Blood-derived Mononuclear Cells Research Articles

Blood Vitamin C Levels of Patients Receiving Immunotherapy and Relationship to Monocyte Subtype and Epigenetic Modification.

The treatment of metastatic melanoma has been revolutionised by immunotherapy, yet a significant number of patients do not respond, and many experience autoimmune adverse events. Associations have been reported between patient outcome and monocyte subsets, whereas vitamin C (ascorbate) has been shown to mediate changes in cancer-stimulated monocytes in vitro. We therefore investigated the relationship of ascorbate with monocyte subsets and epigenetic modifications in patients with metastatic melanoma receiving immunotherapy. Patients receiving immunotherapy were compared to other cancer cohorts and age-matched healthy controls. Ascorbate levels in plasma and peripheral blood-derived mononuclear cells (PBMCs), monocyte subtype and epigenetic markers were measured, and adverse events, tumour response and survival were recorded. A quarter of the immunotherapy cohort had hypovitaminosis C, with plasma and PBMC ascorbate levels significantly lower than those from other cancer patients or healthy controls. PBMCs from the immunotherapy cohort contained similar frequencies of non-classical and classical monocytes. DNA methylation markers and intracellular ascorbate concentration were correlated with monocyte subset frequency in healthy controls, but correlation was lost in immunotherapy patients. No associations between ascorbate status and immune-related adverse events or tumour response or overall survival were apparent.

Comparative analysis of human gut- and blood-derived mononuclear cells: contrasts in function and phenotype.

Alterations in the gut immune system have been implicated in various diseases.The challenge of obtaining gut tissues from healthy individuals, commonly performed via surgical explants, has limited the number of studies describing the phenotype and function of gut-derived immune cells in health. Here, by means of recto-sigmoid colon biopsies obtained during routine care (colon cancer screening in healthy adults), the phenotype and function of immune cells present in the gut were described and compared to those found in blood. The proportion of CD4+, CD8+, MAIT, γδ+ T, and NK cells phenotype, expression of integrins, and ability to produce cytokine in response to stimulation with PMA and ionomycin. T cells in the gut were found to predominantly have a memory phenotype as compared to T cells in blood where a naïve phenotype predominates. Recto-sigmoid mononuclear cells also had higher PD-1 and Ki67 expression. Furthermore, integrin expression and cytokine production varied by cell type and location in blood vs. gut. These findings demonstrate the differences in functionality of these cells when compared to their blood counterparts and validate previous studies on phenotype within gut-derived immune cells in humans (where cells have been obtained through surgical means). This study suggests that recto-sigmoid biopsies collected during colonoscopy can be a reliable yet more accessible sampling method for follow up of alterations of gut derived immune cells in clinical settings.

Preclinical Animal Study and Pilot Clinical Trial of Using Enriched Peripheral Blood-Derived Mononuclear Cells for Intervertebral Disc Degeneration.

Low back pain (LBP) is a leading cause of long-term disability globally. Intervertebral disk degeneration (IVDD) is mainly responsible for discogenic pain in LBP-affected young patients. There is no effective therapy to reverse disease severity and IVDD progression. This study investigates the effect of human peripheral blood-derived mononuclear cells (PBMCs) on pain relief and life quality improvement in IVDD patients. The enriched monocytes of the PBMCs could differentiate into CD14 and CD206 double-positive M2 macrophages in vitro. Preclinical evidence in rats showed that the transplanted PBMCs exhibited anti-inflammatory and moderate tissue-repair effects on controlling IVDD progress in the rat model. The PBMCs significantly steered the aggrecan and type II collagen expressions and attenuated the pro-inflammatory cytokines in the affected disk. Based on the animal results, 36 patients with chronic low back pain (CLBP) were included in clinical trials. The control group was conservative care only, and the experimental group was platelet-rich plasma (PRP) and PBMCs intradiscal injections. We first confirmed the single lumbar disk causing the discogenic pain by provocative discography or magnetic resonance imaging (MRI). Discogenic LBP participants received one intradiscal injection of autologous PBMCs and followed for 6 months. Our clinical trial showed that patients' LBP and disability were significantly ameliorated after the PBMCs transplantation rather than PRP. These preclinical and pilot clinical studies indicate that intradiscal injection of the enriched PBMCs might be a feasible and potential cell therapy to control pain and disability in IVDD patients.

