RNA In Peripheral Blood Mononuclear Cells Research Articles

Small RNA deep sequencing reveals novel miRNAs in peripheral blood mononuclear cells from patients with IgA nephropathy.

Peripheral blood mononuclear cells (PBMCs) contribute to the deposition of immunoglobulin A (IgA) and progression of IgA nephropathy (IgAN). This study was performed to identify novel microRNAs (miRNAs/miRs) associated with IgAN. Small RNAs were isolated from PBMCs collected from 10 healthy participants and 10 patients with IgAN; the RNAs were then subjected to high-throughput small RNA sequencing. The results showed that miRNAs constituted 70.33 and 69.83% of small RNAs in PBMCs from healthy participants and patients with IgAN, respectively. In total, 44 differentially expressed miRNAs were identified, of which 34 were upregulated and 10 were downregulated. Among these differentially expressed miRNAs, most showed novel associations with IgAN, except miR-148a-3p, miR-184 and miR-200a. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the target genes of the differentially expressed miRNAs were primarily enriched in cancer pathways, the PI3K-Akt signaling pathway and MAPK pathways, all of which control cell proliferation and gene expression. Moreover, miR-3121-3p, miR-203a-3p and miR-200a-3p may regulate core 1 synthase, glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) expression by binding to its 3′ untranslated region. In conclusion, 44 differentially expressed miRNAs were discovered, 41 of which were newly found to be associated with IgAN. The differentially expressed miRNAs may regulate the progression of IgAN by controlling the behavior of PBMCs or deposition of IgA via targeting of signaling pathways or expression of C1GALT1. These findings may provide a basis for further research regarding IgAN diagnosis and therapy.

A retrospective case control study identifies peripheral blood mononuclear cell albumin RNA expression as a biomarker for non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) improves after bariatric surgery. The aim of this study was to determine whether peripheral blood mononuclear cell albumin gene expression was related to NAFLD and whether albumin (ALB) and alpha fetoprotein (AFP) expression could be detected in whole blood and visceral adipose tissue. Using a retrospective case control study design, RNA isolated from peripheral blood mononuclear cells from patients prior to undergoing bariatric surgery was used for pooled microarray analysis. Quantitative polymerase chain reaction (QPCR) was used to analyze whole blood and visceral adipose tissue. Liver histology was obtained via intra-operative biopsy and clinical data extracted from the electronic health record. The albumin (ALB) gene was the second most up-regulated found in microarray analysis of peripheral blood mononuclear cell RNA from patients with hepatic lobular inflammation versus normal liver histology. Transcript levels of ALB were significantly different across those with normal (n = 50), steatosis (n = 50), lobular inflammation (n = 50), and peri-sinusoidal fibrosis (n = 50) liver histologies, with lobular inflammation 3.9 times higher than those with normal histology (p < 0.017). Albumin expression levels decreased in 11/13 patients in paired samples obtained prior to and at 1year after Roux-en-Y gastric bypass surgery. ALB expression could be detected in 23 visceral adipose tissue samples obtained intra-operatively and in 18/19 available paired whole blood samples. No significant correlation was found between ALB expression in visceral adipose tissue and whole blood RNA samples. Alpha fetoprotein expression as a marker of early hepatocytic differentiation was detected in 17/17 available VAT RNA samples, but in only 2/17 whole blood RNA samples. Albumin RNA expression from blood cells may serve as a biomarker of NAFLD. Albumin and alpha fetoprotein appear to be ubiquitously expressed in visceral adipose tissue in patients with extreme obesity.

Zika virus infects human blood mononuclear cells

BackgroundZika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV.MethodsPBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry.ResultsWe detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3−CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells.ConclusionsThese findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Evaluation of ERAP1 gene single nucleotide polymorphisms in immunomodulation of pro-inflammatory and anti-inflammatory cytokines profile in ankylosing spondylitis

