Paired Peripheral Blood Mononuclear Cells Research Articles

Studies into the mechanism of measles-associated immune suppression during a measles outbreak in the Netherlands

Measles causes a transient immune suppression, leading to increased susceptibility to opportunistic infections. In experimentally infected non-human primates (NHPs) measles virus (MV) infects and depletes pre-existing memory lymphocytes, causing immune amnesia. A measles outbreak in the Dutch Orthodox Protestant community provided a unique opportunity to study the pathogenesis of measles immune suppression in unvaccinated children. In peripheral blood mononuclear cells (PBMC) of prodromal measles patients, we detected MV-infected memory CD4+ and CD8+ T cells and naive and memory B cells at similar levels as those observed in NHPs. In paired PBMC collected before and after measles we found reduced frequencies of circulating memory B cells and increased frequencies of regulatory T cells and transitional B cells after measles. These data support our immune amnesia hypothesis and offer an explanation for the previously observed long-term effects of measles on host resistance. This study emphasises the importance of maintaining high measles vaccination coverage.

Brief Report: The Anti-HIV-1 ADCC-Mediating Antibodies From Cervicovaginal Secretions of HIV-Infected Women Have an Ability to Mediate Lysing of Autologous CD4+ HIV-Infected Cells.

Fragment crystallizable region of antibody-mediated mechanism such as antibody-dependent cellular cytotoxicity (ADCC) has been identified as an important component of immune protection against HIV. We assessed whether the anti-HIV antibodies mediating ADCC from cervicovaginal lavages (CVLs) of HIV-infected women have an ability to mediate lysing of autologous CD4 HIV-infected cells. The CVLs of 62 HIV-infected (37 long-term slow progressors and 25 with progressive HIV infection: progressors) and 20 HIV-uninfected Indian women with high risk of HIV acquisition were tested for the presence of ADCC-mediating anti-HIV antibodies against HIV-1 C Env in a fluorometric assay. Furthermore, we tested the ability of these antibodies to mediate ADCC-dependent killing of the autologous HIV-infected CD4 T cells using paired peripheral blood mononuclear cells containing target and effector cells. The numbers of ADCC responders were significantly higher in long-term slow progressors (34/37) as compared to the progressor group (9/25) with no significant difference in the magnitude. The magnitude of response was inversely associated with detectable CVL viral load (P < 0.003). The lysis of target cells was significantly higher in enriched IgG fraction as compared to the respective non-IgG fraction. The ADCC antibodies from CVLs significantly reduced the frequency of HIV-1 Env-activated autologous CD4 T cells in the presence of autologous effector cells. The presence of ADCC antibodies in CVLs with an ability to mediate lysing of HIV-infected autologous CD4 T cells provides evidence of their promising contribution to mucosal defense against HIV-1 and has implications in designing prophylactic and immunotherapeutic strategies.

High Expression of FOXP3 mRNA in Blood and Urine as a Predictive Marker in Kidney Transplantation.

Diagnosis of allograft dysfunction by noninvasive biomarker tests is preferable to invasive allograft biopsies and has been extensively considered in recent years. This study aims to evaluate blood and urinary forkhead box P3 (FOXP3) messenger RNA (mRNA) expression in renal transplant recipients in an attempt to determine whether differential diagnosis of graft dysfunction is feasible using mRNA profiles. We analyzed FOXP3 mRNA expression in paired urinary and peripheral blood mononuclear cell (PBMC) samples. A total of 91 kidney transplant recipients enrolled in this study that were classified into 3 groups: biopsy-proven acute rejection (AR; n = 27), chronic allograft nephropathy (n = 19), and well-functioning graft (n = 45). The FOXP3 mRNA expression was quantified by TaqMan probe real-time polymerase chain reaction. Acute rejection patients had a higher expression level of transcription factor FOXP3 compared to the chronic nephropathy and control groups. Analysis of receiver operating characteristic curves showed that rejection could be diagnosed with 100% sensitivity and 96% specificity in urine, and 92% sensitivity and 86% specificity in PBMC samples using the optimal FOXP3 mRNA cutoff value. We subdivided the AR group into progressive and nonprogressive patients, which showed a significant difference in FOXP3 mRNA expression. This result confirmed the role of FOXP3 as a diagnostic marker in predicting transplantation outcomes. Our results suggested that elevated expression of FOXP3 in blood and urine samples from kidney transplant recipients could be a useful noninvasive biomarker to diagnose graft dysfunction.

