RNA Sequencing Of Blood Research Articles

Circulating Adenoid Cystic Carcinoma associated MYB transcripts enable rapid and sensitive detection of metastatic disease in blood liquid biopsies.

Adenoid cystic carcinoma (ACC) is a rare and lethal malignancy that originates in secretory glands of the head and neck. A prominent molecular feature of ACC is the overexpression of the proto-oncogene MYB. ACC has a poor long-term survival due to its high propensity for recurrence and protracted metastasis. Currently, clinical technologies lack the efficiency to distinguish patient prognosis prior to its redevelopment. We hypothesize that metastatic ACC can be detected by monitoring tumor-specific MYB expression in patients' blood. We developed a quantitative polymerase chain reaction (qPCR) assay for MYB transcripts and screened blood samples from four patient cohorts: no history or evidence of ACC (n=23), past history of ACC and no evidence of disease (NED) for greater than three years (n=15), local ACC (n=6), and metastatic ACC (n=5). Our assay detected significantly elevated levels of MYB transcripts in the metastatic ACC cohort (p < 0.01). Receiver operating characteristic (ROC) curves comparing metastatic to NED and metastatic to local disease were significant, with p values < 0.0001 and 0.0008, respectively. Single-cell RNA sequencing (scRNA-seq) of blood from metastatic ACC identified a cluster of circulating tumor cells (CTCs) expressing MYB. Here, we report a sensitive, cost-effective, and minimally invasive diagnostic test that leverages tumor-specific signatures to screen for metastatic ACC disease, potentially enhancing detection earlier than the current clinical standard.

2000P Combining whole blood RNA-sequencing (WB-RNA) and circulating tumor DNA (ctDNA) for the early identification of patients (pts) without clinical benefit to immune checkpoint inhibitors (ICI) in metastatic urothelial cancer (mUC)

2000P Combining whole blood RNA-sequencing (WB-RNA) and circulating tumor DNA (ctDNA) for the early identification of patients (pts) without clinical benefit to immune checkpoint inhibitors (ICI) in metastatic urothelial cancer (mUC)

Bacterial sepsis causes more dramatic pathogenetic changes in the Th1 pathway than does viral (COVID-19) sepsis: a prospective observational study of whole blood transcriptomes

ObjectivesThis study aimed to comprehensively compare host responses of patients with bacterial sepsis and those with viral (COVID-19) sepsis by analyzing messenger RNA (mRNA) and microRNA (miRNA) profiles to shed light on their distinct pathophysiological mechanisms.DesignProspective observational study.SettingWhole blood RNA sequencing was used to analyze mRNA and miRNA profiles of patients diagnosed as having bacterial sepsis or viral (COVID-19) sepsis at the Department of Trauma and Emergency Medicine, Osaka University Graduate School of Medicine.PatientsTwenty-two bacterial sepsis patients, 35 viral (COVID-19) sepsis patients, and 15 healthy subjects admitted to the department were included. We diagnosed bacterial sepsis patients according to the sepsis-3 criterion that the Sequential Organ Failure Assessment score must increase to 2 points or more among patients with suspected infections. Viral (COVID-19) sepsis patients were diagnosed using SARS-CoV-2 RT-PCR testing, and presence of pneumonia was assessed through chest computed tomography scans.InterventionsNone.Measurements and main resultsFor RNA sequencing, 14,500 mRNAs, 1121 miRNAs, and 2556 miRNA-targeted mRNAs were available for analysis in the bacterial sepsis patients. Numbers of genes showing upregulated: downregulated gene expression (false discovery rate < 0.05, |log2 fold change| > 1.5) were 256:2887 for mRNA, 53:5 for miRNA, and 49:2507 for miRNA-targeted mRNA. Similarly, in viral (COVID-19) sepsis patients, 14,500 mRNAs, 1121 miRNAs, and 327 miRNA-targeted mRNAs were analyzed, with numbers of genes exhibiting upregulated: downregulated gene expression of 672:1147 for mRNA, 3:4 for miRNA, and 165:162 for miRNA-targeted mRNA. This analysis revealed significant differences in the numbers of upregulated and downregulated genes expressed and pathways between the bacterial sepsis and viral (COVID-19) sepsis patients. Bacterial sepsis patients showed activation of the PD-1 and PD-L1 cancer immunotherapy signaling pathway and concurrent suppression of Th1 signaling.ConclusionOur study illuminated distinct molecular variances between bacterial sepsis and viral (COVID-19) sepsis. Bacterial sepsis patients had a greater number of upregulated and downregulated genes and pathways compared to viral (COVID-19) sepsis patients. Especially, bacterial sepsis caused more dramatic pathogenetic changes in the Th1 pathway than did viral (COVID-19) sepsis.

