Gene Expression Profiles Of Peripheral Blood Mononuclear Cells Research Articles

Gene Expression Analysis of Immune Regulatory Genes in Circulating Tumour Cells and Peripheral Blood Mononuclear Cells in Patients with Colorectal Carcinoma.

Information regarding genetic alterations of driver cancer genes in circulating tumour cells (CTCs) and their surrounding immune microenvironment nowadays can be employed as a real-time monitoring platform for translational applications such as patient response to therapeutic targets, including immunotherapy. This study aimed to investigate the expression profiling of these genes along with immunotherapeutic target molecules in CTCs and peripheral blood mononuclear cells (PBMCs) in patients with colorectal carcinoma (CRC). Expression of p53, APC, KRAS, c-Myc, and immunotherapeutic target molecules PD-L1, CTLA-4, and CD47 in CTCs and PBMCs were analysed by qPCR. Their expression in high versus low CTC-positive patients with CRC was compared and clinicopathological correlations between these patient groups were analysed. CTCs were detected in 61% (38 of 62) of patients with CRC. The presence of higher numbers of CTCs was significantly correlated with advanced cancer stages (p = 0.045) and the subtypes of adenocarcinoma (conventional vs. mucinous, p = 0.019), while being weakly correlated with tumour size (p = 0.051). Patients with lower numbers of CTCs had higher expression of KRAS. Higher KRAS expression in CTCs was negatively correlated with tumour perforation (p = 0.029), lymph node status (p = 0.037), distant metastasis (p = 0.046) and overall staging (p = 0.004). CTLA-4 was highly expressed in both CTCs and PBMCs. In addition, CTLA-4 expression was positively correlated with KRAS (r = 0.6878, p = 0.002) in the enriched CTC fraction. Dysregulation of KRAS in CTCs might evade the immune system by altering the expression of CTLA-4, providing new insights into the selection of therapeutic targets at the onset of the disease. Monitoring CTCs counts, as well as gene expression profiling of PBMCs, can be helpful in predicting tumour progression, patient outcome and treatment.

Integrated whole-genome gene expression analysis reveals an atlas of dynamic immune landscapes after myocardial infarction.

Myocardial infarction (MI) is a deadly medical condition leading to irreversible damage to the inflicted cardiac tissue. Elevated inflammatory response marks the severity of MI and is associated with the development of heart failure (HF), a long-term adverse outcome of MI. However, the efficacy of anti-inflammatory therapies for MI remains controversial. Deciphering the dynamic transcriptional signatures in peripheral blood mononuclear cells (PBMCs) is a viable and translatable route to better understand post-MI inflammation, which may help guide post-MI anti-inflammatory treatments. In this work, integrated whole-genome gene expression analysis was performed to explore dynamic immune landscapes associated with MI. GSEA and GSVA showed that pathways involved in the inflammatory response and metabolic reprogramming were significantly enriched in PBMCs from MI patients. Based on leukocyte profiles generated by xCell algorithm, the relative abundance of monocytes and neutrophils was significantly increased in PBMCs from MI patients and had positive correlations with typical inflammation-associated transcripts. Mfuzz clustering revealed temporal gene expression profiles of PBMCs during the 6-month post-MI follow-up. Analysis of DEGs and gene sets indicated that PBMCs from HF group were characterized by elevated and lasting expression of genes implicated in inflammation and coagulation. Consensus clustering generated 4 metabolic subtypes of PBMCs with molecular heterogeneity in HF patients. In summary, integrated whole-genome gene expression analysis here outlines a transcriptomic framework that may improve the understanding of dynamic signatures present in PBMCs, as well as the heterogeneity of PBMCs in MI patients with or without long-term clinical outcome of HF. Moreover, the work here uncovers the diversity and heterogeneity of PBMCs from HF patients, providing novel bioinformatic evidence supporting the mechanistic implications of metabolic reprogramming and mitochondrial dysfunction in the post-MI inflammation and HF. Therefore, our work here supports the notion that individualized anti-inflammatory therapies are needed to improve the clinical management of post-MI patients.