BackgroundAnkylosing spondylitis (AS) is a prototype of chronic inflammatory arthritis termed seronegative spondyloarthropathies that typically affects the joints. Among the non-Human leukocyte antigen (HLA) loci, the strongest association has been observed with Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene single nucleotide polymorphisms (SNPs). Moreover, the effect of ERAP1 gene SNPs on the pro-inflammatory and anti-inflammatory cytokines in AS disease has still been poorly elucidated. In this study, we aimed to determine the association of ERAP1 gene SNPs (rs30187 and rs2287987) with AS risk as well as their effect on the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, with emphasis on the immunoregulation of the IL-17/IL-23 pathway, in an Iranian population. MethodsWe performed Single specific primer (SSP)-PCR for genotyping of 160 AS patients and 160 healthy controls. After isolation of peripheral blood mononuclear cells (PBMCs), total RNA of PBMCs was isolated, complementary DNA (cDNA) was synthesized, and quantitative analyses of mRNA expression of cytokines were performed by Real-time PCR for 40 HLA-B27 positive AS patients and 40 healthy individuals as controls. ResultsIt was seen that T allele of rs30187 (OR = 1.54, 95% CI = 1.07–2.22, P = 0.017) and C allele of rs2287987 (OR 1.50, 95% CI 1.05–2.14, P = 0.024) were associated with the risk of AS. Both of these alleles were associated more strongly in the HLA-B27 positive AS patients. There was a significant overexpression of mRNAs of pro-inflammatory (IL-17A, IL-17F, IL-23, TNF-α and IFN-γ), while downregulation of anti-inflammatory cytokines (IL-10 and TGF-β) in PBMCs from 40 HLA-B27 positive AS patients in comparison to controls. AS patients with rs30187 SNP TT genotype expressed mRNA of IL-17A, IL-17F, and IL-23 significantly higher than patents with CT and CC genotypes for this SNP. ConclusionsThis study represented the association of ERAP1 gene rs30187 and rs2287987 polymorphism with the risk of AS. Additionally, it appears that rs30187 polymorphism may be involved in the immunomodulation of the IL-17/IL-23 pathway in the AS disease.

Evaluation of hepatitis C viral RNA persistence in HIV-infected patients with long-term sustained virological response by droplet digital PCR

Several studies have reported the persistence of HCV RNA in liver and/or peripheral blood mononuclear cells (PBMCs) in spite of undetectable viremia in patients who have achieved sustained virological response (SVR). This event, defined as occult HCV infection, remains controversial and low titers of persistent virus may be underestimated because it has not yet been analyzed by a highly sensitive test such as droplet digital PCR (ddPCR). This method provides an alternate ultra-sensitive detection technique for very low numbers of copies of viral RNA or DNA. The aim of this study was to evaluate the persistence of HCV in HIV-coinfected patients with long-term SVR using ddPCR. For each patient, the presence of HCV RNA in serum and PBMCs at baseline was determined by nested RT-ddPCR. Patients with HCV RNA in PBMCs at baseline were followed until the end of the study. One hundred and twenty-three patients were analyzed for persistence of HCV RNA in serum and PBMCs. Persistence of HCV was not found in serum in any patient. HCV RNA was detected in PBMCs in one patient (0.81%; 95% CI: 0.04–3.94) and resolved spontaneously during follow-up. Persistence of HCV RNA in PBMCs is not a common event in HIV/HCV co-infected patients with long-term SVR evaluated by RT-ddPCR.

Silent damage? Occult HCV replication and histological disease may occur following apparent HCV clearance

Silent damage? Occult HCV replication and histological disease may occur following apparent HCV clearance

Immunological biomarkers as indicators for outcome after discontinuation of nucleos(t)ide analogue therapy in patients with HBeAg-negative chronic hepatitis B.

The optimal duration of treatment with nucleos(t)ide analogues (NAs) for patients with HBeAg-negative chronic hepatitis B (CHB) is unknown. The aim of this study was to identify an immune signature associated with off-treatment remission to NA therapy. We performed microarray analysis of peripheral blood mononuclear cell (PBMCs) from six patients with chronic hepatitis B who stopped NA therapy (three with off-treatment remission, three with relapse) and five patients with chronic HBV infection (previously termed 'inactive carriers') served as controls. Results were validated using qRT-PCR on a second group of 21 individuals (17 patients who stopped treatment and four controls). PBMCs from 38 patients on long-term NA treatment were analysed for potential to stop treatment. Microarray analysis indicated that patients with off-treatment remission segregated as a distinct out-group. Twenty-one genes were selected for subsequent validation. Ten of these were expressed at significantly lower levels in the patients with off-treatment remission compared to the patients with relapse and predicted remission with AUC of 0.78-0.92. IFNγ, IL-8, FASLG and CCL4 were the most significant by logistic regression. Twelve (31.6%) of 38 patients on long-term NA therapy had expression levels of all these four genes below cut-off values and hence were candidates for stopping treatment. Our data suggest that patients with HBeAg-negative CHB who remain in off-treatment remission 3years after NA cessation have a distinct immune signature and that PBMC RNA levels of IFNγ, IL-8, FASLG and CCL4 may serve as potential biomarkers for stopping NA therapy.