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. However, efficient differentiation and terminal maturation (symbolized by enucleation) into cRBCs ex vivo highly depend on replenishing human plasma. In addition to cost, this requirement results in variable potency across donors or batches and complicates consistent cRBC production required for clinical translations. Thus, exploration of key factor(s) in plasma and development of a compositionally defined medium would be in need. To investigate the role of human plasma in erythroblast terminal maturation, we first optimized a widely-used differentiation system that we and other have used in the past decade. We found that culture of erythroblasts in a newly developed medium called human plasma like medium (HPLM) greatly improves the yield and consistence of erythrocyte maturation. However, human serum (1-2%) was indispensable for efficient terminal maturation in the absence of plasma. The serum-derived factor or factors (likely a protein or complex) is essential for erythroblast growth assessed at day 3 after induction of terminal differentiation. We used size exclusion chromatography (SEC) and other protein biochemistry approaches to fractionate human serum and assessed the biological effect of each fraction. We found that the activity is fractions containing proteins or complexes of >100K Da. Proteomics revealed that the fractions with biological activities are enriched for low density lipoproteins (LDL)-containing proteins such as apolipoprotein B-100. In addition, we performed RNA-sequencing (RNA-seq) analysis on cultured erythroblasts with or without serum. Transcriptomic data revealed that the genes associated with de novo biosynthesis of steroids and cholesterol are upregulated in the absence of serum, although production of enzymes encoded by these genes alone is insufficient to provide cholesterol required for erythroblast differentiation and maturation. Hence, we hypothesize that cholesterol-carrying LDL that are naturally present in blood is sufficient to replace human serum for erythroblast survival and terminal differentiation ex vivo. We next examined directly whether LDL purified from human blood is sufficient for erythroid commitment and enucleation. Through LDL binding and uptake assays, we confirmed that LDL binds to erythroblasts that expressed the LDL receptor and is transported intracellularly. Furthermore, LDL purified from human plasma can essentially replace 2% unfractionated serum for efficient generation of erythrocytes in culture. In addition to using human erythroblasts derived from peripheral blood mononuclear cells (PBMCs), we also extended this optimized protocol to human erythroblasts from various sources, including cord blood-derived mononuclear cells, CD34+ HSPCs, and extensively expanded erythroblasts after BMI1 gene transduction. After induction of terminal differentiation for 8 days, erythroblasts from all these sources effectively enucleated (>50%) and stably produced reticulocytes with no significant difference compared to the serum-containing system. We are also in progress to substitute LDL by chemical synthetic cholesterol. In sum, we developed a compositionally defined culture medium (LDL-containing Optimized Maturation Medium, LOAM) enabling robust generation of cRBCs from multiple sources of erythroblasts with high enucleation efficiency and higher reticulocyte yield compared to culture in traditional media supplemented with human plasma or serum. Using this advanced human erythroblast differentiation model, we further revealed that cholesterol carried by LDL is critical to erythroblast terminal maturation.

The use of an artificial intelligence algorithm for circulating tumor cell detection in patients with esophageal cancer.