Peripheral blood mononuclear cells (PBMCs), an ideal liquid biopsy approach to evaluate systematic immunity and predict response of neoadjuvant chemo-immunotherapy in resectable NSCLC.

e20618 Background: Immune checkpoint inhibitors (ICIs) dramatically improved clinical outcomes of patients with resectable non-small cell lung cancer (NSCLC). However, ICIs are not the scenario of “One-size fits to all”. It is of great importance to explore biomarkers, especially via liquid biopsy-based approach, to predict or monitor treatment response. Besides comprehensive genomic profiling of primary tumor tissue, this study focused on gene expression profile of PBMCs at baseline as well as one week post 1st immunotherapy administration, and its association with major pathological response (MPR) of neoadjuvant chem-immunotherapy in resectable NSCLC patients. Methods: Pre-treatment tumor biopsies and PBMC at baseline (D0) and 7 days after neoadjuvant chem-immunotherapy (D7) were collected from 27 patients with stage IB-IIIB NSCLC from two prospective clinical trials (NCT04422392 and NCT04762030) between Aug 2019 and July 2021. Tumor biopsies were subjected for comprehensive genomic profiling by an NGS panel covering 571 cancer related genes, and RNA from PBMC was subjected to an RNA sequencing panel covering transcripts of 2660 genes. (Amoy Diagnostics, Xiamen, China). Single sample gene set enrichment analysis (ssGSEA) was used to assess enrichment of inflammatory-related gene sets. CIBERSORT and in-housed established DAISM-DNN algorithm were applied to estimate immune cell populations from PBMC-derived gene expression profile. Results: Patients received chemo-immunotherapy achieved 59.3% of MPR. Stronger signatures including effector T cell and IFN-γ/Effector T-cell were significantly enriched in baseline PBMC samples in MPR patients (p = 0.028, p = 0.042 respectively). In parallel, significant increasing Naïve CD8 positive T cells was observed in PBMC at D7 in MPR patients by both CIBERSORT (p = 0.047) and DAISM-DNN algorithm (p = 0.018). Interestingly, at D7, signature of M0 macrophage was enriched in PBMC from patients with non-MPR. In addition, comprehensive genomic profiling indicated a numerically higher level of tumor mutation burden (TMB) in pretreatment tumor samples in MPR patients (Median TMB, MPR 11.51 mut/Mb vs Non-MPR 7.19 mut/Mb, p = 0.074). Conclusions: Expression profile of baseline PBMC as well as dynamics of PBMC profile in a short period (7 days) may predict the clinical efficacy of neoadjuvant chemo-immunotherapy. More patients are being enrolled into this study. Moreover, to validate these findings, large-scale and prospective investigations are warranted. Clinical trial information: NCT04422392 and NCT04762030.

The Key Genes Underlying Pathophysiology Correlation Between the Acute Myocardial Infarction and COVID-19

IntroductionAccumulating evidences disclose that COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a marked effect on acute myocardial infarction (AMI). Nevertheless, the underlying pathophysiology correlation between the AMI and COVID-19 remains vague.Materials and MethodsBioinformatics analyses of the altered transcriptional profiling of peripheral blood mononuclear cells (PBMCs) in patients with AMI and COVID-19 were implemented, including identification of differentially expressed genes and common genes between AMI and COVID-19, protein–protein interactions, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, TF-genes and miRNA coregulatory networks, to explore their biological functions and potential roles in the pathogenesis of COVID-19-related AMI.ConclusionOur bioinformatic analyses of gene expression profiling of PBMCs in patients with AMI and COVID-19 provide us with a unique view regarding underlying pathophysiology correlation between the two vital diseases.

Gene expression profile of peripheral blood mononuclear cells in mild to moderate obesity in dogs

BackgroundMolecular mechanisms and early diagnosis on the development of mild to moderate of canine obesity are not understood although recent dog obesity is a widespread problem. To understand the differences between normal weight and mild to moderate obesity, the purpose of this study is to investigate the gene expression profiles of peripheral blood mononuclear cells (PBMC) in dogs. MethodsThis study comprised a sample of 12 privately-owned Miniature Dachshund, which were divided into two groups (obese and control) based on body condition scores (BCS). Serum biochemical parameters and PBMC gene expression profiles were compared between groups. ResultsA statistically significant between group differences was recorded for body weight (BW), BCS, serum Insulin and triglyceride (TG) levels (p < 0.05). RNA-seq revealed the upregulated 154 genes and the downregulated 198 genes in obese dogs at more than 3.5-fold change compared with control animals. Hemoglobin subunits alpha- and beta-like were detected in the downregulated genes. RT-PCR analysis showed downregulation of FOLH1, ALAS2 and LOC100855540 genes, and upregulation of BCL2L15 gene, suggesting that the metabolic difference between normal and mild to moderate obesity was involved in the hemoglobin metabolism. ConclusionsThis study revealed significant differences in the gene expression of BCL2L15, FOLH1, ALAS2, and hemoglobin subunits such as LOC100855540 between normal weight and mild to moderate obese dogs, which indicate that these genes may prevent the obesity in dogs and be potentially useful for diagnosis of mild to moderate obesity.