Antihypertensive Potential of Pistacia lentiscus var. Chia: Molecular Insights and Therapeutic Implications.

Background: Hypertension poses a significant global health burden and is associated with cardiovascular morbidity. Chios mastic gum (CMG), derived from Pistacia lentiscus var. Chia, shows potential as a phytotherapeutic agent, due to its multifaceted beneficial effects. However, its anti-hypertensive effects and vascular, circulatory, and renal-related dysfunction, have not been thoroughly investigated. Herein, we aimed to explore the antihypertensive potential of CMG, focusing on vascular and renal endothelium, in vivo. Methods: Two models of hypertension in male rats, induced by Angiotensin II and Deoxycorticosterone acetate (DOCA)-high-salt administration, were utilized. CMG was administered at 220 mg/kg daily for four weeks after hypertension onset and blood pressure was measured non-invasively. Whole blood RNA sequencing, metabolomics, real-time PCR, and Western blot analyses of kidney and aorta tissues were additionally performed. Results: CMG significantly lowered systolic, diastolic, and mean blood pressure in both models. RNA sequencing revealed that CMG modulated immunity in the Angiotensin II model and metabolism in the DOCA-HS model. CMG downregulated genes related to oxidative stress and endothelial dysfunction and upregulated endothelial markers such as Vegfa. Metabolomic analysis indicated improved endothelial homeostasis via lysophosphatidylinositol upregulation. Conclusions: CMG emerges as a potent natural antihypertensive therapy, demonstrating beneficial effects on blood pressure and renal endothelial function.

Direct RNA sequencing of astronaut blood reveals spaceflight-associated m6A increases and hematopoietic transcriptional responses

The advent of civilian spaceflight challenges scientists to precisely describe the effects of spaceflight on human physiology, particularly at the molecular and cellular level. Newer, nanopore-based sequencing technologies can quantitatively map changes in chemical structure and expression at single molecule resolution across entire isoforms. We perform long-read, direct RNA nanopore sequencing, as well as Ultima high-coverage RNA-sequencing, of whole blood sampled longitudinally from four SpaceX Inspiration4 astronauts at seven timepoints, spanning pre-flight, day of return, and post-flight recovery. We report key genetic pathways, including changes in erythrocyte regulation, stress induction, and immune changes affected by spaceflight. We also present the first m6A methylation profiles for a human space mission, suggesting a significant spike in m6A levels immediately post-flight. These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome’s dynamic response to spaceflight.

Blood RNA-sequencing across the continuum of ANA-positive autoimmunity reveals insights into initiating immunopathology

ObjectiveMechanisms underpinning clinical evolution to systemic lupus erythematosus (SLE) from preceding antinuclear antibodies (ANA) positivity are poorly understood. This study aimed to understand blood immune cell transcriptional signatures associated with...

Proteomic, single-cell and bulk transcriptomic analysis of plasma and tumor tissues unveil core proteins in response to anti-PD-L1 immunotherapy in triple negative breast cancer

BackgroundAnti-PD-1/PD-L1 treatment has achieved durable responses in TNBC patients, whereas a fraction of them showed non-sensitivity to the treatment and the mechanism is still unclear. MethodsPre- and post-treatment plasma samples from triple negative breast cancer (TNBC) patients treated with immunotherapy were measured by tandem mass tag (TMT) mass spectrometry. Public proteome data of lung cancer and melanoma treated with immunotherapy were employed to validate the findings. Blood and tissue single-cell RNA sequencing (scRNA-seq) data of TNBC patients treated with or without immunotherapy were analyzed to identify the derivations of plasma proteins. RNA-seq data from IMvigor210 and other cancer types were used to validate plasma proteins in predicting response to immunotherapy. ResultsA random forest model constructed by FAP, LRG1, LBP and COMP could well predict the response to immunotherapy. The activation of complement cascade was observed in responders, whereas FAP and COMP showed a higher abundance in non-responders and negative correlated with the activation of complements. scRNA-seq and bulk RNA-seq analysis suggested that FAP, COMP and complements were derived from fibroblasts of tumor tissues. ConclusionsWe constructe an effective plasma proteomic model in predicting response to immunotherapy, and find that FAP+ and COMP+ fibroblasts are potential targets for reversing immunotherapy resistance.

