Mononuclear Cells Of Patients Research Articles

Immunophenotyping and Function of Peripheral Blood Mononuclear Cells in Patients Undergoing Unrelated Allogeneic Transplantation with Post-Transplantation Cyclophosphamide in Combination with ATG Anti-Graft Versus Host Disease Prophylaxis

Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages > 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p<0.05) and CD8+ cells (60%, p<0.02) from pts receiving PTCy/ATG. Similarly, an increased frequency of LAG-3+ CD8+ cells was observed in the PTCy /ATG pts, in comparison to the no-ATG and ATG groups. Concomitantly, HLA-DR expression on CD56+ NK subset was decreased in PTCy/ATG pts versus the ATG and non-ATG groups (47% vs 61% vs 67%, respectively, p<0.05), indicating reduced activation of NK cells. Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p<0.02). CD57 up-regulation combined with CD28 down-regulation on T cells reflected an exhausted/senescent phenotype; therefore, we further analyzed the frequency of CD28-CD57+ T cells. Significantly elevated expression of CD57 on CD8+CD28- cells was detected in the PTCy/ATG cohort, in comparison to the ATG and no-ATG groups (65% vs 43% vs 21%, respectively, p<0.01). Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p<0.01). Moreover, significantly reduced production of IFNγ in response to stimulation was detected in PTCy/ATG and ATG pts (p<0.01). PBMC from the no-ATG cohort displayed inducible IFNγ production levels comparable with healthy controls. Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.

Investigation of Homing Capacity As Prognostic Marker in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most frequent myeloid malignancy in adults, causing death of about 90% of elderly patients. Cytogenetic and molecular abnormalities are used to categorize AML in favorable, intermediate and adverse molecular risk groups. Classification in one of these groups will directly affect clinical decision-making. Although molecular criteria have significantly improved prognostication and thus AML patient stratification and risk-based treatment, they still do not allow full risk prediction. Using a xenotransplantation model of human AML samples into NOD/SCID/IL2Rgnull(NSG) mice, we have previously shown that favorable risk AML engraft NSG mice after longer latency when compared to intermediate or adverse risk AML (Paczulla et al., 2017). This suggests that this mouse model accurately depicts important features of human AML. To further investigate the relationship between in vivo leukemia latency in NSG mice and AML outcome in patients, we have expanded our analyses to a cohort of n=23 favorable AML patient samples with diverse cytogenetic and molecular features. We hypothesize that the time-to-leukemia induction in mice correlates with the capacity of homing to the bone marrow (BM) as well as with the outcome in patients. AML cells were derived from peripheral blood mononuclear cells of patients, and depleted for CD19-positive B- as well as CD3-positive T-cells by MACS. Patient-derived AML blasts were freshly thawed before each transplantation and intra-femorally transplanted into previously irradiated 6-8 week-old female NSG (500.000 cells per mouse). Transplanted mice were monitored for engraftment at two time points post-transplantation (16 weeks and 6 months) via BM biopsy and detection of human hematopoietic markers (CD33 or CD34, depending on patients features) using flow cytometry. At signs of disease or when more than 80% leukemic blasts were detected in the BM biopsy, mice were euthanized and leukemic infiltration of BM, peripheral blood (PB) and organs investigated using multi-color flow cytometry. Homing capacity was investigated by intravenous transplantation of 1.000.000 CFSE-labeled leukemic blasts in non-irradiated 6-8 week-old male NSG mice. 16 hours later, mice were euthanized and BM and PB analyzed for detection of fluorescent leukemic cells. Interestingly, all transplanted favorable AML samples were able to engraft NSG mice within 54 weeks after transplantations, with very different time-to-leukemia induction between patients. Survival proportion at week 16 was 81.5%, 78.1% at week 26 (6 months) and dropped to 27.3 % at week 40 (9 months). From the BM punctures performed at week 16 (n=65 mice, n=23 AML patients), 44.62% showed more than 1%, 76.92% more than 0.1% and 84.62% more than 0.01 % human leukemic cells. Notably, although some mice showed very low engraftment at week 16, they nevertheless developed leukemia at a later time point. Interestingly, results from BM punctures seem to nicely correlate with long-term survival. Indeed, mice for which more than 1% engraftment was detected at the 16-week time-point showed a 50% survival rate at week 26, 42% at week 26 and 27% at week 40 (in comparison to respectively 92%, 80% and 61% survival for mice with <1% leukemic cells at the same time-point). Furthermore, homing capacity of leukemic cells also seems to correlate with long-term engraftment (n=56 mice, n=20 AML patients). In fact, we could observe a tendency for increased homing to the BM for the samples that resulted in more than 1% engraftment at week 16 in comparison to those with less than 1% (median homing 0.0049% vs. 0.0029%). Taken together, our recent work demonstrates in a large set of patient samples that - in contrast to several previous publications - human AML cells of favorable molecular risk indeed routinely engraft NSG mice. Furthermore this work points out to correlations between homing capacity, BM engraftment at week 16 and long-term survival and time-to-leukemia in NSG mice. Comparative studies using AML cells of intermediate and adverse molecular risk are underway. These findings provide proof-of-principle data for a prospective clinical study analyzing correlations between AML cell homing capacity in mice and outcome of disease in patients. Disclosures No relevant conflicts of interest to declare.