Transcriptomic profiling of peripheral blood mononuclear cells in patients with multiple primary melanoma.

e21558 Background: Patients (PTs) with cutaneous melanoma (MEL) are at increased risk of developing subsequent MEL. The primary objective of the present study was to determine whether the peripheral blood gene expression profile (GEP) of PTs with multiple primary melanoma (MPM) differs from that of patients with single primary melanoma (SPM) and healthy controls (HCs). A secondary objective is to assess if the peripheral blood immune transcriptional profile varies in relation with clinical and immunopathological characteristics of MEL. Methods: Cases with MPM, SPM and HCs were retrieved from the University of Pittsburgh Human Biological Sample and Nevus Image Banking and Analysis Repository. NanoString PanCancer Immune Profiling of 770-immune related genes was used to define GEP in peripheral blood mononuclear cells (PBMCs). Quality control (QC), data normalization, and differential expression analyses were performed using the ROSALIND Analysis System. Genes with fold change ≥1.5 and p-value &lt; 0.05 were considered as differentially-expressed between cohorts. Results: In total, 47 PTs including 10 with SPM, 40 with MPM, and 10 HCs, were included. GEP data from 3 cases did not meet QC requirements and were discarded. The majority of PTs had stage I/II disease (87.2%). The median Breslow thickness was 1 mm, ulceration was present in 6 (12.2%) MELs. Tumor-infiltrating lymphocytes (TILs) were absent, non-brisk, and brisk in 20%, 65%, 15% of cases, respectively. The expression of IFIT1, MX1, ISG15 was decreased in PTs with MPM in comparison to HCs. In comparison to SPMs, PTs with MPM demonstrated downregulation of B-cell receptor signaling pathway genes (CD19, CD22, CD79A, BLK). 14 genes involved in TNF-alpha effects on cytokine activity, cell motility, and apoptosis, and 12 genes in the IL18 signaling pathway were differentially expressed in PTs with Breslow thickness of &gt;1mm vs ≤1mm. The top downregulated genes in &gt;1mm thick MELs were CD83, VEGFA and CXCL3. Ulcerated MEL had decreased expression of CCL2, CCL3, CCL3L1, CCL4. Stage III MEL had decreased expression of GZMH, MHC Class II genes HLA-DMB, HLA-DRB3, lectin receptor family genes KLRC2 and KLRD1, as well as Type-1 immune-associated biomarkers IRF1, SYK, TBX21 in comparison to Stage I/II cases. Tumors deficient in TILs exhibited decreased expression of Granzyme A and B, T-box 21, and IL18RAP (enhances IL18R-mediated signaling) in peripheral blood. Tumors with brisk TILs had increased expression of the B-cell associated transcripts CD19, CD22, CD79A in peripheral blood. Conclusions: The GEP of PBMC's from PTs with MPM was similar overall to that of PTs with SPM/HC, with only a subset of genes exhibiting differentially expression in PTs with MPM vs. PTs with SPM and HCs. GEP varied in relation to Breslow thickness, stage, ulceration, and TIL content in MELs.

Gene expression responses to Zika virus infection in peripheral blood mononuclear cells from pregnant and non-pregnant women.

Congenital Zika syndrome is caused by mother‐to‐fetus transmission of the Zika virus (ZIKV). Peripheral blood mononuclear cells (PBMCs) are permissive to ZIKV infection and may carry ZIKV to the placenta. To identify pregnancy‐related differences in PBMC responses against ZIKV infection, we compared gene expression profiles of ZIKV‐infected and non‐infected PBMCs cultured from pregnant and non‐pregnant women. ZIKV‐infected pregnant conditions generally overexpressed M1‐shifted pro‐inflammatory responses and underexpressed M2‐shifted anti‐inflammatory responses. Additionally, transcripts involved in osteoclast differentiation and cardiac myopathies were upregulated following ZIKV infection. Our results suggest potential roles of pregnancy‐induced immune dysregulation in shaping neonatal pathology associated with ZIKV infection.

Characterization of peripheral blood mononuclear cells gene expression profiles of pediatric Staphylococcus aureus persistent and non-carriers using a targeted assay

Defects in innate immunity affect many different physiologic systems and several studies of patients with primary immunodeficiency disorders demonstrated the importance of innate immune system components in disease prevention or colonization of bacterial pathogens. To assess the role of the innate immune system on nasal colonization with Staphylococcus aureus, innate immune responses in pediatric S. aureus nasal persistent carriers (n = 14) and non-carriers (n = 15) were profiled by analyzing co-clustered gene sets (modules). We stimulated previously frozen peripheral blood mononuclear cells (PBMCs) from these subjects with i) a panel of TLR ligands, ii) live S. aureus (either a mixture of strains or stimulation with respective carriage isolates), or iii) heat-killed S. aureus. We found no difference in responses between carriers and non-carriers when PBMCs were stimulated with a panel of TLR ligands. However, PBMC gene expression profiles differed between persistent and non-S. aureus carriers following stimulation with either live or dead S. aureus. These observations suggest that individuals susceptible to persistent carriage with S. aureus may possess differences in their live/dead bacteria recognition pathway and that innate pathway signaling is different between persistent and non-carriers of S. aureus.