Dl-3-n-butylphthalide improves stroke outcomes after focal ischemic stroke in mouse model by inhibiting the pyroptosis-regulated cell death and ameliorating neuroinflammation

Recent studies have highlighted the involvement of pyroptosis-mediated cell death and neuroinflammation in ischemic stroke (IS) pathogenesis. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of Apium graveolens, possesses a broad range of biological effects. However, the efficacy and the underlying mechanisms of NBP in IS remain contentious. Herein, we investigated the therapeutic effects of NBP and elucidated its potential mechanisms in neuronal cell pyroptosis and microglia inflammatory responses. Adult male mice underwent permanent distal middle cerebral artery occlusion (dMCAO), followed by daily oral gavage of NBP (80 mg/kg) for 1, 7, or 21 consecutive days. Gene Expression Omnibus (GEO) dataset of IS patients peripheral blood RNA sequencing was analyzed to identify differentially expressed pyroptosis-related genes (PRGs) during the ischemic process. Our results suggested that NBP treatment effectively alleviated brain ischemic damage, resulting in decreased neurological deficit scores, reduced infarct volume, and improved neurological and behavioral functions. RNA sequence data from human unveiled upregulated PRGs in IS. Subsequently, we observed that NBP downregulated pyroptosis-associated markers at days 7 and 21 post-modeling, at both the protein and mRNA levels. Additionally, NBP suppressed the co-localization of pyroptosis markers with neuronal cells to variable degrees and simultaneously mitigated the accumulation of activated microglia. Overall, our data provide novel evidence that NBP treatment significantly attenuates ischemic brain damage and promotes recovery of neurological function in the early and recovery phases after IS, probably by negatively regulating the pyroptosis cell death of neuronal cells and inhibiting toxic neuroinflammation in the central nervous system.

Transcriptomic and neuroimaging data integration enhances machine learning classification of schizophrenia.

Schizophrenia is a polygenic disorder associated with changes in brain structure and function. Integrating macroscale brain features with microscale genetic data may provide a more complete overview of the disease etiology and may serve as potential diagnostic markers for schizophrenia. We aim to systematically evaluate the impact of multi-scale neuroimaging and transcriptomic data fusion in schizophrenia classification models. We collected brain imaging data and blood RNA sequencing data from 43 patients with schizophrenia and 60 age- and gender-matched healthy controls, and we extracted multi-omics features of macroscale brain morphology, brain structural and functional connectivity, and gene transcription of schizophrenia risk genes. Multi-scale data fusion was performed using a machine learning integration framework, together with several conventional machine learning methods and neural networks for patient classification. We found that multi-omics data fusion in conventional machine learning models achieved the highest accuracy (AUC ~0.76-0.92) in contrast to the single-modality models, with AUC improvements of 8.88 to 22.64%. Similar findings were observed for the neural network, showing an increase of 16.57% for the multimodal classification model (accuracy71.43%) compared to the single-modal average. In addition, we identified several brain regions in the left posterior cingulate and right frontal pole that made a major contribution to disease classification. We provide empirical evidence for the increased accuracy achieved by imaging genetic data integration in schizophrenia classification. Multi-scale data fusion holds promise for enhancing diagnostic precision, facilitating early detection and personalizing treatment regimens in schizophrenia.

Abstract 5001: Personalized neoantigen/cancer-testis antigen nanovaccine for advanced solid tumors, a single-arm, open-label pilot study