Effect of miR-214 on Fludarabine Resistance in Chronic Lymphocytic Leukemia

To investigate effect and mechanism of miR-214 in fludarabine resistance of chronic lympho-cytic leukemia (CLL). A total of 10 patients with CLL resistante to fludarabine (Flu) and 10 healthy persons admitted to Hematology Department of our hospital in August 2014 - July 2018 were selected. Expression level of miR-214 in mononuclear cells in patients with CLL and healthy persons were determined by RT-PCR. Primary CLL cells from patients with CLL were divided into normal control group (control group), negative control group (miR-214-NC group) and viral transinfection group (miR-214-ASO group). After 24 h-transfection, CLL cells were cultured with different con-centration of Flu for 48 h, then the cell proliferation and apoptosis were detected, and the levels of down-stream genes and proteins releted with PTEN and PI3K/AKT signialing pathway were determined. The expression level of miR-214 in mononuclear cells of CLL patients significantly increased in comparison with healthy persons(P<0.05); the expression level of miR-214 in miR-214-ASO group significantly decreased (P<0.05); Absorbance in control group at Flu concentration of 3, 10 and 30 μmol/L was significantly decreased (P<0.05). Apoptosis rate in miR-214-ASO group at Flu concentration of 10 mmol/L significantly increased (P<0.05). At Flu concentration of 10 mmol/L, mRNA levels PTEN and BAD in miR-214-ASO group significantly increased (P<0.05), but mRNA levels of MDM2 and NF-κB significantly decreased (P<0.05). At Flu concentration of 10 mmol/L, protein levels of PTEN and p-BAD in miR-214-ASO group significantly increased (P<0.05), but protein levels of MDM2 and NF-κB significantly decreased (P<0.05). Inhibition of miR-214 can enhance the sensitivity of drug-resistant CLL cells to fludarabine, which may be raleted with the promotion of cell apotosis and regulation of down-stream molecules expression of PTEN/AKT signaling pathway.

Altered expression of long noncoding RNAs in peripheral blood mononuclear cells in patients with impaired leptomeningeal collaterals after acute anterior large vessel occlusions.

In the event of acute ischemic stroke (AIS) due to anterior large vessel occlusion (aLVO), leptomeningeal collaterals (LMCs) status is a key factor to define the severity and functional prognosis of this disease. However, the extent of LMCs exhibits substantial variability among the patients, which is genetic determined. Long non-coding RNAs (lncRNAs) expression profiles in human peripheral blood have been found to be altered after AIS. But whether there are specific lncRNAs correlated with LMC status in aLVO has not yet been investigated. Differential lncRNA expression panels in peripheral blood mononuclear cells (PBMCs) were assessed by microarray analysis and individual quantitative real-time polymerase chain reaction (RT-PCR) in three independent sets consist of 134 patients with aLVO and 73 healthy controls (HCs). LMCs Status in those patients was assessed based on baseline computed tomographic angiography (CTA). Microarray analysis showed 23 differentially expressed lncRNAs in patients with poor LMCs status. After independent validations by RT-PCR, lncRNA ENST00000422956 was found to be significantly downregulated in patients with poor LMCs status. Receiver-operating characteristic (ROC) analysis revealed the area under the ROC curve (AUC) for ENST00000422956 to predict poor LMCs status was 0.749. Moreover, ENST00000422956 expression level and baseline National Institutes of Health Stroke Scale (NIHSS) score were identified as independent predictors for impaired LMCs, and a significantly positive correlation was observed between ENST00000422956 expression level and LMCs status. Via cis-regulatory analysis, paired box 8 (Pax8) was identified as the target gene for ENST00000422956. The dysregulated lncRNA ENST00000422956 in PBMCs was associated with impairment of LMCs in patients with aLVO, suggesting that measurement of circulatory lncRNAs might be included as possible biomarkers for evaluation of LMCs status in AIS. More importantly, this might be the foundation for understand the potential roles of lncRNAs in LMCs formation after ischemic stroke.