A-105 NO EVIDENCE FOR HIV REPLICATION IN LYMPH NODES DURING ART

To better understand the mechanisms of HIV persistence and to further investigate the question on ongoing viral replication in lymph nodes, we characterized HIV proviral populations, their levels of expression, and their sites of host integration in paired lymph node mononuclear cells (LNMC) and peripheral blood mononuclear cells (PBMC) collected after long-term ART and compared to them to the HIV populations prior to ART initiation. Three donors initiated ART in chronic infection and had viremia suppressed for 4-12 years; one donor initiated ART in acute infection and had viremia suppressed for 18 years. Proviral populations and expression were characterized by single-cell analyses and expanded clones were identified using the integration sites assay (ISA). Populations were compared phylogenetically, using a test for panmixia (well-mixed or divergent), by determining the fraction of expressing vs. latent proviruses, determining the levels of expression in single cells with actively-transcribing proviruses, and by comparing the expanded clonal populations. Proviruses in LNMC and PBMC were well mixed and were not significantly divergence from the pre-ART populations. The fraction of proviruses that were latent vs. actively transcribing during ART were not different (5%-8% in the PBMC and 2%-20% in the LNMC) (p=0.4). The levels of expression in actively-infected cells were low in both compartments, typical of suppression of viral replication. The same clonal populations were detected in PBMC and LNMC (p=0.8). These findings are not consistent with continued viral replication during ART in either the peripheral blood or the lymph nodes. Our results also suggest that infected cells migrate freely between the peripheral blood and the lymph nodes and demonstrate that the HIV reservoir is long-lived and proliferating populations of cells that were infected prior to initiating ART.

Chemotherapy Immunophenoprofiles in Non-Small-Cell Lung Cancer by Personalized Membrane Proteomics.

No study has addressed how the immune status at the molecular level is affected by first-line pemetrexed and cisplatin (PEM-CIS) combination therapy in patients with non-small-cell lung cancer (NSCLC). Thus, we aimed to identify the immune status from membrane proteome alterations in patients with NSCLC upon PEM-CIS treatment. The paired peripheral blood mononuclear cells (PBMCs) were collected from four patients with lung adenocarcinoma before and after the first regimen of PEM-CIS treatment and applied quantitative membrane proteomics analysis. In the personalized PBMC membrane proteome profiles, 2424 proteins were identified as displaying patient-specific responsive patterns. We discovered an elevated neutrophil activity and a more suppressive T-cell phenotype with the downregulation of cytotoxic T lymphocyte antigen 4 degradation and the upregulation of type 2 T-helper and T-regulatory cells in the patient with the highest progression-free survival (PFS) of 14.5 months. Patients with a PFS of 2 months showed higher expressions of T-cell subsets, MHC class II pathways, and T-cell receptor signaling, which indicated an activated immune status. Without the additional isolation of specific immune cell populations, our study demonstrated that PEM-CIS chemotherapy altered patients' immune system in terms of neutrophils, T cells, and antigen presentation pathways.

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells

GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.

Expression of Immune Checkpoint Receptors in Multiple Myeloma Patients

Abstract Multiple myeloma is a malignancy of plasma cells and the second most common hematologic cancer. High-dose chemotherapy plus autologous stem cell transplantation has been performed to achieve remission, but almost all the patients will finally relapse. Alternative therapeutic options are necessary for the multiple myeloma patients. Immune checkpoint inhibitors, such as anti-PD-1 have shown practice-changing therapeutic effectiveness for advanced non-small cell lung cancer, as well as Hodgkin’s lymphoma. To investigate T cell exhaustion in multiple myeloma patients, we assessed the expression of various immune checkpoint receptors, such as PD-1, Tim-3, LAG-3 and TIGIT, in the paired peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC), by multicolor flow cytometry. In BMMC, PD-1+, and PD-1+TIGIT+ were increased in CD8+ T cells, compared to the paired PBMC. Only a few patients showed Tim-3+ or LAG-3+ T cell population, and the most identified Tim-3+ or LAG-3+ T cell populations were observed in PD-1hiCD8+ T cells in the BMMC. There was significantly increased PD-1+, and PD-1+TIGIT+ expression in CD69+CCR7−CD8+ population compared to CD69− CCR7−CD8+ in BMMC. On direct ex vivo MHC multimer analysis of BMMC, tumor-associated-antigen specific CD8+ T cell showed increased PD-1+, TIGIT+, PD-1+TIGIT+ and CD69+ expression. PD-L1 was also expressed on malignant plasma cell, and the level of expression was correlated with the disease burden. Collectively, our data show that the immune checkpoint receptors-expressing T cells are enriched in bone marrow, where malignant plasma cells reside. Functional restoration of these T cells by immune checkpoint blockers will be investigated in further study.