Abstract Background: Neoantigens, derived from somatic mutations in tumor cells, have been identified as promising targets of immunotherapy. A personalized neoantigen/cancer-testis antigen (CTA) nanovaccine (PNVAC) platform has been established by us previously, and demonstrated its feasibility, safety and efficacy in preventing recurrence of high-risk resected gastric/gastroesophageal junction (G/GEJ) cancer in both preclinical and clinical studies. This study aims to explore the universality of PNVAC monotherapy or combined with anti-angiogenesis drugs or anti-programmed death 1 (PD-1) in patients with multiple advanced solid malignancies. Methods: Patient-specific neoantigens were selected based on tumor-specific mutations identified by whole-exome sequencing (WES) and RNA sequencing of paired blood and tumor tissues. Bioinformatic analysis for neoantigen prediction, including sequencing read filtering, human leukocyte antigen (HLA) typing and neoantigen filtering was performed. PNVAC is an amphiphiles nanovaccine loaded with multiple personalized neoantigens designed to induce specific T cell responses. PNVAC is administrated to patients with metastatic solid tumors on days 1, 4, 8, 15, 22, 43 (prime phase) and 64, 85 and 169 (boost phase) alone, or combined with anti-angiogenesis or anti-PD-1 drugs. Safety, immunogenicity and clinical efficacy are evaluated. Results: Of the 30 enrolled patients, no treatment-related severe adverse events (AEs) occurred and the vast majority of AEs were limited to grade 1-2, only 1 patient developed grade 3 thrombocytopenia. The objective response rate (ORR) was 26.7% (8/30), including 2 cases of complete response (CR) and 6 cases of partial response (PR), and a disease control rate (DCR) of 66.7% achieved. The median progression-free survival (PFS) was 9.90 months (95% CI, 3.46-16.34 months), while the median overall survival (OS) was not reached (range, 0.80-43.53 months), and the estimated 1- and 2-year survival rates were 86.2% and 60.6%. Notably, among the 21 patients who had relapsed disease after previous ICB and/or anti-angiogenesis, disease control was asserted in 14 (66.7%) of them (7 of SD, 5 of PR, 2 of CR). ORR of this set was 33.3% (7/21), higher than that of all the enrolled patients (26.7%), and the set without prior ICB and/or anti-angiogenesis treatment (11.1%). For immune analysis, PNVAC elicited robust and persistent immune responses against neoantigens/CTAs. Additionally, peripheral T cells with a cytotoxic phenotype tended to increase after vaccination, and immune memory was also detected in some representative patients. Conclusions: These data supported the safety, immunogenicity, and efficacy of this regimen in patients with advanced solid malignancies, thus broadening the application and combined strategies of neoantigen-based vaccines. Clinical trial information: ChiCTR1800017319. Citation Format: Jia Wei, Qin Liu, Lijing Zhu, Jie Shao, Ju Yang, Guanghui Xu, Nandie Wu, Hanbing Wang, Rutian Li, Huizi Sha, Qiuping Xu, Jie Shen, Li Xie, Lifeng Wang, Juan Du, Lanqi Cen, Manman Tian, Lixia Yu, Baorui Liu. Personalized neoantigen/cancer-testis antigen nanovaccine for advanced solid tumors, a single-arm, open-label pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5001.

Abstract 2661: Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade

Abstract Background: Tertiary lymphoid structures (TLS) are ectopic lymphoid follicles that arise in non-lymphoid tissue. TLS may contribute to response to immune checkpoint blockade (ICB) in solid tumors, but understanding of the life cycle of these structures, particularly the circumstances of their resolution and the functional contribution of this stage to the adaptive immune response, remains incomplete. Methods: We employed a multi-omics approach to evaluate TLS in the tumors of patients with hepatocellular carcinoma (HCC) treated with neoadjuvant ICB (n = 19) and untreated controls (n = 14). TLS density was assessed by immunohistochemistry and correlated with pathologic response, relapse free survival, and overall survival. Imaging mass cytometry was used to characterize distinct TLS morphologies in areas of tumor regression bed and viable tumor. Individual TLS from treated tumors were microdissected and characterized by T cell receptor (TCR) and immunoglobulin heavy chain (IGH) sequencing, and TCR clonotypes identified in TLS were further characterized by comparison against matched single cell RNA and TCR sequencing of peripheral blood and TIL. Results: Intratumoral TLS density was significantly increased in HCC tumors treated with neoadjuvant ICB compared to untreated controls (p = 0.05). Tumors with major or complete pathologic response to treatment had significantly higher intratumoral TLS density compared to tumors with partial pathologic response (p = 0.000246) and non-response (p = 0.0129). In the treatment group, patients with tumors in the top tertile of intratumoral TLS density had significantly longer relapse free survival (p = 0.021 by log-rank test). In the regression bed of treated tumors, an involuted TLS morphology was identified which was notable for dispersion of the B cell germinal center, retention of a T cell zone enriched for T-DC interactions, and increased expression of markers of T cell memory. In a subset of patients, immune repertoire sequencing demonstrated increased TCRβ clonality and BCR somatic hypermutation at involuted TLS. Highly expanded T cell clonotypes identified in TLS were found in the GZMK+ and GZMB+ CD8 T effector memory clusters in matched single cell data. Conclusions: These data suggest that in HCC tumors treated with neoadjuvant ICB, treatment may induce formation of intratumoral TLS, which are associated with superior pathologic response and relapse free survival. An involuted TLS morphology is identified in tumor regression bed with features consistent with dissolution of the B cell germinal center, persistent antigen presentation to T cells, and increased T cell memory formation. Taken together, these findings suggest a potential role for late-stage TLS in supporting consolidation of the intratumoral T cell repertoire after elimination of viable tumor. Citation Format: Daniel H. Shu, Won Jin Ho, Luciane T. Kagohara, Alexander Girgis, Sarah M. Shin, Ludmila Danilova, Jae W. Lee, Dimitrios N. N. Sidiropoulos, Sarah Mitchell, Kabeer Munjal, Kathryn Howe, Kayla J. Bendinelli, Hanfei Qi, Guanglan Mo, Janelle Montagne, Tamara Y. Lopez-Vidal, Qingfeng Zhu, Amanda L. Huff, Xuan Yuan, Alexei Hernandez, Erin M. Coyne, Neeha Zaidi, Daniel J. Zabransky, Logan L. Engle, Aleksandra Ogurtsova, Marina Baretti, Daniel Laheru, Jennifer N. Durham, Hao Wang, Robert Anders, Elizabeth M. Jaffee, Elana J. Fertig, Mark Yarchoan. Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2661.