Full - transcriptome analysis of miRNA expression in mononuclear cells in patients with acute decompensation of chronic heart failure of various etiologies

Analysis of miRNA expression levels in mononuclear blood cells (MBC) of patients with acute decompensation f chronic heart failure (CHF) of various etiologies and evaluation of the possibility of their use as a biological marker. 7 male patients with acute decompensation of CHF with a reduced ejection fraction (EF), NYHA functional class II-IV (FC) according to NYHA [mean (M) EF 29.2%, standard deviation (SD) 13.27%] in age 38 to 65 years old [median (Me) 58 years]. In 3 patients, heart failure developed as a result of dilated cardiomyopathy (DCMP), in 4 patients - against the background of post - infarction cardiosclerosis of the ischemic nature [group of patients with coronary heart disease (CHD)]. The control group - 5 age - matched (from 41 to 57 years old, Me 49 years old) healthy male volunteers. A complete transcript analysis of miRNA expression in MNCs was performed for all patients and healthy volunteers. Differentially expressed miRNAs were determined in patients with CHF (regardless of etiology) compared with healthy individuals: miR-182, miR-144, miR-183, miR-486-5p, miR-143 (log2FC >1, FDR p - value.

Expression and function of Toll‑like receptors in peripheral blood mononuclear cells in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common chronic inflammatory autoimmune disease. Toll-like receptors (TLRs) are involved in non-specific immunity. In the present study, the roles of TLRs in AS were investigated. The levels of inflammatory cytokines were detected by ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of TLRs and nuclear factor-κB (NF-κB) signaling-associated factors were determined via RT-qPCR and western blot analyses. It was observed that the levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), C-reactive protein, TLR4 and TLR5 were increased in patients with AS, whereas those of IL-10 and TLR3 were decreased. Pomalidomide, a TNF-α release inhibitor, reduced the expression of IL-6, TNF-α, TLR4, TLR5 and phosphorylated-p65, and upregulated that of IL-10, TLR3 and p65 in peripheral blood mononuclear cells from patients with AS. Treatment of patients with infliximab, an anti-TNF-α monoclonal antibody, induced similar effects in vivo. In conclusion, it was revealed that inhibition of TNF-α suppressed inflammatory responses in patients with AS, increased the expression of TLR3 and decreased NF-κB signaling, and the expression of TLR4 and TLR5. The results indicated that TLRs and the NF-κB signaling pathway were involved in the regulation of inflammatory responses in AS. These findings provided insight into the mechanisms underlying the development of AS and potential novel therapeutic approaches.

Prevalence and effect of occult hepatitis C infection in patients with persistent liver enzyme elevation after achieving 24 weeks of sustained virological response

EnBackgroundDespite achieving sustained virological response (SVR) of chronic hepatitis C infection, some of the treated patients have persistent elevations of transaminases. Occult hepatitis C infection (OCI) could be one of the causes.AimThe aim of this study was to detect OCI in peripheral blood mononuclear cells in patients who achieved 24-week SVR with persistent elevations of transaminases.MethodsWe included 998 naïve chronic HCV-infected patients who received treatment at our hospital. Patients with elevated liver enzymes after achieving SVR were determined. HCV RNA PCR in peripheral blood mononuclear cells was done for those patients (group 1) and was compared with a group with normal levels of enzymes, which was matched in age and sex (group 2).ResultsNine hundred and sixty-five patients achieved SVR (96.69%). Seventy-four (7.7%) patients of them had elevated enzymes. OCI was detected in 14/74 (18.9%) patients of group 1, whereas it was seen in 4/67 (5.9%) in group 2. Cirrhosis, OCI, and obesity were associated with this enzymes elevation (P=0.005, 0.024, and <0.001). By multivariate analysis, none of these three parameters were independently associated with the enzyme elevation. The presence of OCI was not significantly associated with the presence of cirrhosis or obesity.ConclusionOCI is not infrequent in patients with persistent transaminase elevations despite obtaining 24 weeks of SVR. Liver cirrhosis, OCI, and obesity could have synergistic effects and should be considered as important risk factors of this persistent enzyme elevation.

