biopsies from a segment of the ileum or colon required to yield reproducible results has not been rigorously evaluated. We examined intestinal biopsies from patients with Crohn's disease to validate methods for detecting changes in inflammatory gene expression. METHODS: To evaluate the reproducibility of gene expression measurements, intestinal biopsies were obtained from designated segments from 6 healthy controls, 6 patients with active Crohn's disease, and 6 patients with inactive Crohn's disease. Disease activity was based on the simple endoscopic score for Crohn's disease. Expression of 7 proinflammatory genes was measured from each biopsy using quantitative polymerase chain reaction. Using a linear mixed effects model, the power to detect transcriptional changes corresponding to active and inactive Crohn's disease was calculated. RESULTS: Total simple endoscopic score for Crohn's disease score corresponds with expression of most inflammatory biomarkers. For most genes, 2 to 5 biopsies are needed to reduce sampling error to <25% for most genes. To measure changes in mRNA expression corresponding to active versus inactive Crohn's disease, 1 to 2 intestinal biopsies from 3 patients before and after treatment are needed to yield power of at least 80%. CONCLUSIONS: Measuring proinflammatory gene expression from mucosal biopsies from patients with Crohn's disease is practicable and provides objective biomarkers that can be used in proof-of-concept and mechanism-of-action trials to assess response to therapy.