Despite recent advances in multidisciplinary treatments of esophageal squamous cell carcinoma (ESCC), patients frequently suffer from distant metastasis after surgery. For numerous types of cancer, circulating tumor cells (CTCs) are considered predictors of distant metastasis, therapeutic response and prognosis. However, as more markers of cytopathological heterogeneity are discovered, the overall detection process for the expression of these markers in CTCs becomes increasingly complex and time consuming. In the present study, the use of a convolutional neural network (CNN)-based artificial intelligence (AI) for CTC detection was assessed using KYSE ESCC cell lines and blood samples from patients with ESCC. The AI algorithm distinguished KYSE cells from peripheral blood-derived mononuclear cells (PBMCs) from healthy volunteers, accompanied with epithelial cell adhesion molecule (EpCAM) and nuclear DAPI staining, with an accuracy of >99.8% when the AI was trained on the same KYSE cell line. In addition, AI trained on KYSE520 distinguished KYSE30 from PBMCs with an accuracy of 99.8%, despite the marked differences in EpCAM expression between the two KYSE cell lines. The average accuracy of distinguishing KYSE cells from PBMCs for the AI and four researchers was 100 and 91.8%, respectively (P=0.011). The average time to complete cell classification for 100 images by the AI and researchers was 0.74 and 630.4 sec, respectively (P=0.012). The average number of EpCAM-positive/DAPI-positive cells detected in blood samples by the AI was 44.5 over 10 patients with ESCC and 2.4 over 5 healthy volunteers (P=0.019). These results indicated that the CNN-based image processing algorithm for CTC detection provides a higher accuracy and shorter analysis time compared to humans, suggesting its applicability for clinical use in patients with ESCC. Moreover, the finding that AI accurately identified even EpCAM-negative KYSEs suggested that the AI algorithm may distinguish CTCs based on as yet unknown features, independent of known marker expression.

Inhibition of Cyclin-Dependent Kinases 8/19 Restricts Bacterial and Virus-Induced Inflammatory Responses in Monocytes.

Hyperactivation of the immune system remains a dramatic, life-threatening complication of viral and bacterial infections, particularly during pneumonia. Therapeutic approaches to counteract local and systemic outbreaks of cytokine storm and to prevent tissue damage remain limited. Cyclin-dependent kinases 8 and 19 (CDK8/19) potentiate transcriptional responses to the altered microenvironment, but CDK8/19 potential in immunoregulation is not fully understood. In the present study, we investigated how a selective CDK8/19 inhibitor, Senexin B, impacts the immunogenic profiles of monocytic cells stimulated using influenza virus H1N1 or bacterial lipopolysaccharides. Senexin B was able to prevent the induction of gene expression of proinflammatory cytokines in THP1 and U937 cell lines and in human peripheral blood-derived mononuclear cells. Moreover, Senexin B substantially reduced functional manifestations of inflammation, including clustering and chemokine-dependent migration of THP1 monocytes and human pulmonary fibroblasts (HPF).

Qualification of a multidonor mixed lymphocyte reaction assay for the functional characterization of immunomodulatory extracellular vesicles

Background aimsExtracellular vesicles (EVs), including exosomes and microvesicles, are released by almost all cells and found in all body fluids. Unknown proportions of EVs transmit specific information from their cells of origin to specific target cells and are key mediators in intercellular communication processes. Depending on their origin, EVs can modulate immune responses, either acting as pro- or anti-inflammatory. With the aim to analyze the immunomodulating activities of EV preparations, especially those from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed for its reproducibility. MethodsTo this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions, in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance can be modulated by EVs. More quantifiable, the strength of the allogenic reaction is reflected by the content of activated CD4 and CD8 T cells being recognized by means of their CD25 and CD54 expression. ResultsOf note, connected to the use of primary cells, independent multi-donor PBMC pools differed in their capability to activate their cultured T cells. Thus, throughout the study, only pooled PBMC batches were used whose activated T-cell contents exceeded 25% of the total T-cell population at culture day 5 and whose contents were reproducibly reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation–suppressing effects of the MSC-EV preparations tested were in all cases accompanied by the impact on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were harvested from mdMLR cultures than in their absence. Furthermore, in the absence of immunomodulatory EVs, most monocytes appeared as non-classical (CD14+CD16+) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical (CD14++CD16–) and intermediate (CD14++CD16+) monocyte subpopulations. ConclusionsOverall, the obtained results qualify the mdMLR assay as a robust experimental tool for the evaluation of immunomodulatory potentials of given MSC-EV samples. However, further assay development is required to develop and qualify an authority-acceptable potency assay for clinically applicable MSC-EV products.