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include Up-Regulated Innate Immune Modulators

Formation of pathological anti-FVIII antibodies, or “inhibitors,” is the most serious complication of therapeutic FVIII infusions, affecting up to 1/3 of severe Hemophilia A (HA) patients. Inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development vs. immune tolerance, achieved with or without Immune Tolerance Induction (ITI) therapy, are not well-understood. To address these questions, temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) was carried out for HA subjects with and without a current or historic inhibitor using RNA-Seq. PBMCs were isolated from 40 subjects in the following groups: HA with an inhibitor that resolved either following ITI or spontaneously; HA with a current inhibitor; HA with no inhibitor history and non-HA controls. PBMCs were stimulated with 5 nM FVIII and RNA was isolated 4, 16, 24, and 48 h following stimulation. Time-series differential expression analysis was performed and distinct transcriptional signatures were identified for each group, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. Subjects with a current inhibitor showed differential expression of 56 genes and a clustering analysis identified three major temporal profiles. Interestingly, gene ontology enrichments featured innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A, and COLEC12. NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1β and TNFα, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. RNA-Seq results were validated by RT-qPCR, ELISAs, multiplex cytokine analysis, and flow cytometry. The inflammatory status of HA patients suffering from an ongoing inhibitor includes up-regulated innate immune modulators, which may act as ongoing danger signals that influence the responses to, and eventual outcomes of, ITI therapy.

Comprehensive Transcriptome Profiling of Peripheral Blood Mononuclear Cells from Patients with Sepsis

Background: Sepsis, as a clinical emergency, usually causes multiorgan dysfunction and can lead to high mortality. Establishment of specific and sensitive biomarkers for early diagnosis is critical to identify patients who would benefit from targeted therapy. In this study, we investigated this syndrome by analyzing the transcriptome of peripheral blood mononuclear cells (PBMCs) from patients with sepsis and identified sepsis-specific biomarkers.Methods: In this study, a total of 87 patients with sepsis and 40 healthy controls from a prospective multicenter cohort were enrolled. Samples from 44 subjects (24 patients with sepsis and 20 healthy controls) were sequenced and the remaining patients were included in the validation group. Using high-throughput sequencing, a gene expression profile of PBMCs from patients with sepsis was generated to elucidate the pathophysiology of sepsis and identify sepsis-specific biomarkers.Results: Principal component analysis (PCA) and unsupervised hierarchical cluster analysis showed that patients with sepsis separated from healthy controls. A total of 1639 differentially expressed genes (DEGs) were identified (|log2 fold change|>2, adjusted P value <0.05) between these two groups, with 1278 (78.0%) upregulated and 361 (22.0%) downregulated in patients with sepsis. Gene Ontology (GO) analysis of the upregulated DEGs identified 194 GO terms that were clustered into 27 groups, and analysis of the downregulated DEGs identified 20 GO terms that were clustered into 4 groups. Four unique genes were identified that could be predictive of patients with sepsis. External validation of the four genes using quantitative real-time polymerase chain reaction (qRT-PCR) was consistent with the results of mRNA sequencing, revealing their potential in sepsis diagnosis.Conclusions: The transcriptome characteristics of PBMCs, which were significantly altered in sepsis patients, provide new insights into sepsis pathogenesis. The four identified gene expression changes differentiated patients with sepsis from healthy subjects, which could serve as a convenient tool contributing to sepsis diagnosis.