Abstract 5309: ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model

Abstract ATOR-4066 is a preclinical stage bispecific antibody targeting CD40 and CEACAM5, based on Alligator’s novel Neo-X-Prime™ antibody technology platform. ATOR-4066 induces a neoantigen specific T-cell response by simultaneous binding to CD40 on myeloid cells such as antigen presenting cells (APCs) and to CEACAM5 on tumor cells. CEACAM5 is a tumor-associated antigen highly expressed on several cancer indications such as colorectal and gastric cancer, as well as on tumor-derived material. ATOR-4066 treatment results in (1) CEACAM5-conditional CD40-mediated activation of APCs and (2) enhanced uptake of tumor-derived material, leading to cross-presentation of neoantigen and priming of neoantigen-specific T cells, resulting in enhanced tumor cell killing. We have previously shown evidence of a potent anti-tumor effect of ATOR-4066 in vivo, superior to the effects observed with the corresponding CD40 mAb. Moreover, we have demonstrated CEACAM5-dependent activation of CD40-expressing cells in vitro, and an ability to mediate co-localization of CEACAM5-expressing tumor debris and CD40 expressing APCs. In addition, ATOR-4066 was shown to induce CEACAM5-conditional activation of tumor-infiltrating CD40-expressing immune cells in dissociated primary human tumor material from colorectal and gastric cancer patients. Here we present data further elucidating the mode of action of ATOR-4066 in vivo. Following ATOR-4066 treatment, tumors and lymph nodes were isolated from human CD40 transgenic mice bearing MC38 tumors expressing human CEACAM5. Flow cytometry analysis shows an increased number of tumor-infiltrating immune cells in mice treated with ATOR-4066 compared to treatments with CD40 mAb or vehicle. The study also reveals upregulation of co-stimulatory molecules such as CD86 on DCs and macrophages after treatment with ATOR-4066 compared to groups treated with CD40 mAb or vehicle. The effect was localized to the tumor microenvironment and not observed in tumor draining lymph nodes, confirming previous in vitro results demonstrating CEACAM5-conditional immune activation. Moreover, RNA-sequencing of tumor samples displayed distinct transcriptomic alterations following ATOR-4066 treatment vs. CD40 mAb or vehicle treatment in vivo. In contrast, RNA-sequencing of peripheral blood in ATOR-4066 treated mice showed only small transcriptomic differences compared to the vehicle treated group, but distinct from mice treated with a CD40 mAb, further demonstrating the CEACAM5 dependent response of ATOR-4066. In summary, these preclinical data demonstrate that ATOR-4066 activates infiltrating myeloid cells resulting in increases in activated tumor infiltrating T cells, thus creating a pro-inflammatory tumor microenvironment, which strongly supports further development and clinical testing of ATOR-4066. Citation Format: Hampus Andersson, Ida Uddbäck, Mona Celander, Maria Johansson, Lill Ljung, Anneli Nilsson, Amulya Krishna Shetty, David Gomez Jimenez, Mattias Levin, Sara Fritzell, Laura von Schantz, Malin Lindstedt, Karin Hägerbrand, Anette Sundstedt, Peter Ellmark. ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5309.

Host gene expression signatures to identify infection type and organ dysfunction in children evaluated for sepsis: a multicentre cohort study