Abstract 2791: Biomarker strategy for a phase 1 study of ORIC-944, a potent and selective allosteric PRC2 inhibitor, in patients with metastatic prostate cancer

Abstract Background: Polycomb repressive complex 2 (PRC2) tri-methylates histone H3 at lysine 27 (H3K27me3) leading to transcriptionally silenced genes. ORIC-944 is a potent, highly selective allosteric small molecule inhibitor of PRC2’s embryonic ectoderm development (EED) subunit that is undergoing clinical development as an orally bioavailable monotherapy in patients with metastatic prostate cancer. We previously reported strong tumor growth inhibition with ORIC-944 in enzalutamide-resistant 22Rv1 prostate cancer xenografts. Here, to devise and implement a biomarker strategy aimed at ORIC-944 dose selection in the ongoing phase 1 study (NCT05413421), we undertook preclinical pharmacology studies and assay development. Methods: Naïve and 22Rv1-tumor bearing male mice were treated with vehicle, 10, 30 or 100 mg/kg ORIC-944 PO for up to 37 days. H3K27me3 was evaluated in dorsal skin (epidermal layer) and peripheral monocytes by IHC and an alphaLISA test, respectively. Since dying tumor cells release nucleosomes to circulation, cell-free (cf)-nucleosomal H3K27me3 levels normalized to cf-H3.1 were also assessed in plasma using a proprietary magnetic bead-based sandwich immunoassay. Tumors were profiled by RNA-sequencing and H3K27me3 chromatin immunoprecipitation sequencing. Results: In the epidermal skin layer, H3K27me3 was present in vehicle-treated naïve animals but strongly depleted in ORIC-944-treated mice in a dose-dependent manner. Likewise in monocytes, H3K27me3 was reduced to undetectable levels in 100 mg/kg ORIC-944-treated mice. In plasma, normalized cf-nucleosomal H3K27me3 levels significantly decreased in response to ORIC-944 in a dose- and time-dependent manner, selectively in 22Rv1-tumor bearing mice compared to non-tumor bearing mice. Putative PRC2 target genes were identified in 22Rv1 xenografts by focusing on those genes with H3K27me3 binding in the promoter, whose expression was de-repressed by ORIC-944 in a time-dependent manner and then re-expressed after dose suspension. In the ongoing phase 1 study, H3K27me3 levels are being evaluated in the stratum spinosum of skin and in blood-derived monocytes using a proprietary IHC assay and alphaLISA test, respectively. Modulation in the expression of the putative PRC2 target genes is being assessed in blood-derived peripheral blood mononuclear cells by RNA-sequencing. Conclusion: This comprehensive biomarker strategy enables us to establish target engagement and capture exposure-dependent pharmacodynamics in the ongoing phase 1 study evaluating ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer. Citation Format: Anneleen Daemen, Natalie Yuen, Aleksandr Pankov, Eric A. Ariazi, Subhash D. Katewa, Frank L. Duong, Amber Wang, Shravani Barkund, Shelly Kaushik, Jessica D. Sun, Lori S. Friedman, Melissa R. Junttila. Biomarker strategy for a phase 1 study of ORIC-944, a potent and selective allosteric PRC2 inhibitor, in patients with metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2791.

Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells

To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Retinoid homeostasis in major depressive disorder

The small, hormone-like molecule retinoic acid (RA) is a vital regulator in several neurobiological processes that are affected in depression. Next to its involvement in dopaminergic signal transduction, neuroinflammation, and neuroendocrine regulation, recent studies highlight the role of RA in homeostatic synaptic plasticity and its link to neuropsychiatric disorders. Furthermore, experimental studies and epidemiological evidence point to the dysregulation of retinoid homeostasis in depression. Based on this evidence, the present study investigated the putative link between retinoid homeostasis and depression in a cohort of 109 patients with major depressive disorder (MDD) and healthy controls. Retinoid homeostasis was defined by several parameters. Serum concentrations of the biologically most active Vitamin A metabolite, all-trans RA (at-RA), and its precursor retinol (ROL) were quantified and the individual in vitro at-RA synthesis and degradation activity was assessed in microsomes of peripheral blood-derived mononuclear cells (PBMC). Additionally, the mRNA expression of enzymes relevant to retinoid signaling, transport, and metabolism were assessed. Patients with MDD had significantly higher ROL serum levels and greater at-RA synthesis activity than healthy controls providing evidence of altered retinoid homeostasis in MDD. Furthermore, MDD-associated alterations in retinoid homeostasis differed between men and women. This study is the first to investigate peripheral retinoid homeostasis in a well-matched cohort of MDD patients and healthy controls, complementing a wealth of preclinical and epidemiological findings that point to a central role of the retinoid system in depression.

