Peripheral Blood Mononuclear Cell Targets Research Articles

PS-B11-8: ONE NOVEL MIRNA WITH 4 MRNA TARGETS WAS IDENTIFIED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HYPERTENSIVE PATIENTS WITH METABOLIC SYNDROME

Objective: Hypertension (HTN), metabolic syndrome (MetS) and chronic kidney disease (CKD) are global health disorders that are epidemiologically associated. The immune system plays a role in hypertension and target organ damage. MicroRNAs are crucial post-transcriptional regulators of immune cell development and function. We hypothesized that microRNAs play a role in activation of immune cells in hypertension with target organ damage in humans. Design and method: Normotensive subjects (NTN) and patients with HTN associated or not with at least 2 other features of the MetS or with CKD were studied (n = 15–16). Peripheral blood mononuclear cells (PBMCs) were isolated, RNA extracted, small and total RNA sequencing (RNA-seq) done using Illumina HiSeq-2500 and data analyzed using a systems biology approach. Differentially expressed (DE) microRNAs and mRNAs were identified with fold change (FC) > 2 and > 1.5, respectively, and P < 0.005. The most representative DE miRNA isoforms with RNA-seq count number (CN) > 500, and with predicted targets by TargetScan with CN> 300 were validated by reverse transcription-quantitative PCR (RT-qPCR) in PBMCs. Results: RNA-seq identified DE microRNAs and mRNAs in HTN (22 and 19), MetS (57 and 401) and CKD (6 and 26) compared to NTN. Three of 14 selected miRNAs were validated by RT-qPCR. miR-409–5p and miR-411–5p were decreased respectively by 46% (P < 0.05) and 60% (P < 0.001), whereas the novel miR-pl-86 was increased 2-fold (P < 0.05) in MetS vs NTN. RT-qPCR data showed that tubulin alpha 1a (TUBA1A) predicted as mRNA target for miR-409–5p and miR-411–5p was increased 1.9-fold in MetS vs NTN group (P < 0.05). However, there was no correlation between RT-qPCR and RNA-seq data for TUBA1A. RT-qPCR data validated the decrease of 4 predicted miR-pl-86 targets. WD repeat domain 89 (WDR89), Dmx like 1 (DMXL1), zinc finger protein 600 (ZNF600) and NOC3-like DNA replication regulator (NOC3L) were decreased by ≧ 50% in MetS vs NTN (P < 0.05). RT-qPCR data demonstrated a correlation with RNA-seq data for miR-pl-86 (R2 = 0.37, P < 5.5E-07, n = 56), WDR89 (R2 = 0.29, P < 1.4E-05, n = 57), DMXL1 (R2 = 0.33, P < 2.7E-06, n = 57), ZNF600 (R2 = 0.25, P < 7.9E-05, n = 57), and NOC3L (R2 = 0.27, P < 3.5E-05, n = 57). Conclusion: This study identified one up-regulated novel microRNA and 4 down-regulated mRNA targets in PBMCs of patients with HTN and MetS. The role of this miRNA and mRNAs in immune cells of HTN patients with MetS needs to be determined.

Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic.

ClinicalTrials.gov Identifier NCT05080920.

Abstract 070: ONE UP-REGULATED NOVEL MICRORNA AND 4 DOWN-REGULATED MRNA TARGETS WERE IDENTIFIED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HYPERTENSIVE PATIENTS WITH METABOLIC SYNDROME