Sepsis is defined as dysregulated host response to infection that leads to life-threatening organ dysfunction. Biomarkers characterising the dysregulated host response in sepsis are lacking. We aimed to develop host gene expression signatures to predict organ dysfunction in children with bacterial or viral infection. This cohort study was done in emergency departments and intensive care units of four hospitals in Queensland, Australia, and recruited children aged 1 month to 17 years who, upon admission, underwent a diagnostic test, including blood cultures, for suspected sepsis. Whole-blood RNA sequencing of blood was performed with Illumina NovaSeq (San Diego, CA, USA). Samples with completed phenotyping, monitoring, and RNA extraction by March 31, 2020, were included in the discovery cohort; samples collected or completed thereafter and by Oct 27, 2021, constituted the Rapid Paediatric Infection Diagnosis in Sepsis (RAPIDS) internal validation cohort. An external validation cohort was assembled from RNA sequencing gene expression count data from the observational European Childhood Life-threatening Infectious Disease Study (EUCLIDS), which recruited children with severe infection in nine European countries between 2012 and 2016. Feature selection approaches were applied to derive novel gene signatures for disease class (bacterial vs viral infection) and disease severity (presence vs absence of organ dysfunction 24 h post-sampling). The primary endpoint was the presence of organ dysfunction 24 h after blood sampling in the presence of confirmed bacterial versus viral infection. Gene signature performance is reported as area under the receiver operating characteristic curves (AUCs) and 95% CI. Between Sept 25, 2017, and Oct 27, 2021, 907 patients were enrolled. Blood samples from 595 patients were included in the discovery cohort, and samples from 312 children were included in the RAPIDS validation cohort. We derived a ten-gene disease class signature that achieved an AUC of 94·1% (95% CI 90·6-97·7) in distinguishing bacterial from viral infections in the RAPIDS validation cohort. A ten-gene disease severity signature achieved an AUC of 82·2% (95% CI 76·3-88·1) in predicting organ dysfunction within 24 h of sampling in the RAPIDS validation cohort. Used in tandem, the disease class and disease severity signatures predicted organ dysfunction within 24 h of sampling with an AUC of 90·5% (95% CI 83·3-97·6) for patients with predicted bacterial infection and 94·7% (87·8-100·0) for patients with predicted viral infection. In the external EUCLIDS validation dataset (n=362), the disease class and disease severity predicted organ dysfunction at time of sampling with an AUC of 70·1% (95% CI 44·1-96·2) for patients with predicted bacterial infection and 69·6% (53·1-86·0) for patients with predicted viral infection. In children evaluated for sepsis, novel host transcriptomic signatures specific for bacterial and viral infection can identify dysregulated host response leading to organ dysfunction. Australian Government Medical Research Future Fund Genomic Health Futures Mission, Children's Hospital Foundation Queensland, Brisbane Diamantina Health Partners, Emergency Medicine Foundation, Gold Coast Hospital Foundation, Far North Queensland Foundation, Townsville Hospital and Health Services SERTA Grant, and Australian Infectious Diseases Research Centre.

Novel homozygous ADK out-of-frame deletion causes adenosine kinase deficiency with rare phenotypes of sepsis, metabolites disruption and neutrophil dysfunction

Adenosine kinase deficiency (OMIM #614300) is a type of inborn errors of metabolism with multiorgan symptoms primarily neurological disorders, hepatic impairment, global developmental delay, and mild dysmorphism. The genetic causes of adenosine kinase deficiency are homozygous or compound heterozygous loss-of-function variants of ADK. To date, fewer than 25 cases of adenosine kinase deficiency have been reported worldwide and none have been reported in China. In this research, trio whole-exome sequencing (Trio-WES) identified a novel homozygous ADK (NM_001123.4) out-of-frame deletion, c.518_519delCA (p.Thr173Serfs*15), in a Chinese patient with rare phenotypes of sepsis, metabolites disruption and neutrophil dysfunction. This variant was dysfunctional, with marked reduction of ADK level in both the patient’s peripheral blood and cells transfected with the corresponding variant. Additionally, metabolomics detected by high-throughput mass spectrometry showed disturbances in the methionine (Met) and energy pathway. RNA sequencing (RNA-seq) of the patient’s peripheral blood suggested a defective anti-inflammatory response characterized by impaired neutrophil activation, migration, and degranulation, which might be the primary cause for the sepsis. To our knowledge, we identified the first Chinese patient of adenosine kinase deficiency with a novel homozygous out-of-frame deletion in ADK causing multiorgan disorders, metabolites disruption, rare phenotypes of sepsis, and neutrophil dysfunction. Our findings broaden the genetic spectrum and pathogenic mechanisms of adenosine kinase deficiency.