The trajectory of osteoblast progenitor cells in patients with type 2 diabetes and the predictive model for their osteogenic differentiation ability

The fate of osteoprogenitor cells along with the progression of type 2 diabetes (T2DM) and factors determining the fate of those cells remains to be elucidated. This cross-sectional study included 18 normoglycemic, 27 prediabetic, and 73 T2DM to determine osteogenic differentiation across the continuum of dysglycemia and to construct a model to predict the fate of osteoprogenitor cells. This study demonstrated a preserved osteogenic differentiation ability of peripheral blood-derived mononuclear cells (PBMC) isolated from normoglycemic and prediabetic but a progressive decline in their osteogenic differentiation during the progression of T2DM. The rate of osteogenic differentiation rapidly declined by 4–7% annually during the first 10 years of diabetes and then slowed down. A predictive model composed of three independent risk factors, including age, duration of diabetes, and glomerular filtration rate, demonstrated an AuROC of 0.834. With a proposed cut-off of 21.25, this model had 72.0% sensitivity, 87.5% specificity, and 78.9% accuracy in predicting the fate of osteoprogenitor cells. In conclusion, this study provided a perspective on the osteogenic differentiation ability of the osteoprogenitor cells across a continuum of dysglycemia and a predictive model with good diagnostic performance for the prediction of the fate of osteoprogenitor cells in patients with T2DM.

Enriched Peripheral Blood-Derived Mononuclear Cells for Treating Knee Osteoarthritis.

Osteoarthritis (OA) is a common chronic skeletal disease in the elderly. There is no effective therapy to reverse disease severity and knee OA (KOA) progression, particularly at the late stage. This study aims to examine the effect of peripheral blood-derived mononuclear cells (PBMNCs) on pain and motor function rescue in patients with Kellgren-Lawrence (KL) grade II to IV KOA. Participants received one intra-articular (IA) injection of autologous PBMNCs. The mononuclear cells were isolated from peripheral blood, enriched by a specialized medium (MoFi medium), and separated by Ficoll-Paque solution. The isolated and enriched PBMNCs could differentiate into M1 and M2 macrophages in vitro. The in vivo anti-inflammatory effect of the PBMNCs was similar to that of bone marrow mesenchymal stem cells, evaluated by complete Freund's adjuvant-induced arthritis in rodents. A single-arm and open-label pilot study showed that patients' knee pain and motor dysfunction were significantly attenuated after the cell transplantation, assessed by visual analogue scale (VAS) and Knee injury and Osteoarthritis Outcome Score (KOOS) at 6 and 12 months post-treatment. Notably, the therapeutic effect of the PBMNCs treatment can be stably maintained for 24 months, as revealed by the KOOS scores. These preclinical and pilot clinical data suggest that IA injection of MoFi-PBMNCs might serve as a novel medical technology to control the pain and the progress of KOA.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1β and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1β and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

Clozapine-specific proliferative response of peripheral blood-derived mononuclear cells in Japanese patients with clozapine-induced agranulocytosis.

Although clozapine-induced granulocytopenia (CIG) is less severe than clozapine-induced agranulocytosis (CIA), and some patients with CIG may not go on to develop serious complications, clozapine is discontinued in cases of both CIA and CIG. Understanding the pathogenic mechanisms of CIA/CIG could provide better management of clozapine therapy. Recently, as a mechanistic insight into adaptive immune systems, European groups reported clozapine-specific proliferative responses and clozapine-specific T cells using blood taken from patients with CIA and/or CIG. The aims of our study are to support this mechanistic evidence and to investigate the difference in the lymphocyte response to clozapine between patients with CIG and those with CIA. Lymphocyte stimulation tests (LSTs) were conducted using CD25-positive cell-depleted peripheral blood-derived mononuclear cells (PBMCs) isolated from blood of four Japanese patients with CIA, four patients with CIG, and nine clozapine-tolerant subjects. Three of four patients with CIA and one of four patients with CIG showed proliferative responses to clozapine with a stimulation index of greater than 2. In contrast, none of the nine clozapine-tolerant subjects showed any response to clozapine. Olanzapine did not stimulate PBMCs of patients with CIA, patients with CIG, or clozapine-tolerant subjects. Clozapine- and CIA-specific lymphocyte reactions in a Japanese population provided supportive evidence that the pathogenesis of CIA is based on adaptive immune reactions. In addition, patients with CIG who show a positive response to an LST may at the very least not be chosen for clozapine-rechallenge and further prospective studies are desirable to verify this hypothesis.