Extracellular vesicles with ubiquitinated adenosine A2A receptor in plasma of patients with coronary artery disease.

Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A2A receptor (A2AR), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A2AR. Interestingly, the presence of ubiquitinated A2AR in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A2AR expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A2AR in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A2AR‐containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A2AR in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.

The transcriptome of peripheral blood mononuclear cells in patients with clinical subtypes of late age-related macular degeneration

BackgroundPeripheral blood mononuclear cells (PBMCs) are implicated in the pathogenesis of age-related macular degeneration (AMD). We here mapped the global gene transcriptome of PBMCs from patients with different clinical subtypes of late AMD.ResultsWe sampled fresh venous blood from patients with geographic atrophy (GA) secondary to AMD without choroidal neovascularizations (n = 19), patients with neovascular AMD without GA (n = 38), patients with polypoidal choroidal vasculopathy (PCV) (n = 19), and aged control individuals with healthy retinae (n = 20). We isolated PBMCs, extracted RNA, and used microarray to investigate gene expression. Volcano plots identified statistically significant differentially expressed genes (P < 0.05) at a high magnitude (≥30% higher/lower) for GA (62 genes), neovascular AMD (41 genes), and PCV (41 genes). These clinical subtypes differed substantially across gene expression and the following pathways identified in enrichment analyses. In a subgroup analysis, we investigated presence vs. absence of subretinal fibrosis and found 826 differentially expressed genes (≥30% higher/lower, P < 0.05) with relation to mRNA splicing, endothelial migration, and interleukin-1 signaling.ConclusionsWe here map the global gene transcriptome of PBMCs related to clinical subtypes of late AMD and find evidence of subtype-specific immunological involvement. Our findings provide a transcriptomic insight into the systemic immunity associated with AMD.

ASSESSMENT OF MICRORNA-21 GENE EXPRESSION LEVELS IN RELATION WITH THE DETERIORATION OF LEFT VENTRICULAR STRAIN IN HYPERTENSIVE PATIENTS

Objective: MicroRNAs (miRs) modulate cardiovascular development and disease by post-transcriptional gene expression regulation and thus they are emerging as potential biomarkers and promising therapeutic targets in cardiovascular disease. miR-21 is found to play important roles in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions.. We evaluated miR-21 gene expression levels in peripheral blood mononuclear cells in patients with essential hypertension in relation to left ventricular global longitudinal peak strain (GLPS). Design and method: We included 50 patients with newly diagnosed essential hypertension stage 1 and 2 (31 men, mean age 69.5 ± 11.8 years, mean systolic blood pressure (SBP) 167 ± 19 mmHg and mean diastolic blood pressure (DBP) 107 ± 11 mmHg). All patients underwent a serial assessment with standard conventional transthoracic and a two-dimensional speckle tracking echocardiography at baseline and at 24 months follow-up. miR-21 gene expression levels in peripheral blood mononuclear cells were quantified by real-time reverse transcription polymerase chain reaction. Results: Systolic and diastolic blood pressure was significantly reduced in the 12 months follow up under antihypertensive medication (SBP: from 167 ± 19 mmHg to 147 ± 15 mmHg and DBP: from 107 ± 11 mmHg to 95 ± 15 mmHg, p < 0.05 for both). GLPS showed a significant reduction during the 24 months follow-up (from −18.3 ± −4.9 % at baseline to −16.5 ± −6.9%, p = 0.04). miR-21 gene expression levels at baseline revealed a significant positive correlation with the reduction of GLPS (r = 0.52, p < 0.001). This correlation was independent of the patients’ clinical parameters Conclusions: Our data reveal that miR-21 might have a prognostic value in the deterioration of left ventricular GLPS in essential hypertension. In addition, it may be involved in the pathophysiology of in hypertensive heart disease and may be a promising therapeutic target.