Peripheral blood mononuclear cells hepatitis C virus RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate

Background Eradication of hepatitis C virus (HCV) infection is the goal of direct-acting antivirals with a high rate of sustained virological response (SVR). Currently, SVR is determined by negative serum HCV RNA by real-time (RT)-PCR that may not give any information about intracellular HCV replication. Aim To study peripheral blood mononuclear cells (PBMCs) HCV RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients. Patients and methods In all, 150 patients with chronic HCV, classified into 100 easy-to-treat patients (group I) and 50 difficult-to-treat patients (group II), who achieved SVR to sofosbuvir plus daclatasvir with or without ribavirin were enrolled in the study. HCV RNA was evaluated in both sera and isolated PBMCs using the polymerase chain at 3 and 12 months from the end of treatment (EOT). Results As regards HCV RNA in peripheral blood mononuclear cells (PBMCs) we found that no case was positive in the easy to treat (group I). In difficult to treat (group II), six (4%) patients were positive at 3 months and 24 (16%) patients at 12 months after EOT. However, there is no virological, clinical, or biochemical relapse noted during the follow-up period among positive cases. All positive cases were cirrhotic, with significantly lower platelets count and albumen level but higher bilirubin than group I. Conclusion All easy-to-treat groups were HCV RNA in PBMCs negative at EOT and during the follow-up period (1 year). Follow-up of cirrhotic patients with positive HCV RNA in PBMCs showed no clinical, biochemical, or virological relapse.

Quantification of Human Swiprosin-1/EFhd2 Expression on Protein and RNA Level.

Many Ca2+-binding proteins are differentially regulated under pro-inflammatory conditions in different organs. Specific quantification of RNA and protein expression of those proteins demands validated protocols. Peripheral blood mononuclear cells (PBMC) can mirror an inflammatory status originating from several organs and can therefore be an important diagnostic tool. Swiprosin-1/EFhd2 (EFhd2) is a ~30kDa Ca2+ and F-actin binding, cytoskeletal protein with two central EF hands and a C-terminal coiled-coil domain. Unbiased gene expression analyses and proteomics revealed that EFhd2 is regulated under pro-inflammatory conditions in several cell types and tissues. Here we describe validated protocols to quantify the expression of the human orthologue of Swiprosin-1/EFhd2 on RNA and protein level in PBMC. Both methods reveal that EFhd2 is stronger expressed in monocytes than in B cells of healthy donors. Thus, initial experiments relying on qPCR are likely to provide results with functional relevance. The higher expression of EFhd2 in monocytes could be related to monocyte migration under inflammatory conditions.

IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality

BackgroundUncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). MethodsPBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their “diagnostic performance” in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. ResultsWe established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify:(1)EDTA PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times >48 h with 98% sensitivity and 87% specificity at a cutoff of 57.(2)citrate PBMC samples or RNA extracted from these PBMCs with RT precentrifugation times of >48 h with 92% sensitivity and 84% specificity at a cutoff of 348. ConclusionThe proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.

Evaluation of ERAP1 Gene Single Nucleotide Polymorphism in Impressing the Inflammatory Cytokine Profile of Ankylosing Spondylitis Patients