MiR-23a-5p alleviates chronic obstructive pulmonary disease through targeted regulation of RAGE-ROS pathway

BackgroundChronic obstructive pulmonary disease (COPD) is a common respiratory disease and represents the third leading cause of death worldwide. This study aimed to investigate miRNA regulation of Receptor for Advanced Glycation End-products (RAGE), a causal receptor in the pathogenesis of cigarette smoke (CS)-related COPD, to guide development of therapeutic strategies.MethodsRAGE expression was quantified in lung tissue of COPD patients and healthy controls, and in mice with CS-induced COPD. RNA-sequencing of peripheral blood from COPD patients with binding site prediction was used to screen differentially expressed miRNAs that may interact with RAGE. Investigation of miR-23a-5p as a potential regulator of COPD progression was conducted with miR-23a-5p agomir in COPD mice in vivo using histology and SCIREQ functional assays, while miR-23a-5p mimics or RAGE inhibitor were applied in 16-HBE human bronchial epithelial cells in vitro. RNA-sequencing, ELISA, and standard molecular techniques were used to characterize downstream signaling pathways in COPD mice and 16-HBE cells treated with cigarette smoke extract (CSE).ResultsRAGE expression is significantly increased in lung tissue of COPD patients, COPD model mice, and CSE-treated 16-HBE cells, while inhibiting RAGE expression significantly reduces COPD severity in mice. RNA-seq analysis of peripheral blood from COPD patients identified miR-23a-5p as the most significant candidate miRNA interaction partner of RAGE, and miR-23a-5p is significantly downregulated in mice and cells treated with CS or CSE, respectively. Injection of miR-23a-5p agomir leads to significantly reduced airway inflammation and alleviation of symptoms in COPD mice, while overexpressing miR-23a-5p leads to improved lung function. RNA-seq with validation confirmed that reactive oxygen species (ROS) signaling is increased under CSE-induced aberrant upregulation of RAGE, and suppressed in CSE-stimulated cells treated with miR-23a-5p mimics or overexpression. ERK phosphorylation and subsequent cytokine production was also increased under RAGE activation, but inhibited by increasing miR-23a-5p levels, implying that the miR-23a-5p/RAGE/ROS axis mediates COPD pathogenesis via ERK activation.ConclusionsThis study identifies a miR-23a-5p/RAGE/ROS signaling axis required for pathogenesis of COPD. MiR-23a-5p functions as a negative regulator of RAGE and downstream activation of ROS signaling, and can inhibit COPD progression in vitro and in vivo, suggesting therapeutic targets to improve COPD treatment.Graphical

Plasma Protein Biomarkers Distinguish Multisystem Inflammatory Syndrome in Children From Other Pediatric Infectious and Inflammatory Diseases.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but serious hyperinflammatory complication following infection with severe acute respiratory syndrome coronavirus 2. The mechanisms underpinning the pathophysiology of MIS-C are poorly understood. Moreover, clinically distinguishing MIS-C from other childhood infectious and inflammatory conditions, such as Kawasaki disease or severe bacterial and viral infections, is challenging due to overlapping clinical and laboratory features. We aimed to determine a set of plasma protein biomarkers that could discriminate MIS-C from those other diseases. Seven candidate protein biomarkers for MIS-C were selected based on literature and from whole blood RNA sequencing data from patients with MIS-C and other diseases. Plasma concentrations of ARG1, CCL20, CD163, CORIN, CXCL9, PCSK9 and ADAMTS2 were quantified in MIS-C (n = 22), Kawasaki disease (n = 23), definite bacterial (n = 28) and viral (n = 27) disease and healthy controls (n = 8). Logistic regression models were used to determine the discriminatory ability of individual proteins and protein combinations to identify MIS-C and association with severity of illness. Plasma levels of CD163, CXCL9 and PCSK9 were significantly elevated in MIS-C with a combined area under the receiver operating characteristic curve of 85.7% (95% confidence interval: 76.6%-94.8%) for discriminating MIS-C from other childhood diseases. Lower ARG1 and CORIN plasma levels were significantly associated with severe MIS-C cases requiring inotropes, pediatric intensive care unit admission or with shock. Our findings demonstrate the feasibility of a host protein biomarker signature for MIS-C and may provide new insight into its pathophysiology.

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we propose that RNA-seq should be considered a diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers insights into a patient’s immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 196 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that—combined with sequence alignments and BLASTp—they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Host transcriptomics and machine learning for secondary bacterial infections in patients with COVID-19: a prospective, observational cohort study