High PD-1 expression on regulatory and effector T-cells in lung cancer draining lymph nodes.

The treatment of advanced nonsmall cell lung cancer (NSCLC) with PD-1/PD-L1 immune checkpoint inhibitors has improved clinical outcome for a proportion of patients. The current challenge is to find better biomarkers than PD-L1 immunohistochemistry (IHC) that will identify patients likely to benefit from this therapy. In this exploratory study we assessed the differences in T-cell subsets and PD-1 expression levels on T-cells in tumour-draining lymph nodes (TDLNs) and peripheral blood mononuclear cells (PBMCs).To evaluate this, flow cytometric analyses were performed on endobronchial ultrasound-guided (EBUS) fine-needle aspirates (FNA) from TDLNs of patients with NSCLC, and the results were compared to paired PBMC samples. For a select number of patients, we were also able to obtain cells from a non-TDLN (NTDLN) sample.Our data show that the frequency of PD-1+ CD4+ and CD8+ T-cells, as well as the PD-1 expression level on activated regulatory T (aTreg) and CD4+ and CD8+ T-cells, are higher in TDLNs than in PBMCs and, in a small sub-analysis, NTDLNs.These elevated PD-1 expression levels in TDLNs may reflect tumour-specific T-cell priming and conditioning, and may serve as a predictive or early-response biomarker during PD-1 checkpoint blockade.

Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) (“early ART”), and 7 participants started ART >14 months after EDI (“late ART”). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2–3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

Network-based analysis of differentially expressed genes in cerebrospinal fluid (CSF) and blood reveals new candidate genes for multiple sclerosis.

BackgroundThe involvement of multiple genes and missing heritability, which are dominant in complex diseases such as multiple sclerosis (MS), entail using network biology to better elucidate their molecular basis and genetic factors. We therefore aimed to integrate interactome (protein–protein interaction (PPI)) and transcriptomes data to construct and analyze PPI networks for MS disease.MethodsGene expression profiles in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) samples from MS patients, sampled in relapse or remission and controls, were analyzed. Differentially expressed genes which determined only in CSF (MS vs. control) and PBMCs (relapse vs. remission) separately integrated with PPI data to construct the Query-Query PPI (QQPPI) networks. The networks were further analyzed to investigate more central genes, functional modules and complexes involved in MS progression.ResultsThe networks were analyzed and high centrality genes were identified. Exploration of functional modules and complexes showed that the majority of high centrality genes incorporated in biological pathways driving MS pathogenesis. Proteasome and spliceosome were also noticeable in enriched pathways in PBMCs (relapse vs. remission) which were identified by both modularity and clique analyses. Finally, STK4, RB1, CDKN1A, CDK1, RAC1, EZH2, SDCBP genes in CSF (MS vs. control) and CDC37, MAP3K3, MYC genes in PBMCs (relapse vs. remission) were identified as potential candidate genes for MS, which were the more central genes involved in biological pathways.DiscussionThis study showed that network-based analysis could explicate the complex interplay between biological processes underlying MS. Furthermore, an experimental validation of candidate genes can lead to identification of potential therapeutic targets.

Malignant Mesothelioma Effusions Are Infiltrated by CD3+ T Cells Highly Expressing PD-L1 and the PD-L1+ Tumor Cells within These Effusions Are Susceptible to ADCC by the Anti–PD-L1 Antibody Avelumab