Introduction: Hypertension is associated with target organ damage such as kidney injury. The immune system plays a role in hypertension and target organ damage. Activation of T cells has been reported among peripheral blood mononuclear cells (PBMCs) of patients with HTN. MicroRNAs are crucial post-transcriptional regulators of immune cell development and function. Hypothesis: MicroRNAs play a role in the activation of immune cells in hypertension with target organ damage in humans. Methods: Normotensive subjects (NTN) and patients with hypertension (HTN) associated or not with at least 2 other features of the metabolic syndrome (MetS) or chronic kidney disease (CKD) were studied (n=15-16). PBMCs were isolated from blood, RNA extracted, small and total RNA sequencing (RNA-seq) using an Illumina HiSeq-2500 and data were analyzed using a systems biology approach. Differentially expressed (DE) microRNAs and mRNAs were identified with fold change (FC) >2 and >1.5, respectively, and P <0.005. DE miRNAs with RNA-seq count number (CN) >500, and predicted targets by TargetScan with CN>300 were validated by reverse transcription-quantitative PCR (RT-qPCR) in PBMCs. Results: RNA-seq identified DE microRNAs and mRNAs in HTN (22 and 19), MetS (57 and 401) and CKD (6 and 26) compared to NTN. RT-qPCR validated a novel miRNA (miR-pl-86 [2-fold up]) and 4 predicted mRNA targets (WD repeat domain 89 [ WDR89 , 51% down], Dmx like 1 [ DMXL1 , 52% down], zinc finger protein 600 [ ZNF600 , 63% down] and NOC3 like DNA replication regulator [ NOC3L , 61% down]) in MetS vs NTN ( P <0.05). RNA-seq results were correlated with RT-qPCR data for miR-pl-86 (R 2 =0.37, P <5.5E-07, n=56) , WDR89 (R 2 =0.29, P <1.4E-05, n=57) , DMXL1 (R 2 =0.33, P <2.7E-06, n=57) , ZNF600 (R 2 =0.25, P <7.9E-05, n=57) , and NOC3L (R 2 =0.27, P <3.5E-05, n=57). Conclusion: This study identified one up-regulated novel microRNA and 4 down-regulated mRNA targets in PBMCs of patients with HTN and MetS.

Abstract MP44: A Novel And 2 Known Differentially Expressed MicroRNAs Were Identified In Peripheral Blood Mononuclear Cells Of Patients With Hypertension Associated With Metabolic Syndrome

Background: Hypertension (HTN) is associated with subclinical target organ damage including cardiac, vascular and kidney injury. The immune system plays a role in hypertension and target organ damage. Activation of T cells has been reported among peripheral blood mononuclear cells (PBMCs) of patients with HTN. MicroRNAs (miRNAs) are crucial post-transcriptional regulators of immune cells. Whether miRNAs play a role in the activation of immune cells in hypertension complicated by target organ damage in humans remains unknown. We aimed to address this question by identifying differentially expressed (DE) miRNAs and their mRNA targets in PBMCs of patients with hypertension complicated or not with metabolic syndrome (MetS) or chronic kidney disease (CKD). Methods: Normotensive subjects and patients with hypertension (HTN) associated or not with at least 2 other features of MetS or CKD were studied (n=15-16). PBMCs were isolated from blood, RNA extracted for small and total RNA sequencing (RNA-seq) using Illumina HiSeq-2500 and data were analyzed using a systems biology approach. MiRDeep2 was used for novel miRNAs prediction, miRNA annotation and counting. TargetScan 7.07 was used to predict DE miRNA targets with weighted context score percentile >50%. DE genes miRNAs and mRNAs were identified with fold change (FC) >1.5 and P <0.005. DE miRNAs with FC>2 and mean read count number (MRCM) >500, and with predicted targets with MRCM>300 were validated by reverse transcription-quantitative PCR (RT-qPCR). Results: DE miRNAs, mRNAs and non-coding RNAs were identified in HTN (22, 19 and 0), MetS (57, 401 and 11) and CKD (6, 26 and 2) compared to NTN. TargetScan predicted that 7 miRNAs target 3 mRNAs in NTN, 57 miRNAs target 55 mRNAs in MetS and 3 miRNAs target 2 mRNAs in CKD. DE miR-409-5p (FC: 0.54±0.10 vs 1.00±0.09, P <0.05), miR-411-5p (FC: 0.40±0.06, vs 1.00±0.11, P <0.001) and the novel miR-pl-86 (FC: 1.96±0.17 vs 1.00±0.15, P <0.05) in MetS vs NTN were validated by RT-qPCR. RNA-seq data were correlated with RT-qPCR for miR-409-5p (R 2 =0.40, P <2.4E-07, n=55), miR-411-5p (R 2 =0.55, P <1.1E-10, n=55), miR-pl-86 (R 2 =0.37, P <5.5E-07, n=56). Conclusion: This study showed that DE miR-409-5p, miR-411-5p and miR-pl-86 may play a role in HTN associated with MetS.