Identification of Serum Exosome-Derived circRNA-miRNA-TF-mRNA Regulatory Network in Postmenopausal Osteoporosis Using Bioinformatics Analysis and Validation in Peripheral Blood-Derived Mononuclear Cells.

BackgroundOsteoporosis is one of the most common systemic metabolic bone diseases, especially in postmenopausal women. Circular RNA (circRNA) has been implicated in various human diseases. However, the potential role of circRNAs in postmenopausal osteoporosis (PMOP) remains largely unknown. The study aims to identify potential biomarkers and further understand the mechanism of PMOP by constructing a circRNA-associated ceRNA network.MethodsThe PMOP-related datasets GSE161361, GSE64433, and GSE56116 were downloaded from the Gene Expression Omnibus (GEO) database and were used to obtain differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to determine possible relevant functions of differentially expressed messenger RNAs (mRNAs). The TRRUST database was used to predict differential transcription factor (TF)-mRNA regulatory pairs. Afterwards, combined CircBank and miRTarBase, circRNA-miRNA as well as miRNA-TF pairs were constructed. Then, a circRNA-miRNA-TF-mRNA network was established. Next, the correlation of mRNAs, TFs, and PMOP was verified by the Comparative Toxicogenomics Database. And expression levels of key genes, including circRNAs, miRNAs, TFs, and mRNAs in the ceRNA network were further validated by quantitative real-time PCR (qRT-PCR). Furthermore, to screen out signaling pathways related to key mRNAs of the ceRNA network, Gene Set Enrichment Analysis (GSEA) was performed.ResultsA total of 1201 DE mRNAs, 44 DE miRNAs, and 1613 DE circRNAs associated with PMOP were obtained. GO function annotation showed DE mRNAs were mainly related to inflammatory responses. KEGG analysis revealed DE mRNAs were mainly enriched in osteoclast differentiation, rheumatoid arthritis, hematopoietic cell lineage, and cytokine-cytokine receptor interaction pathways. We first identified 26 TFs and their target mRNAs. Combining DE miRNAs, miRNA-TF/mRNA pairs were obtained. Combining DE circRNAs, we constructed the ceRNA network contained 6 circRNAs, 4 miRNAs, 4 TFs, and 12 mRNAs. The expression levels of most genes detected by qRT-PCR were generally consistent with the microarray results. Combined with the qRT-PCR validation results, we eventually identified the ceRNA network that contained 4 circRNAs, 3 miRNAs, 3 TFs, and 9 mRNAs. The GSEA revealed that 9 mRNAs participate in many important signaling pathways, such as “olfactory transduction”, “T cell receptor signaling pathway”, and “neuroactive ligand-receptor interaction”. These pathways have been reported to the occurrence and development of PMOP. To sum up, key mRNAs in the ceRNA network may participate in the development of osteoporosis by regulating related signal pathways.ConclusionsA circRNA-associated ceRNA network containing TFs was established for PMOP. The study may help further explore the molecular mechanisms and may serve as potential biomarkers or therapeutic targets for PMOP.

Optimization of Peripheral Blood Mononuclear Cell Extraction from Small Volume of Blood Samples: Potential Implications for Children-Related Diseases.