PBMC gene expression profiles of female Bangladeshi adults chronically exposed to arsenic-contaminated drinking water

Arsenic, a class I human carcinogen, is ubiquitously found throughout the environment and around the globe, posing a great public health concern. Notably, Bangladesh and regions of West Bengal have been found to have high levels (0.5–4600 μg/L) of arsenic drinking water contamination, and approximately 50 million of the world’s 200 million people chronically exposed to arsenic in Bangladesh alone. This study was carried out to examine genome-wide gene expression changes in individuals chronically exposed to arsenic-contaminated drinking water. Our study population includes twenty-nine Bangladeshi female participants with urinary arsenic levels ranging from 22.32 to 1828.12 μg/g creatinine. RNA extracted from peripheral blood mononuclear cells (PBMCs) were evaluated using RNA-Sequencing analysis. Our results indicate that a total of 1,054 genes were significantly associated with increasing urinary arsenic levels (FDR p < 0.05), which include 418 down-regulated and 636 up-regulated genes. Further Ingenuity Pathway Analysis revealed potential target genes (DAPK1, EGR2, APP), microRNAs (miR-155, -338, −210) and pathways (NOTCH signaling pathway) related to arsenic carcinogenesis. The selection of female-only participants provides a homogenous study population since arsenic has significant sex dependent effects, and the wide exposure range provides new insight for key gene expression changes that correlate with increasing urinary arsenic levels.

Whole blood vs PBMC: compartmental differences in gene expression profiling exemplified in asthma

BackgroundBlood has proven to be a useful resource for molecular analysis in numerous biomedical studies, with peripheral blood mononuclear cells (PBMCs) and whole blood being the major specimen types. However, comparative analyses between these two major compartments (PBMCs and whole blood) are few and far between. In this study, we compared gene expression profiles of PBMCs and whole blood samples obtained from research subjects with or without mild allergic asthma.MethodsWhole blood (PAXgene) and PBMC samples were obtained from 5 mild allergic asthmatics and 5 healthy controls. RNA from both sample types was measured for expression of 730 immune-related genes using the NanoString nCounter platform.ResultsWe identified 64 uniquely expressed transcripts in whole blood that reflected a variety of innate, humoral, and adaptive immune processes, and 13 uniquely expressed transcripts in PBMCs which were representative of T-cell and monocyte-mediated processes. Furthermore, analysis of mild allergic asthmatics versus non-asthmatics revealed 47 differentially expressed transcripts in whole blood compared to 1 differentially expressed transcript in PBMCs (FDR < 0.25). Finally, through simultaneous measurement of PBMC proteins on the nCounter assay, we identified CD28 and OX40 (TNFRSF4), both of which are critical co-stimulatory molecules during T-cell activation, as significantly upregulated in asthmatics.ConclusionsWhole blood RNA preserved in PAXgene tubes is excellent for producing gene expression data with minimal variability and good sensitivity, suggesting its utility in multi-centre studies requiring measurement of blood gene expression.

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include up-Regulated Innate Immune Modulators

The formation of pathological anti-FVIII antibodies, referred to as "inhibitors", is the most serious complication of therapeutic FVIII infusions, affecting up to one third of severe Hemophilia A (HA) patients. Intensive FVIII therapy, i.e. "Immune Tolerance Induction" (ITI), enables ~2/3 of treated patients to achieve peripheral tolerance to FVIII. FVIII inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development versus immune tolerance, achieved with or without ITI therapy, are not well understood. To address these questions, we carried out temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) from HA subjects with and without a current or historic inhibitor using RNA-seq. PBMCs were isolated from 40 subjects in the following groups: (A) HA with an inhibitor that resolved either following ITI or spontaneously; (B) HA with a current inhibitor; (C) HA with no inhibitor history and (D) non-HA healthy controls. PBMCs were rested overnight and then stimulated with 5 nM FVIII, and total RNA was isolated 4, 16, 24 and 48 hours following stimulation. RNA from unstimulated cells at t = 4 hrs served as a negative control. Time-series differential expression analysis was performed with DESeq2 and genes with a log likelihood ratio test FDR &amp;lt;0.05 and an absolute fold change &amp;gt;1.25 at at least one stimulation time point compared to control were deemed significant. Subjects with a resolved past inhibitor (Group A) showed differential expression of only 15 genes. In contrast, subjects with a current inhibitor (Group B) showed differential expression of 56 genes. A clustering analysis divided the temporal trajectories of Group B genes into 3 distinct clusters. Twenty-three genes were up-regulated at 16 hr and 21 genes at 48 hr post-stimulation, respectively. Interestingly, gene ontology (GO) enrichment analysis of these genes revealed enrichments for innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A and COLEC12.NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1beta and TNF-alpha, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. Expression levels of NLRP3, TLR8, CLEC10A and IL32 transcripts were validated by real time PCR, and changes in RNA transcript abundances correlated well with the RNA-seq results. IL-32 results were validated by both RT-qPCR on an aliquot of the original RNA sample and ELISA to measure the cytokine in supernatants at t=48 hrs. HA subjects with no inhibitor history (Group C) had 195 differentially expressed genes whose temporal profiles fell into 4 distinct clusters. GO enrichment analysis revealed biological processes related to epithelial cell proliferation, responses to toxic substances, and positive/negative regulation of cytokine secretion (TNF, NQO1, PMEPA1). The non-HA healthy control subjects (Group D) also showed cellular responses to ex vivo FVIII stimulation. A total of 63 differentially regulated genes fell into 4 distinct clusters. GO analysis identified expression patterns associated with leukocyte-mediated immunity, T-cell activation, and a hypoxia response. Overall, distinct transcriptional signatures were identified for each of the four groups, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. We are currently characterizing PBMC immune cell subsets, e.g. macrophages and CD4+ T cells, to identify specific cell types responsible for the differentially regulated genes. Cellular responses of tolerized HA subjects and healthy non-HA controls were consistent with the known immunogenicity of FVIII, including persistence of FVIII-specific CD4+ T cells even in individuals with no measurable FVIII inhibitor. The inflammatory status of HA patients suffering from an ongoing inhibitor clearly includes up-regulation of innate immune modulators, some of which may act as ongoing danger signals that influence the responses to, and eventual outcome of, ITI therapy. Disclosures Pratt: Grifols, Inc: Research Funding; Bloodworks NW: Patents &amp; Royalties: inventor on patents related to FVIII immunogenicity.