IntroductionThe functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. MethodsTumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry, and its prognostic significance was examined. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1–positive and PD-L1–positive infiltrating lymphocytes and their role in inducing PD-L1 expression in tumor cells. Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines, was evaluated in presence of autologous and allogeneic natural killer cells. ResultsOf 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1–positive, which was associated with slightly inferior overall survival compared to patients with PD-L1–negative tumors (median 23.0 versus 33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples testing positive, respectively. In nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients with paired malignant effusion and peripheral blood mononuclear cell (PBMC) samples, PD-L1 expression was significantly higher on CD3-positive T cells present in malignant effusions as compared with PBMCs (p = 0.016). In addition, the numbers of CD14-positive PD-1–positive cells were increased in malignant effusions compared with PBMCs (p = 0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced interferon-γ–mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab-mediated ADCC in the presence of autologous natural killer cells. ConclusionsMost pleural as well as peritoneal mesotheliomas express PD-L1. Malignant effusions in this disease are characterized by the presence of tumor cells and CD3-positive T cells that highly express PD-L1. In addition, mesothelioma tumor cells are susceptible to ADCC by the anti–PD-L1 antibody avelumab.

Abstract 5142: Correlation of peripheral blood S100A9+ myeloid-derived suppressor cells and tumor-associated macrophages in lung adenocarcinoma patient

Abstract Background: Peripheral blood S100A9+ monocytic myeloid derived suppressor cells (MDSCs) is a predictive factor for treatment response of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) of lung adenocarcinoma patients. Tumor associated macrophages (TAMs), which might derived from peripheral blood monocyte, is also a predictive factor to treatment response of EGFR-TKI. However, the correlation of S100A9+ monocytic MDSCs and TAMs is not well understood in lung adenocarcinoma patients. Methods: Stage IV lung adenocarcinoma patients with paired formalin fixed paraffin embedded (FFPE) tumor tissue and peripheral blood mononuclear cell (PBMCs) were enrolled. CD68+ cells were calculated in 3 different 200X microscopic field of FFPE as TAMs, and S100A9+ cells also calculated with same method as MDSCs in tumor microenvironment. S100A9+ MDSCs percentage was calculated by flow cytometry from CD14+S100A9+ in PBMC. Results: Twenty-four lung adenocarcinoma patients with paired FFPE tissue and PBMC data were enrolled. PBMC S100A9+ MDSCs percentage is 17.9± 13.6; average number of S100A9+ cells and CD68+ TAMs in tumor tissue is 96.8±45.7 and 79.3±13.3, respectively. The correlation between S100A9+ MDSCs percentage and tissue S100A9+ cells and CD68+ TAMs is significant (Spearman r = 0.54, p = 0.003 for S100A9+; Spearman r = 0.03, p = 0.03 for CD68; n = 24) CD68+ and S100A9+ cells were also correlated (Spearman r = 0.41, p = 0.04, n = 24). Clinically, high or low (cutoff value as median number) tissue S100A9+ cells and CD68+ TAMs were both predictive factors for treatment PFS for EGFR-TKI. (For CD68, median: 56, high vs low TAM PFS = 7.4 vs 15.4 months, p = 0.04; For S100A9+ cells, median: 76, high vs low S100A9+ cells PFS = 7.2 vs 13.6 months, p = 0.05) Conclusion: Peripheral blood S100A9+ MDSCs is well correlated to tumor tissue S100A9+ cells and CD68+ TAMs. Both tissue S100A9+ cells and CD68+ TAMs were predictive factors for EGFR-TKI PFS. Our study suggests that peripheral blood S100A9+ monocytic MDSC might be one of the origin of tumor tissue CD68+ TAMs. Citation Format: Kang-Yun Lee, Po-Hao Feng. Correlation of peripheral blood S100A9+ myeloid-derived suppressor cells and tumor-associated macrophages in lung adenocarcinoma patient. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5142.

Epstein–Barr virus load in transplant patients: Early detection of post-transplant lymphoproliferative disorders

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection.Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1–4), advanced PTLD (categories 2–4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units.The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/105 PBMC or 2.30; 2.60; 4.47loggEq/105 OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma.The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression.