Managing medical procedures for children with problematic disorders is a challenging approach, especially in the case of blood withdrawal for autism spectrum disorder-affected children. Peripheral blood mononuclear cells (PBMC) represent an important cellular model to study immune responses and drug toxicity. The monocytic cells, a fraction of PBMC, are strongly involved in some pathophysiological processes, such as inflammation and immune system changes. Here, we propose a simple, reliable protocol for obtaining peripheral blood-derived mononuclear cells from small volumes of blood samples.

Abstract P041: Initial results from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer

Abstract Background: Upregulation of the glucocorticoid receptor (GR) is a potential mechanism of resistance to enzalutamide and other androgen receptor (AR) modulators in prostate cancer. Preclinical studies have demonstrated that GR activation can bypass enzalutamide-mediated AR inhibition and support prostate cancer cell growth. Overexpression of GR is associated with poor outcomes in castration-resistant prostate cancer patients (CRPC) treated with enzalutamide. ORIC-101 is a potent and selective orally bioavailable, small molecule antagonist of GR. Mechanistically, ORIC-101 inhibits GR transcriptional activity and blocks the pro-survival signals mediated by the activated nuclear hormone receptor. Methods: A modified interval 3+3 (i3+3) design was used to assess safety, pharmacokinetics (PK), and pharmacodynamics (PD) to select the Recommended Phase 2 Dose (RP2D) of ORIC-101 in combination with enzalutamide in patients with metastatic CRPC progressing on enzalutamide 160 mg, dosed once daily (NCT04033328). ORIC-101, at doses ranging from 80 to 240 mg once daily, given in a continuous dosing regimen, was added to enzalutamide at the time of disease progression. Plasma PK and PD biomarkers were assessed on multiple days and times before and after dosing. PD modulation in blood-derived peripheral blood mononuclear cells (PBMCs) was assessed by RT-qPCR for GR target genes. Antitumor activity was assessed by Prostate Cancer Clinical Trials Working Group 3 (PCWG3) and RECIST 1.1. Results: 10 patients were treated in 3 cohorts in the dose escalation portion of the study. ORIC-101 exposure increased with dose and no drug-drug interaction (DDI) was observed that necessitated reduction from the standard enzalutamide dose of 160 mg. No dose limiting toxicities were observed at any dose level. Based upon plasma exposure and PD modulation, the RP2D was established as 240 mg ORIC-101 plus 160 mg enzalutamide, both dosed once daily continuously in 28-day cycles. All adverse events (AEs) were Grade 1 or 2 with the most common (&amp;gt;15%), treatment-related AEs being fatigue (40%), nausea (30%), constipation (20%), decreased appetite (20%), high aspartate aminotransferase (20%), high alkaline phosphatase (20%), and headache (20%). There were no Grade ≥3 treatment-related AEs. Biomarker data demonstrated ORIC-101 induced reduction in GR target gene expression in PBMCs, indicating PD modulation across dose levels of ORIC-101. Data will be updated at the time of the presentation. Conclusions: Preliminary evidence suggests that ORIC-101 effectively modulates GR and has an acceptable tolerability profile when combined with enzalutamide. Dose expansion is ongoing at the RP2D. Citation Format: Wassim Abida, Neeraj Agarwal, Andrew W. Hahn, Neal Shore, Paul Sieber, Tanya Dorff, Mathew Rettig, Mathew Smith, Paul Monk, Rongda Xu, Ann Johnson, Anneleen Daemen, Edna Chow Maneval, Pratik S. Multani, Rupal Patel, Michael J. Morris. Initial results from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P041.