Two Distinct Subtypes Revealed in Blood Transcriptome of Breast Cancer Patients With an Unsupervised Analysis.

Background: Breast cancer (BC) is a highly heterogeneous cancer. The interaction between immune system and BC is complex, widespread yet unclear. In this study, we aimed to reveal the heterogeneity of host systemic immune response to BC and understand the possible mechanisms that may drive the heterogeneity using transcriptomic data from peripheral blood mononuclear cells (PBMCs).Methods: Transcriptome-wide gene expressions of PBMCs in 33 BC patients were generated by RNA sequencing. An unsupervised clustering algorithm was employed to discover PBMC transcriptome subtypes among BC patients. Association analysis between PBMC subtypes and age, clinical stage, abundance of immune cells, and other clinical factors was performed to understand the underlying biological processes that may drive this heterogeneity. Immune gene signature identification and in silico survival analysis were performed to investigate the potential clinical implications of these PBMC subtypes. The findings were validated using the whole blood transcriptomes of an independent cohort.Results: We observed that established BC subtypes were not associated with PBMC gene expression profiles. Instead, we discovered and validated two new BC subtypes using PBMC transcriptome, which have distinct immune cell proportions, especially for lymphocytes (P = 5.22 × 10−12) and neutrophils (P = 1.13 × 10−14). Enrichment analysis of differentially expressed genes revealed that these two subtypes had distinct patterns of immune responses, including osteoclast differentiation and interleukin-10 signaling pathway. We developed two immune gene signatures that can differentiate these two BC PBMC subtypes. Further analysis suggested they had the ability to predict the clinical outcome of BC patients.Conclusions: PBMC transcriptome profiles can classify BC patients into two distinct subtypes. These two subtypes are mainly shaped by different immune cell abundance, which may have implications on clinical outcomes.

Differential transcriptional response following glucocorticoid activation in cultured blood immune cells: a novel approach to PTSD biomarker development

Post-traumatic stress disorder (PTSD) is a condition of stress reactivity, whose clinical manifestations are evident when patients are triggered following exposure to a traumatic event. While baseline differences in gene expression of glucocorticoid signaling and inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) have been associated with PTSD, these alterations do not fully recapitulate the molecular response to physiological triggers, such as stress hormones. Therefore, it is critical to develop new techniques that will capture the dynamic transcriptional response associated with stress-activated conditions relative to baseline conditions. To achieve this goal, cultured PBMCs from combat-exposed veterans with PTSD(+) (n = 10) and without PTSD(−) (n = 10) were incubated with increasing concentrations (vehicle, 2.5 nM, 5 nM, 50 nM) of dexamethasone (DEX). Across diagnosis and dosage, several genes and gene networks were reliable markers of glucocorticoid stimulation (FDR < 5%), including enhanced expression of FKPB5, VIPR1, NR1I3, and apoptosis-related pathways, and reduced expression of NR3C1, STAT1, IRF1, and related inflammatory and cellular stress-responsive pathways. Dose-dependent differential transcriptional changes in several genes were also identified between PTSD+ and PTSD−. Robust changes in expression were observed at 2.5 nM DEX in PTSD− but not PTSD+ participants; whereas, with increasing concentrations (5 nM and 50 nM), several genes were identified to be uniquely up-regulated in PTSD+ but not PTSD− participants. Collectively, these preliminary findings suggest that genome-wide gene expression profiling of DEX-stimulated PBMCs is a promising method for the exploration of the dynamic differential molecular responses to stress hormones in PTSD, and may identify novel markers of altered glucocorticoid signaling and responsivity in PTSD.