Ankylosing spondylitis (AS), an autoinflammatory disease, has been associated with impaired Endoplasmic reticulum aminopeptidase (ERAP) 1 activity, which is involved in priming antigenic peptides. The purpose of this study was to evaluate if the genetic variant of ERAP1 gene could impress the inflammation status of the AS patients. For genotyping, 140 AS cases and 140 healthy controls were enrolled. After isolation of peripheral blood mononuclear cells (PBMCs) and DNA extraction, all the subjects were genotyped for rs27044 polymorphism using SSP-PCR assay. Total RNA of PBMCs was isolated, cDNA was synthesized, and quantitative analyses of mRNA expression of cytokines were performed via Real-time PCR using the SYBR Green Gene Expression MasterMix. To measure the concentration of cytokines in serum of subjects, Enzyme-linked immunosorbent assay (ELISA) was used. It was observed that the G allele of rs27044 polymorphism was significantly prevalent in AS patients. Moreover, the GG genotype and the GG+GC dominant model had significantly different distribution between study groups. There was a significant overexpression of mRNAs of IL-17A, IL-6, IL-33, TNF-α, and IFN-γ, while IL-10 was significantly downregulated in AS patients. The ELISA results were in line with that of the gene expression analysis. No significant differences in mRNA expression and concentration of cytokine were identified among AS patients with three genotypes for rs27044 SNP. This study replicated the association of polymorphisms in ERAP1 gene with the risk of AS in a population from Iranian. However, it did not directly determine the inflammatory profile of the AS patients.

PBMC fixation and processing for Chromium single-cell RNA sequencing

BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.MethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform.ResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones.ConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.

Identifying differentially expressed long non-coding RNAs in PBMCs in response to the infection of multidrug-resistant tuberculosis

PurposeThe aim of this paper was to identify differentially expressed long non-coding RNAs (lncRNAs) in peripheral blood mononuclear cells (PBMCs) influenced by the infection of multidrug-resistant tuberculosis (MDR-TB).Materials and methodsIncRNA and mRNA expression profiles in PBMCs derived from healthy controls (HCs) and individuals with MDR-TB and drug-sensitive tuberculosis (DS-TB) were analyzed and compared by microarray assay. Six lncRNAs were randomly selected for validation by using real-time quantitative polymerase chain reaction (RT-qPCR). The biological functions and signaling pathways affected by the differentially expressed mRNAs were investigated by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based approaches.ResultsCompared with the HC group, 1,429 lncRNAs (983 mRNAs) and 2,040 lncRNAs (1,407 mRNAs) were identified to be deregulated in the MDR-TB group and in the DS-TB group, respectively, and 1,511 lncRNAs and 1,047 mRNAs were identified to be differentially expressed in both MDR-TB and DS-TB groups. Between the three groups, 22 lncRNAs and 38 mRNAs were found deregulated. Most deregulated lncRNAs were from intergenic regions (~55% of the total), natural antisense to protein-coding loci (~32% of the total), or intronic antisense to protein-coding loci (~5% of the total). Significantly enriched signaling pathways regulated by the deregulated mRNAs were mainly associated with natural killer cell-mediated cytotoxicity, antigen processing and presentation, graft-vs-host disease, the transforming growth factor-β signaling pathway, and the Hippo signaling pathway.ConclusionThis study is the first to report differentially expressed lncRNAs in PBMCs in response to MDR-TB infection. It revealed that some lncRNAs might be associated with regulating host immune response to MDR-TB infection. Further elucidation of the potential of these deregulated lncRNAs in MDR-TB and its reactivation requires further study.

HCV Therapy Follow-up Fractionation (CTF2) by Intra-PBMC Nested RNA PCR Recognizes Early Virologic Response and Relapse.

Background and Aims: Sustained virologic response is evaluated by single-step reverse transcription (SRT) PCR assay, which assesses hepatitis C virus (HCV) clearance from plasma but not from tissues such as peripheral blood mononuclear cells (PBMCs). Persistence of HCV RNA in PBMCs beyond end of treatment (EOT) is associated with nonresponse. Our goal was to measure intra-PBMC HCV RNA levels during oral antiviral therapy according to the HCV therapy follow-up fractionation (CTF2) protocol.Methods: Compensated chronic HCV patients (n = 2 78 SRT-PCR positive) were scheduled to receive oral antiviral therapy. Subjects were followed-up by SRT and intra-PBMCs HCV RNA PCR at the end of the 2nd, 6th, 10th, 14th, 18th and 24th weeks to evaluate virus clearance from plasma and PBMCs, respectively. The CTF2 protocol evaluated SRT and PBMC PCR status at each follow-up point for determining therapy continuation or interruption to address cost effectiveness.Results: All patients tested negative by SRT PCR after therapy for 2 weeks. Application of the CTF2 protocol revealed: a) increasing HCV clearance rate from 75.9% at the end of 10th week to 90.3% at the end of 24th week (p < 0.00001); b) faster clearance of HCV from plasma compared to PBMCs at each point of follow-up until the 18th week (p < 0.05); c) higher viral elimination rates diagnosed by PBMC HCV RNA PCR(−) compared to PBMC HCV RNA PCR(+) from the 6th to 24th week of treatment (p < 0.0001); d) higher over-time increase curve of combined plasma and PBMC HCV RNA determined negativity compared to the decline in positivity curves by PBMC PCR at the 6th-18th week compared to the 24th week (p < 0.01)—these results validated treatment continuation; and e) solitary evaluation of EOT sustained HCV infection and relapses by PBMC HCV RNA (p < 0.001).Conclusions: Early elimination of serum and tissue (PBMC) HCV infection by oral antiviral therapy can be achieved and evaluated during a cost-effective CTF2 protocol application.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.