Integrative Studies of Human Cord Blood Derived Mononuclear Cells and Umbilical Cord Derived Mesenchyme Stem Cells in Ameliorating Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a common pulmonary complication observed in preterm infants that is composed of multifactorial pathogenesis. Current strategies, albeit successful in moderately reducing morbidity and mortality of BPD, failed to draw overall satisfactory conclusion. Here, using a typical mouse model mimicking hallmarks of BPD, we revealed that both cord blood-derived mononuclear cells (CB-MNCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) are efficient in alleviating BPD. Notably, infusion of CB-MNCs has more prominent effects in preventing alveolar simplification and pulmonary vessel loss, restoring pulmonary respiratory functions and balancing inflammatory responses. To further elucidate the underlying mechanisms within the divergent therapeutic effects of UC-MSC and CB-MNC, we systematically investigated the long noncoding RNA (lncRNA)–microRNA (miRNA)–messenger RNA (mRNA) and circular RNA (circRNA)–miRNA–mRNA networks by whole-transcriptome sequencing. Importantly, pathway analysis integrating Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG)/gene set enrichment analysis (GSEA) method indicates that the competing endogenous RNA (ceRNA) network is mainly related to the regulation of GTPase activity (GO: 0043087), extracellular signal-regulated kinase 1 (ERK1) and ERK2 signal cascade (GO: 0070371), chromosome regulation (GO: 0007059), and cell cycle control (GO: 0044770). Through rigorous selection of the lncRNA/circRNA-based ceRNA network, we demonstrated that the hub genes reside in UC-MSC- and CB-MNC-infused networks directed to the function of cell adhesion, motor transportation (Cdk13, Lrrn2), immune homeostasis balance, and autophagy (Homer3, Prkcd) relatively. Our studies illustrate the first comprehensive mRNA–miRNA–lncRNA and mRNA–miRNA–circRNA networks in stem cell-infused BPD model, which will be valuable in identifying reliable biomarkers or therapeutic targets for BPD pathogenesis and shed new light in the priming and conditioning of UC-MSCs or CB-MNCs in the treatment of neonatal lung injury.

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

873 S-531011, a novel anti-human CCR8 antibody: anti-tumor responses through depletion of tumor-infiltrating CCR8-positive Tregs

BackgroundRegulatory T cells (Tregs) are suppressive immune cells required for the maintenance of immune homeostasis, but tumor-infiltrating Tregs are known to suppress the antitumor immune system and promote tumor progression. Therefore, selective reduction of tumor-infiltrating Tregs is anticipated to reinvigorate antitumor immunity without inducing autoimmunity. S-531011 is a novel anti-human IgG1 antibody targeting human CCR8 (C-C motif chemokine receptor 8) which is selectively expressed in tumor-infiltrating Tregs, with both in vitro antibody dependent cellular cytotoxicity (ADCC) against CCR8-expressing cells and neutralizing activity against CCL1-CCR8 signaling. Here, to evaluate antitumor activities and safety aspects of S-531011, we conducted non-clinical pharmacology studies of S-531011 using human CCR8 knock-in (KI) mice and human tissues.MethodsS-531011 was administrated to CT26WT tumor-bearing hCCR8-KI mice, and the effect on the presence of tumor-infiltrating CCR8+ Treg and tumor growth were evaluated. We also investigated the antitumor efficacy of S-531011 in combination with anti-mouse PD-1 antibody. Next, human lung cancer tissues and human NK-cells were co-cultured, and the ex vivo ADCC against tumor-infiltrating Tregs by S-531011 was verified. We also incubated human peripheral blood-derived mononuclear cells (PBMC) from healthy individuals with S-531011 to investigate the effects on the proportion of Tregs in human PBMC.ResultsIntravenous administration of S-531011 to CT26WT tumor-bearing hCCR8-KI mice significantly reduced tumor-infiltrating CCR8+ Tregs and markedly suppressed tumor growth. Furthermore, the combined therapy of S-531011 with anti-mouse PD-1 antibody showed greater anti-tumor effect than monotherapy without any apparent side effects. Ex vivo ADCC studies using human lung cancer tissues and FCM analysis of CCR8 expression in tumor-infiltrating Tregs suggested that most of the tumor-infiltrating CCR8+ Tregs were depleted by S-531011. On the other hand, S-531011 didn’t reduce Tregs in human PBMC.ConclusionsS-531011 is a promising drug which has a strong antitumor effect by depleting tumor-infiltrating CCR8+ Tregs, as a not only monotherapy but also combination therapy with other immune checkpoint inhibitors.Ethics ApprovalThe present study was approved by the Institutional Ethics Committee of Osaka University Hospital (approved number: 13266-15) and Shionogi Co., Ltd. (approved number: 021-003). Animal studies were approved by the Institutional Animal Care and Use Committee (approved number: S20093D, S20197D and S20198D).