DIFFERENTIALLY EXPRESSED MICRORNAS AND THEIR TARGETS WERE IDENTIFIED IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF PATIENTS WITH HYPERTENSION ASSOCIATED OR NOT WITH TARGET ORGAN DAMAGE

Objective: Hypertension is associated with subclinical target organ damage including cardiac, vascular and kidney injury. The immune system plays a role in HTN and target organ damage in animal models. Activation of T cells has been reported among peripheral blood mononuclear cells (PBMCs) of patients with HTN. MicroRNAs are crucial post-transcriptional regulators of immune cells. Whether microRNAs play a role in the activation of immune cells in HTN complicated by target organ damage in humans remains unknown. We aimed to address this question by identifying differentially expressed (DE) microRNAs and their mRNA and non-coding RNA (ncRNA) targets in PBMCs of patients with HTN complicated or not with target organ damage. mRNA targets of DE microRNAs were predicted with TargetScan using the inversely related DE mRNAs as a filter to improve prediction efficiency. Design and method: Normotensive (NTN), hypertensive patients (HTN) and patients with HTN associated with at least 2 other features of the metabolic syndrome (MetS) or with chronic kidney disease (CKD) grades 3-4 were studied (n = 15-16 in each group). PBMCs were isolated from blood, RNA extracted for RNA sequencing (RNA-seq) using Illumina HiSeq-2500 and data were analyzed using a systems biology approach. DE genes microRNAs and mRNAs were identified with fold change (FC) >1.5 and P < 0.005. DE miRNAs with FC >2 and RNA-seq count number (CM) >500, and with predicted targets with CM >300 were validated by reverse transcription-quantitative PCR (RT-qPCR). Results: DE microRNAs, mRNAs and other ncRNAs were identified by RNA-seq in HTN (22, 19 and 0), MetS (57, 401 and 11) and CKD (6, 26 and 2) compared to NTN. TargetScan predicted that 7 microRNAs target 3 mRNAs in NTN, 57 microRNAs target 55 mRNAs in MetS and 3 microRNA target 2 mRNAs in CKD. Three of 14 selected miRNAs were validated by RT-qPCR: miR-409-5p (46% down, P < 0.05) and miR-411-5p (60% down, P < 0.001) and novel miR-pl-86 (2-fold up, P < 0.05) in MetS vs NTN. Conclusions: RNA-seq and RT-qPCR validation showed that DE miR-409-5p, miR-411-5p and the novel miR-pl-86 may play a role in HTN associated with MetS.

Concomitant overexpression of mir-182-5p and mir-182-3p raises the possibility of IL-17-producing Treg formation in breast cancer by targeting CD3d, ITK, FOXO1, and NFATs: A meta-analysis and experimental study.