Abstract 4994: Differential gene expression analysis reveals pathways involved in anti-pd-1 therapy response prediction

Abstract The goal of this study is to evaluate the potential use of gene expression profile of peripheral blood mononuclear cells (PBMCs) as a non-invasive biomarker for predicting the response of anti-PD-1 therapy in non-small cell lung cancer (NSCLC) subjects. Peripheral blood of NSCLC stage 4 subjects treated with an anti-PD-1 monoclonal antibody was collected prior to treatment (week 0) and subsequent infusions. Total RNA was extracted, and libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit and sequenced by Illumina NextSeq. The paired-end reads were mapped to human reference hg38 using STAR and gene expressions were calculated using RSEM. Preliminary analysis of differential gene expression (DE) was performed among the responders at respectively (T1) weeks 3-4, (T2) weeks 6-9 and (T3) weeks 12-16 after treatment, compared with the baseline at T0. Further DE analysis was performed between the baseline samples of responders and non-responders. Genes with FDR-adjusted p values less than 0.05 were selected for pathway enrichment analysis with DAVID. Altogether 30 RNA-Seq samples of seven subjects were collected at multiple time points before and after treatment. According to standard RECIST 1.1 criteria, five subjects had a partial response to the therapy, whereas two subjects had stable disease or no response. In addition, liquid biopsy results were available for selected samples. Among the responders, there are 203 DE genes for T1 with log2 fold change (LFC) of gene expression from -2.4 to 3.5, 859 DE genes for T2 with LFC from -3.4 to 3.3, and 395 DE genes for T3 with LFC from -3 to 6.8. Among the DE genes for the three time points, 155 genes are common to T1/T2, 250 common to T2/T3, and 104 common to T1/T2/T3. Pathway enrichment test using Gene Ontology Biological Process terms shows that the strongest DE pathways for initial response after treatment at T1 are regulation of immune response (FDR adjusted p = 2.5E-5), oxygen transport (p = 5.5E-3) and T cell costimulation (p = 3.9E-3). Based on the 104 common DE genes, oxygen transport (p = 0.029) is the only pathway consistently perturbed due to an increase in hemoglobin. When comparing the baseline of responders and non-responders, we identified a candidate gene signature, with some of the genes also reported in other studies. However, more samples are required to establish its statistical significance. Systemic immune response after first dose of anti-PD1 treatment is reflected by DE genes in blood samples collected in weeks 3-4. Tumor regression might be indicated by the consistent increase in hemoglobin level. The results of these analyses show promise of developing a predictive signature for the response of anti-PD-1 immunotherapy in NSCLC subjects based on the gene expression profile of PBMCs. While these are preliminary results, we continue to recruit subjects, and will present the up-to-date results at the conference. Citation Format: Yee Him Cheung, Jie Wu, Weihua Huang, Changhong Yin, Katherine Linder, Kaushal Parikh, Ke Tang, Christopher Kasbek, Jun Huang, Zeil Rosenberg, Michael Fanucchi, John Fallon, Nevenka Dimitrova. Differential gene expression analysis reveals pathways involved in anti-pd-1 therapy response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4994.

Early transcriptome profile of goat peripheral blood mononuclear cells (PBMCs) infected with peste des petits ruminant's vaccine virus (Sungri/96) revealed induction of antiviral response in an interferon independent manner

Sungri/96 vaccine strain is considered the most potent vaccine providing long-term immunity against peste des petits ruminants (PPR) in India. Previous studies in our laboratory highlighted induction of robust antiviral response in an interferon independent manner at 48 h and 120 h post infection (p.i.). However, immune response at the earliest time point 6 h p.i. (time taken to complete one PPRV life cycle), in PBMCs infected with Sungri/96 vaccine virus has not been investigated. This study was taken up to understand the global gene expression profiling of goat PBMCs after Sungri/96 PPRV vaccine strain infection at 6 h post infection (p.i.). A total of 1926 differentially expressed genes (DEGs) were identified with 616 - upregulated and 1310 - downregulated. TLR7/TLR3, IRF7/IRF1, ISG20, IFIT1/IFIT2, IFITM3, IL27 and TREX1 were identified as key immune sensors and antiviral candidate genes. Interestingly, type I interferons (IFNα/β) were not differentially expressed at this time point as well. TREX1, an exonuclease which inhibits type I interferons at the early stage of virus infection was found to be highly upregulated. IL27, an important antiviral host immune factor was significantly upregulated. ISG20, an antiviral interferon induced gene with exonuclease activity specific to ssRNA viruses was highly expressed. Functional profiling of DEGs showed significant enrichment of immune system processes with 233 genes indicating initiation of immune defense response in host cells. Protein interaction network showed important innate immune molecules in the immune network with high connectivity. The study highlights important immune and antiviral genes at the earliest time point.