Viral respiratory tract infections are frequently complicated by secondary bacterial infections. This studyaimed to use machine learning to predict the risk of bacterial superinfection in SARS-CoV-2-positive individuals. In this prospective, multicentre, observational cohort study done in nine centres in six countries (Australia, Indonesia, Singapore, Italy, Czechia, and France) blood samples and RNA sequencing were used to develop a robust model of predicting secondary bacterial infections in the respiratory tract of patients with COVID-19. Eligible participants were older than 18 years, had known or suspected COVID-19, and symptoms of a recent respiratory infection. A control cohort of participants without COVID-19 who were older than 18 years and with no infection symptoms was also recruited from one Australian centre. In the pre-analysis phase, data were filtered to include only individuals with complete blood transcriptomics and patient data (ie, age, sex, location, and WHO severity score at the time of sample collection). The dataset was then divided randomly (4:1) into a training set (80%) and a test set (20%). Gene expression data in the training set and control cohort were used for differential expression analysis. Differentially expressed genes, along with WHO severity score, location, age, and sex, were used for feature selection with least absolute shrinkage and selection operator (LASSO) in the training set. For LASSO analysis, samples were excluded if gene expression data were not obtained at study admission, no longitudinal clinical information was available, a bacterial infection at the time of study admission was present, or a fungal infection in the absence of a bacterial infection was detected. LASSO regression was performed using three subsets of predictor variables: patient data alone, gene expression data alone, or a combination of patient data and gene expression data. The accuracy of the resultant models was tested on data from the test set. Between March, 2020, and October, 2021, we recruited 536 SARS-CoV-2-positive individuals and between June, 2013, and January, 2020, we recruited 74 participants into the control cohort. After prefiltering analysis and other exclusions, samples from 158 individuals were analysed in the training set and 47 in the test set. The expression of seven host genes (DAPP1, CST3, FGL2, GCH1, CIITA, UPP1, and RN7SL1) in the blood at the time of study admission was identified by LASSO as predictive of the risk of developing a secondary bacterial infection of the respiratory tract more than 24 h after study admission. Specifically, the expression of these genes in combination with a patient's WHO severity score at the time of study enrolment resulted in an area under the curve of 0·98 (95% CI 0·89-1·00), a true positive rate (sensitivity) of 1·00 (95% CI 1·00-1·00), and a true negative rate (specificity) of 0·94 (95% CI 0·89-1·00) in the test cohort. The combination of patient data and host transcriptomics at hospital admission identified all seven individuals in the training and test sets who developed a bacterial infection of the respiratory tract 5-9 days after hospital admission. These data raise the possibility that host transcriptomics at the time of clinical presentation, together with machine learning, can forward predict the risk of secondary bacterial infections and allow for the more targeted use of antibiotics in viral infection. Snow Medical Research Foundation, the National Health and Medical Research Council, the Jack Ma Foundation, the Helmholtz-Association, the A2 Milk Company, National Institute of Allergy and Infectious Disease, and the Fondazione AIRC Associazione Italiana per la Ricerca contro il Cancro.

Whole blood transcriptome analysis for age- and gender-specific gene expression profiling in Japanese individuals.

Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20-30s and 60-70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population.

Single-cell RNA sequencing in donor and end-stage heart failure patients identifies NLRP3 as a therapeutic target for arrhythmogenic right ventricular cardiomyopathy

BackgroundDilation may be the first right ventricular change and accelerates the progression of threatening ventricular tachyarrhythmias and heart failure for patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), but the treatment for right ventricular dilation remains limited.MethodsSingle-cell RNA sequencing (scRNA-seq) of blood and biventricular myocardium from 8 study participants was performed, including 6 end-stage heart failure patients with ARVC and 2 normal controls. ScRNA-seq data was then deeply analyzed, including cluster annotation, cellular proportion calculation, and characterization of cellular developmental trajectories and interactions. An integrative analysis of our single-cell data and published genome-wide association study-based data provided insights into the cell-specific contributions to the cardiac arrhythmia phenotype of ARVC. Desmoglein 2 (Dsg2)mut/mut mice were used as the ARVC model to verify the therapeutic effects of pharmacological intervention on identified cellular cluster.ResultsRight ventricle of ARVC was enriched of CCL3+ proinflammatory macrophages and TNMD+ fibroblasts. Fibroblasts were preferentially affected in ARVC and perturbations associated with ARVC overlap with those reside in genetic variants associated with cardiac arrhythmia. Proinflammatory macrophages strongly interact with fibroblast. Pharmacological inhibition of Nod-like receptor protein 3 (NLRP3), a transcriptional factor predominantly expressed by the CCL3+ proinflammatory macrophages and several other myeloid subclusters, could significantly alleviate right ventricular dilation and dysfunction in Dsg2mut/mut mice (an ARVC mouse model).ConclusionsThis study provided a comprehensive analysis of the lineage-specific changes in the blood and myocardium from ARVC patients at a single-cell resolution. Pharmacological inhibition of NLRP3 could prevent right ventricular dilation and dysfunction of mice with ARVC.