T cells are polarized toward regulatory T cells (Tregs) in tumor microenvironment by the shuttling of microRNAs that target T cell–activating signaling pathways. We evaluated the expression of the miR‐182 cluster (miR‐96, 182, and 183) in peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC), and T cell polarization by the expression of FOXO1, NFATs, ITK, TCR/CD3 complex, and IL‐2/IL‐2RA. Twenty‐six microRNAs overexpressed in tumor tissues and sera of these patients were extracted by a meta‐analysis. Then, the expression of the miR‐182 cluster was investigated in PBMCs and sera of these patients and correlated with their targets in PBMCs. Finally, miR‐182 was cloned into Jurkat cells to evaluate its effects on T cell polarization. FOXO1, CD3d, ITK, NFATc3, NFATc4, and IL‐2RA were targeted by miR‐182, due to which their expression decreased in PBMCs of patients. Although IL‐6, IL‐17, and TGF‐β increased after miR‐182 transduction, IL‐2 dramatically decreased. We revealed CD4+FOXP3+ T cell differentiation in the miR‐182–transduced group. Although miR‐182 has inhibitory effects on T cells by the inhibition of FOXO1, TCR/CD3 complex, NFATs, and IL‐2/IL‐2RA signaling pathways, it increases FOXP3, TGF‐β, and IL‐17 expression to possibly drive T cell deviation toward the transitional state of IL‐17–producing Tregs and Treg formation in the end.

Rho kinase cascade activation in circulating leukocytes in patients with diabetes mellitus type 2

BackgroundThe intracellular ROCK signaling pathway is an important modulator of blood pressure and of cardiovascular and renal remodeling when Rho-kinase activity is increased. Besides, in preclinical models of diabetes, ROCK activation has also a role in abnormal glucose metabolism as well as in subsequent vascular and myocardial dysfunction. In humans, there are a few data assessing ROCK activation in patients with type 2 diabetes mellitus (T2D) and no studies assessing upstream/downstream components of the ROCK pathway. We assessed here levels of ROCK activation and some of the RhoA/ROCK cascade molecules in peripheral blood mononuclear cells (PBMCs) in T2D patients under current treatment.MethodsCross-sectional observational study comparing 28 T2D patients under current antidiabetic treatment with 31 consecutive healthy subjects, matched by age and gender. Circulating levels of malondialdehyde, angiotensin II and inflammatory cytokines IL-6 and IL-8 were determined in all subjects. ROCK activation in PMBCs, upstream and downstream cascade proteins, and levels of the proinflammatory molecules VCAM, ICAM-1 and IL-8 were determined in their PMBCs by Western blot.ResultsCompared to healthy controls, ROCK activation in T2D patients measured by 2 direct ROCK targets in PBMCs was increased by 420 and 570% (p < 0001) and it correlated significantly with serum glucose levels. p38 MAPK phosphorylation (downstream from ROCK) and JAK-2 (upstream from ROCK) were significantly higher in the T2D patients by 580% and 220%, respectively. In T2D patients, significantly increased PBMC levels of the proinflammatory molecules VCAM-1, ICAM-1 and IL-8 were observed compared to control subjects (by 180%, 360% and 260%, respectively). Circulating levels of Ang II and MDA were significantly higher in T2D patients by 29 and 63%, respectively.ConclusionsT2D patients under treatment with glucose-lowering drugs, antihypertensive treatment as well as with statins have significantly increased ROCK activation in their circulating leukocytes along with higher phosphorylation of downstream cascade proteins despite pharmacologic treatment, along with increased plasma angiotensin II and MDA levels. ROCK inhibition might have an additional role in the prevention and treatment of T2D.

Abstract P158: MicroRNA Profiling in Peripheral Blood Mononuclear Cells From Hypertensive Patients With or Without Chronic Kidney Disease