Gene expression profile of peripheral blood mononuclear cells may contribute to the identification and immunological classification of breast cancer patients.

It has been reported that the gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits a unique gene expression signature in several types of cancer. In this study, we aimed to explore the breast cancer patient-specific gene expression profile of PBMCs and discuss immunological insight on host antitumor immune responses. We comprehensively analyzed the gene expression of PBMCs by RNA sequencing in the breast cancer patients as compared to that of healthy volunteers (HVs). Pathway enrichment analysis was performed on MetaCoretm to search the molecular pathways associated with the gene expression profile of PBMCs in cancer patients compared with HVs. We found a significant unique gene expression signature, such as the Toll-like receptor (TLR) 3- and TLR4-induced Toll/interleukin-1 receptor domain-containing adapter molecule 1 (TICAM1)-specific signaling pathway in the breast cancer patients as compared to that of healthy volunteers. Distinctive immunological gene expression profiles also showed the possibility of classifying breast cancer patients into subgroups such as T-cell inhibitory and monocyte-activating groups independent of known phenotypes of breast cancer. These preliminary findings suggest that evaluation of gene expression patterns of PBMCs might be both a less invasive diagnostic procedure and a useful way to reveal immunological insight of breast cancer, including biomarkers for cancer immunotherapy, such as immune checkpoint inhibitor therapy.

In vitro proinflammatory gene expression predicts in vivo telomere shortening: A preliminary study

The chronic psychological stress of caregiving leads to higher risks for many diseases. One of the mechanisms through which caregiving is associated with disease risk is chronic inflammation. Chronic inflammation may accelerate cellular aging via telomere dysfunction and cell senescence, although this has not been examined in human cells from healthy people. We examined peripheral blood mononuclear cells (PBMCs) from 20 healthy mothers of children with autism (caregivers) and 19 mothers of neurotypical children (controls) in an in vitro culture system where PBMCs were stimulated with phytohaemagglutinin (PHA). We measured RNA expression levels of a panel of immune function genes before and after PHA stimulation, as well as telomere length from PBMCs collected from the participants at baseline and 15 months later. Caregivers and controls had similar gene expression profiles in unstimulated PBMCs, but after PHA stimulation, caregivers had increased RNA levels of the master inflammatory regulator NF-κB and its proinflammatory cytokine targets IL-1β, IL-6 and its receptor IL-6R as well as inflammatory chemokines IL-8, CXCL1 and CXCL2. Gene expression analysis suggested caregivers have increased Treg and Th17 T cell differentiation. Additionally, key signaling molecules involved in the upregulation of COX-2, a critical enzyme in the synthesis of the inflammatory mediator prostaglandin, were elevated. When both groups were examined together, higher expression levels of proinflammatory genes were associated with shorter telomere length in PBMCs from blood drawn 15 months later, independent of baseline telomere length. Taken together, these results suggest that chronic stress is associated with an exaggerated inflammatory response in PBMCs, which in turn is associated with shorter telomere length measured from PBMCs collected 15 months later. To our knowledge, this is the first human study that shows increased proinflammatory expression predicts future telomere shortening.

Proinflammatory cytokines and response to molds in mononuclear cells of patients with Meniere disease

Epidemiological studies have found a higher prevalence of allergic symptoms and positive prick tests in patients with Meniere’s disease (MD); however the effect of allergenic extracts in MD has not been established. Thus, this study aims to determine the effect of Aspergillus and Penicillium stimulation in cytokine release and gene expression profile in MD. Patients with MD showed higher basal levels of IL-1β, IL-1RA, IL-6 and TNF-α when compared to healthy controls. We observed that IL-1β levels had a bimodal distribution suggesting two different subgroups of patients, with low and high basal levels of cytokines. Gene expression profile in peripheral blood mononuclear cells (PBMC) showed significant differences in patients with high and low basal levels of IL-1β. We found that both mold extracts triggered a significant release of TNF-α in MD patients, which were not found in controls. Moreover, after mold stimulation, MD patients showed a different gene expression profile in PBMC, according to the basal levels of IL-1β. The results indicate that a subset of MD patients have higher basal levels of proinflammatory cytokines and the exposure to Aspergillus and Penicillium extracts may trigger additional TNF-α release and contribute to exacerbate inflammation.