Compartmental HBV evolution and replication in liver and extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers

BackgroundHepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence. ObjectivesTo investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy. Study DesignA total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N=7 baseline, N=5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver. ResultsCompare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2–0.08 copies/hepatocyte, p>0.05) or in PBMC 0.003–0.02 copies/PBMC, p>0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma. ConclusionContinuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy.

Abstract 5706: Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test

Abstract It is reported that gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits unique gene expression signature in cancer patients including renal cell carcinoma, pancreatic cancer and lung cancer. Since pancreatic cancer diagnosis is difficult in not only early detection of the disease but also in the diagnosis itself, development of novel diagnostic tools in addition to conventional diagnostic strategy has been awaited. On the other hand, in breast cancer, because early diagnosis by mammography and ultra sound examination on breast is established successfully, exploration of gene expression profile of PBMCs may be important in terms of insight to host antitumor immune response aspects. In the current study, we performed RNA sequencing (RNA-seq) analysis on RNA of PBMCs from 3 healthy volunteers, 6 early and 7 metastatic breast cancer patients including all phenotypes defined by ER, PgR and HER2. Genes that showed FDR&amp;lt;0.05 and fold change of &amp;lt;0.5 and 2.0&amp;lt; in comparison of healthy volunteers and breast cancer patients were applied to clustering analysis by Ward method. We found significant unique gene expression signature between healthy volunteers and breast cancer patients. Furthermore, it was shown that HER2 overexpressing breast cancer patients, which breast cancer patients were divided into 3 clusters including HER2 type and other 2 different groups, seemed to be classified into healthy group different from other phenotypes of breast cancer patients. These findings suggested that evaluation of gene expression patterns of PBMCs of breast cancer patients might distinguish breast cancer patients from healthy subjects and the gene expression signature of PBMCs which divided breast cancer patients into 3 groups might reveal immunologically important biologic properties such as response prediction of cancer immunotherapy including immune checkpoint inhibition treatment. Citation Format: Eiji Suzuki, Kosuke Kawaguchi, Masahiro Sugimoto, Fengling Pu, Ryuji Uozumi, Ayane Yamaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Satoshi Morita, Masakazu Toi. Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5706. doi:10.1158/1538-7445.AM2017-5706

Suppressive Effect of 1α,25-Dihydroxyvitamin D3 on Th17-Immune Responses in Kidney Transplant Recipients With Tacrolimus-Based Immunosuppression.

The aim of this study was to investigate whether 1α,25-dihydroxyvitamin D3 can regulate Th17-related immune responses in kidney transplant recipients (KTRs) being treated with tacrolimus (Tac)-based immunosuppression. First, we evaluated the effect of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on Th17-immune responses in an in vitro study using peripheral blood mononuclear cells (PBMCs) from healthy volunteers or KTRs. Next, we investigated mammalian target of rapamycin/STAT3 signaling as a mechanism by which 1,25(OH)2D3 exerted its effect on T cells using the Jurkat cell line. Third, we investigated Th17-cytokine levels or Th17 cell percentage in PBMCs according to the serum 25-hydroxyvitamin D (25(OH)D) level in 81 KTRs, and we performed a prospective study to assess whether 1,25(OH)2D3 (calcitriol) treatment decreased Th17 cytokine levels (IL-17, IL-22) in 42 KTRs. In the in vitro study, we observed that the addition of 1,25(OH)2D3 to Tac significantly inhibited the appearance of IL-17-positive cells in culture. The expression of IL-17 and IL-22 messenger RNA in PBMCs was also decreased by the addition of 1,25(OH)2D3. In the Jurkat cell line, the mTOR/STAT3 pathway was further downregulated with the addition of 1,25(OH)2D3 to Tac. In the 81 KTRs, the 25(OH)D level was inversely correlated with the Th17 cytokine levels or the proportion of Th17 cell out of CD4 T cells. Treatment with calcitriol for 6 months significantly decreased Th17 cytokine levels compared with the baseline values in another 42 KTRs. Treatment with 1,25(OH)2D3 may have immunologic benefits by effectively suppressing the Th17-related immune responses in KTRs on Tac-based immunosuppression.