Background: Hypertension (HTN) and chronic kidney disease (CKD) are global health disorders that are epidemiologically associated. The immune system has been shown to play a role in HTN and CKD in animal models. Activation of T cells has been observed in peripheral blood mononuclear cells (PBMCs) of patients with HTN. MicroRNAs (miRNAs) are crucial post-transcriptional regulators of immune cells. Whether miRNAs play a role in the activation of immune cells in HTN and CKD in humans remains unknown. We aimed to address this question by identifying differentially expressed (DE) miRNAs and their mRNA targets in PBMCs of HTN and CKD patients. Methods and Results: Normotensive, HTN (systolic blood pressure (BP) &gt;135 mm Hg or diastolic BP of 85-115 mm Hg with BpTRU) and CKD subjects (estimated glomerular filtration rate &lt;60mL/min/m 2 ) (n=15-16) were studied. PBMCs were isolated from 30 ml of whole blood and used for total RNA extraction with the mirVana miRNA isolation kit. cDNA libraries were prepared using the TruSeq small RNA prep kit and the TruSeq stranded total RNA prep kit, and were sequenced by Illumina HiSeq 2500. DE miRNAs ( P &lt;0.05) were identified using EdgeR, which found 41 up- and 38 down-regulated miRNAs, as well as 101 up- and 316 down-regulated mRNAs uniquely associated with the hypertensive group, while 11 up- and 18 down-regulated miRNAs, as well as 153 up- and 73 down-regulated mRNAs were found uniquely associated with the CKD group. Meanwhile, 4 up- and 8 down-regulated miRNAs, as well as 13 up- and 19 down-regulated mRNAs were found in both groups. Target Scan was used to predict DE miRNA targets in the DE mRNAs. Enrichment analysis showed that the HTN-associated DE miRNA-targeting DE genes were highly enriched in gene ontology (GO) terms involved in cytosolic transport, protein kinase B (PKB) signalling and RNA 3’ processing ( q &lt;0.001), while the CKD-associated DE miRNA-targeting DE genes were highly enriched in GO terms involved in immune response, ribosomal process and metal ion homeostasis ( q &lt;0.001). Conclusions: DE miRNAs were identified in PBMCs of HTN and CKD patients. Enrichment analysis in DE miRNA-targeting DE mRNAs revealed GO terms that could be linked to different degrees of immune cell activation in HTN and CKD.

Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling

Cultured primary peripheral blood mononuclear cells (PBMC) represent a potentially physiologic in vitro model of HIV-1 infection, but assessment of antibody-mediated HIV-1 neutralization using PBMC has been hindered by donor variability and lack of a sustainable individual PBMC source. To advance this model for HIV vaccine evaluation, intra- and inter-assay variability were assessed using monoclonal and polyclonal antibodies and PBMC targets from multiple HIV-seronegative donors. Inter-assay variability was introduced by using different PBMC for virus propagation, and more substantially, for assay targets. Neutralization titers varied by as much as 4 logs when using different individual donor PBMC as targets; variability was antibody-specific, with the greatest variation observed using an individual polyclonal plasma. Pooling of multiple PBMC donors significantly reduced median inter-assay variation to the level of intra-assay variation, suggesting a pathway forward for establishing a uniform, sustainable and standardized approach to the assessment of antibody function using a PBMC model.

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.

Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates

The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.

HIV NEUTRALIZATION ASSAY USING POLYMERASE CHAIN REACTION-DERIVED MOLECULAR SIGNALS

Characterization of the capacity of human polyclonal antibody to neutralize wild-type patient isolates has important implications for vaccine development. We report the development of a polymerase chain reaction-based neutralization assay that quantitatively measures each infection using HIV proviral formation. These molecular end points identified the absence or quantitative diminution of DNA provirus formation as well as a delay in the kinetics of HIV DNA provirus formation. Using both laboratory strain prototype isolates (HIV-1-MN, HIV-IIIb) and primary wild-type patients' isolates, neutralization end points were reproducibly determined. End points were reached within 72 h, thereby minimizing the impact of subsequent rounds of infection on interpretation of results. Although the neutralization titer of polyclonal sera was usually comparable using standard technology, this assay did find isolate-dependent variation in the relationship between p24 production and HIV proviral DNA formation. Finally, we noted the disparity between the ability of human sera to neutralize prototype and wild-type isolates in primary peripheral blood mononuclear cell targets. We believe this assay provides unique opportunities to characterize the initial events of virus-antibody interaction and will help to elucidate clinically relevant neutralization immunoregulatory mechanisms.