BackgroundRheumatoid arthritis (RA) is often characterized by the presence of rheumatoid factor, anti-citrullinated protein antibodies, and bone erosions. Current therapies can compromise immunity, leading to risk of infection. The interleukin-20 receptor (IL-20R) axis comprising IL-19, IL-20, and IL-24 and their shared receptors activates tissue homeostasis processes but not the immune system. Consequently, modulation of the IL-20R axis may not lead to immunosuppression, making it an interesting drug target. We evaluated the role of the IL-20R axis in RA and associations between plasma cytokine levels and clinical disease.MethodsPlasma IL-19, IL-20, and IL-24 levels were measured in early RA patients during a treat-to-target strategy by enzyme-linked immunosorbent assays. The IL-20R1 and IL-22R1 levels in paired peripheral blood mononuclear cells and synovial fluid mononuclear cells from a different cohort of RA patients were evaluated by flow cytometry and confocal microscopy. Monocytes/macrophages were stimulated with heat-aggregated human immunoglobulin immune complexes and immune complexes containing citrullinated fibrinogen, and osteoclasts were incubated with IL-19, IL-20, and IL-24.ResultsThe plasma concentrations of IL-20 and IL-24 (but not IL-19) were increased in early RA patients compared with healthy controls (both P < 0.002) and decreased after 6 months of treatment (both P < 0.0001). The expression of IL-22R1 (but not IL-20R1) was increased on monocytes from RA synovial fluid compared with monocytes from both RA and healthy control peripheral blood. The plasma concentrations of IL-20 and IL-24 were increased in rheumatoid factor and anti-citrullinated protein antibody positive compared with negative early RA patients (all P < 0.0001). Immune complexes stimulated the production of the IL-20R cytokines by monocytes/macrophages. Increased baseline plasma concentrations of IL-20 and IL-24 were associated with Sharp-van der Heijde score progression after 24 months (Spearman’s rho = 0.19 and 0.26, both P < 0.05) in the early RA patients. The IL-22R1 was expressed by osteoclast precursors and in multinucleated osteoclasts. IL-20 and IL-24 increased the secretion of monocyte chemoattractant protein 1 by these cells.ConclusionsThis study suggests that IL-20 and IL-24 link RA-associated autoantibodies with radiographic progression via the IL-22R1. Modulation of this axis holds promise as feasible anti-erosive treatment modalities in seropositive RA.

Molecular Characterisation and HIV Drug Resistance Patterns of HIV-1 Variants in Plasma and Peripheral Blood Mononuclear Cells Sample Pairs

Objective: Plasma is currently the specimen of choice for routine HIV drug resistance (HIVDR) testing. However, HIV compartmentalization has been well documented in different reservoirs, including peripheral blood mononuclear cells (PBMCs). We molecularly characterized paired plasma and PBMC sample pairs and compared their HIVDR mutation patterns. Methods: To compare HIVDR mutation patterns in plasma and PBMC sample pairs, whole blood was collected for plasma and PBMC isolation from 43 treatment-naive and 10 treatment-experienced individuals, and HIVDR profiles determined by sequencing the pol gene. Treatment-naive individuals were initiated on Efavirenz/Emtricitabine/ Tenofovir fixed dose combination therapy and follow up HIV-1 viral loads were performed after at least 6 months of treatment. Results: HIVDR mutation prevalence in the treatment-naive group was 5.1% in plasma samples compared to 10.3% when both plasma and PBMC sequences were considered. Variable amino acid positions were detected in 21% and 12% of the protease and reverse transcriptase genes, respectively. These were generally not in HIVDR positions and did not form signature patterns. The subset of patients with additional resistance associated sequence variations detected in PBMCs had undetectable HIV-1 viral loads after at least 6 months of anti-retroviral therapy. Conclusions: The sequencing of paired plasma and PBMC specimens provided additional HIVDR data which were not detected when only plasma samples were tested. However the resistant variants detected in the PBMCs did not seem to negatively affect treatment outcome at 6 months as viral suppression was achieved. These data highlight proviral HIVDR mutations in HIV infected individuals and longer follow up on patients on treatment may be needed to determine their clinical impact.

Differential expression of microRNAs in renal transplant patients with acute T-cell mediated rejection

BackgroundMicroRNAs (miRNAs) regulate most of encoding genes and protein. In this study, we aimed to investigate the expression levels of miR-142-5p, miR-142-3p, miR-155 and miR-223 in paired biopsy and peripheral blood mononuclear cell (PBMC) samples of renal allograft recipients with acute T-cell mediated rejection (ATCMR), compared with normal allografts (NA). MethodsIn this study, the expression levels of individual miRNAs were determined in biopsy and PBMC samples of 17 recipients with ATCMR and 18 recipients with NA. ResultsOur results showed that the intragraft expression levels of all studied miRNAs were significantly higher in ATCMR than NA. However, regarding the PBMC samples, miR-142-3p and miR-223 were significantly increased in ATCMR than NA. Receiver operating characteristic (ROC) analysis showed that miR-142-5p, miR-142-3p, miR-155 and miR-223 in biopsy samples and miR-142-3p and miR-223 in PBMC samples could discriminate ATCMR from NA recipients. ConclusionIt has been reported that high intragraft expressions of miRNAs have a profound role in the pathogenesis of ATCMR process. Our results showed that high expression of all the studied miRNAs in biopsies and miR-142-3p and miR-223 in PBMC samples could be used as suggestive diagnostic tools to discriminate ATCMR patients from NA.