Assessment of Hepatitis C Virus RNA in Peripheral Blood Mononuclear Cells as a Predictor of Response to Pegylated-Interferon and Ribavirin: A Cohort Study

Background: Sustained virologic response (SVR) to pegylated-interferon (PegIFN) and ribavirin (RBV) in hepatitis C virus (HCV)-infected patients could be predicted by detection of serum HCV RNA whereas detection of HCV RNA in other reservoirs such as peripheral blood mononuclear cells (PBMCs) for prediction of treatment response is still a mystery. Objectives: This study aimed at assessing the prediction of SVR by detection of HCV RNA in PBMCs or serum in patients during treatment. Methods: In a cohort study (2011 to 2014), 100 chronic HCV patients at Tehran Hepatitis Center were treated with PegIFN and RBV. Serum HCV RNA level was measured at baseline, 4, 12, and 24 weeks during treatment and at 24 weeks after termination of treatment. Meanwhile, HCV RNA was evaluated in PBMCs at weeks 4, 12, and 24 during the treatment. Results: Out of 100 patients treated in this study, 91 completed the course of treatment. Most patients were young males infected with HCV genotype 1. Cirrhosis and previous history of treatment was found in 16.5% and 26.5% of patients. Sustained virologic response was achieved in 65 (71.4%) patients. Among baseline parameters, only female gender was significantly associated with SVR. Undetectable serum HCV RNA at week 4 (OR = 4.74) and week 12 (OR = 11.63) of treatment predicted SVR rate while the same was not true for detection of serum HCV RNA at week 24 of treatment. Moreover, detection of HCV RNA in PBMCs at weeks 4 and 12 of treatment was not associated with the rate of SVR, while absence of HCV RNA in PBMCs at week 24 of treatment was associated with SVR (OR = 4.55). Conclusions: Detection of HCV RNA in PBMCs, especially at week 24 of treatment with PegIFN and RBV, could be considered as an additional marker for prediction of treatment response. It is recommended to assess HCV on-treatment kinetic in PBMCs of patients treated with direct-acting antiviral agents for prediction of treatment response.

Expression dynamics of HSP90 and nitric oxide synthase (NOS) isoforms during heat stress acclimation in Tharparkar cattle.

Six male Tharparkar cattle of 2-3years old were selected for the study. After 15-day acclimation at thermoneutral zone (TNZ) in psychrometric chamber, animals were exposed at 42°C for 6h up to 23days followed by 12days of recovery period. Blood samples were collected during control period at TNZ (days 1, 5, and 12), after heat stress exposure (day 1, immediate heat stress acclimation (IHSA); days 2 to 10, short-term heat stress acclimation (STHSA); days 15 to 23, long-term heat stress acclimation (LTHSA); days 7 and 12, recovery period), and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. The messenger RNA (mRNA) and protein expression in PBMCs were determined by qPCR and western blot, respectively. Samples at TNZ were taken as control. The mRNA expression of HSP90, iNOS, and eNOS was significantly upregulated (P<0.05) on day 1 (ISHA) as compared to control, remained consistent during STHSA, again increased during LTHSA, and finally reduced to basal level during recovery period. The protein expression of HSP90, iNOS, and eNOS were akin to their transcript pattern. PBMC culture study was conducted to study transcriptional abundance of HSP90, iNOS, and eNOS at different temperature-time combinations. The present findings indicate that HSP90, iNOS, and eNOS could possibly play an important role in mitigating thermal insults and confer thermotolerance during long-term heat stress exposure in Tharparkar cattle.