In vivo Investigations of the Effect of Short- and Long-Term Recombinant Growth Hormone Treatment on DNA-Methylation in Humans

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

Correlation of low CD73 expression on synovial lymphocytes with reduced adenosine generation and higher disease severity in juvenile idiopathic arthritis.

ObjectiveTo investigate the expression and adenosine‐generating activity of the ecto‐5′‐nucleotidase CD73 on synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from children with juvenile idiopathic arthritis (JIA).MethodsGiven the role of CD73 protein in the production of antiinflammatory adenosine and its intersection with inflammatory biologic pathways, the expression of CD73 on SF and PB lymphocytes from patients with JIA and PB lymphocytes from healthy control subjects was determined by flow cytometry. The AMPase activity of CD73 on PBMCs and SFMCs was measured by high‐performance liquid chromatography. The effects of cell activation on CD73 expression were examined by in vitro culture of PBMCs.ResultsCD8+ and CD19+ SFMCs from patients with JIA expressed decreased levels of CD73 when compared to paired PBMCs from JIA patients and PBMCs from healthy controls. When the percentages of CD73+ synovial lymphocytes were compared between the 2 clinical forms of oligoarthritis, children with extended oligoarthritis showed lower CD73 expression compared to those with the milder form of the disease. CD8+ SFMCs had a lower ability to produce adenosine from etheno‐AMP compared to CD8+ PBMCs. T cell activation through the T cell receptor (TLR) of CD8+CD73+ cells and B cell activation through TLR‐9 resulted in reduced expression of CD73. This down‐regulation occurred on dividing cells.ConclusionThese findings show that low CD73 expression on T and B cells in the inflamed site is related to cell proliferation and is correlated with the clinical severity of oligoarticular JIA. The decreased CD73 expression on SFMCs, in turn, results in reduced adenosine production, which leads to a decreased potential for antiinflammatory activity.

Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases.

Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ~20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mononuclear cells of immune-active CHB carriers, compared with other disease phases. During all CHB disease phases, differences exist between HBV variants found in peripheral blood mononuclear cells and plasma. Moreover, these data indicate that HBV evolution occurs in a compartment and disease phase-specific fashion.

Differential impact of APOBEC3-driven mutagenesis on HIV evolution in diverse anatomical compartments

Previous studies on HIV quasispecies have revealed HIV compartmentalization in various tissues within an infected individual. Such HIV variation is a result of a combination of factors including high replication and mutation rates, recombination, and APOBEC3-host selective pressure. To evaluate the differential impact of APOBEC3 editing in HIV-1 compartments, we analyzed the level of G-to-A hypermutation in HIV-1 protease and reverse transcriptase sequences among 30 HAART-treated patients for whom peripheral blood mononuclear cells and body tissues or fluids [cerebral spinal fluid (CSF), rectal tissue, or renal tissue] were collected on the same day. APOBEC3-mediated hypermutation was identified in 36% (11/30) of participants in at least one viral reservoir. HIV hypermutated sequences were often observed in viral sanctuaries (total n = 10; CSF, n = 6; renal tissue, n = 1; rectal tissue n = 3) compared with peripheral blood (total n = 4). Accordingly, APOBEC3 editing generated more G-to-A drug resistance mutations in sanctuaries: three patients' CSF (i.e. G73S in protease; M184I, M230I in reverse transcriptase) and two other patients' rectal tissues (M184I, M230I in reverse transcriptase) while such mutations were absent from paired peripheral blood mononuclear cells. APOBEC3-induced mutations observed in peripheral blood underestimate the overall proportion of hypermutated viruses in anatomical compartments. The resulting mutations may favor escape to antiretrovirals in these compartments in conjunction with a lower penetration of drugs in some sanctuaries. On the other side, because hypermutated sequences often harbor inactivating mutations, our results suggest that accumulation of defective viruses may be more dominant in sanctuaries than in peripheral blood of patients on effective HAART.