IL-8 and IL-23 Levels in Peripheral Blood Mononuclear Cells of Patients with Cutaneous Leishmaniasis Caused by Leishmania major: A Case-Control Study

Background: Several cytokines contribute to cutaneous leishmaniasis outcomes. IL-23 is a key cytokine in inflammation and a central cytokine in controlling Th17. IL-8, also known as a neutrophil chemotactic factor in the lesion site of cutaneous leishmaniasis, indicates disease promotion. Objectives: This study aimed to evaluate the relationship of Leishmania major with IL-8 and IL-23 expression. Methods: This case-control study was performed on 25 healthy individuals and 25 cutaneous leishmaniasis patients who lived in Southwestern Iran, in 2017. Peripheral blood mononuclear cells (PBMCs) were first isolated. Total RNA was extracted and reverse-transcribed into cDNA. The expression levels of IL-8 and IL-23 were measured by the real-time PCR. The data were statistically analyzed using SPSS version 16. Results: Our findings indicated that the expression of IL-8 was significantly higher in the leishmaniasis group than in the control group with the median (IQR) of 0.39 (0.92) and 0.03 (0.11), respectively (P = 0.02). The expression of IL-23 was higher in the leishmaniasis group than in the control group with the median (IQR) of 0.13 (0.43) and 0.11 (0.61), respectively (P = 0.48). The increased IL-8 expression accompanied the location sites in face and hand and the increased number of skin lesions. Conclusions: The expression of IL-8 in patients with cutaneous leishmaniasis is an index of increased activity of local neutrophils that can contribute to leishmaniasis survival or inflammation increase. However, IL-23 appears to be less important in the inflammatory reaction than IL-8.

Interleukin-25 is upregulated in patients with systemic lupus erythematosus and ameliorates murine lupus by inhibiting inflammatory cytokine production

Interleukin-25 (IL-25), an anti-inflammatory member of the IL-17 family of cytokines, has been extensively investigated in multiple autoimmune and inflammatory diseases. However, its pathogenic role in systemic lupus erythematosus (SLE) remains largely unknown. This study aimed to explore the expression and clinical significance of IL-25 in patients with SLE as well as its pathogenic role in lupus-prone MRL/lpr mice. The results showed that IL-25 mRNA and serum levels were increased in patients with SLE compared with those in healthy controls. Higher IL-25 mRNA and serum levels were found in patients with an active disease. IL-25 levels were positively associated with SLEDAI, anti-dsDNA, and IgG but negatively associated with C3 and C4. Ex vivo assay showed that IL-25 could inhibit the production of the inflammatory cytokines IL-1β, IL-17, IL-6, and IFN-γ as well as TNF-α in the peripheral blood mononuclear cells in patients with SLE. In vivo studies revealed that treatment with IL-25 significantly ameliorated lupus symptoms in lupus-prone MRL/lpr mice by suppressing the production of inflammatory cytokines, including IL-1α, IL-1β, IL-6, IL-12p70, IL-17A, and IFN-β. Cumulatively, our results suggest that IL-25 levels are increased in patients with SLE and associated with disease activity; IL-25 plays a potent immunosuppressive role in the pathogenesis of SLE by suppressing the production of inflammatory cytokines. IL-25 could potentially be used as a diagnostic and therapeutic target for SLE treatment.

1696-P: PBMC of Recent-Onset Patients with Type 1 Diabetes Present a Differential Gene Expression Profile

Introduction: Type 1 diabetes is characterized by the autoimmune destruction of insulin-secreting β-cells, mediated by T auto reactive infiltrating cells, inflammatory cytokines and immunological mechanisms. However, little is known about the expression of genes and pathways dysregulated in peripheral blood mononuclear cells of patients with T1D. Objective: to investigate the gene expression profiles of circulating PBMC in recent-onset T1D patients (up to 6 months of diagnosis) in comparison with controls in the context of putative disease-related pathobiological processes and pathways. Methods: expression of mRNA from PBMC of 20 T1D patients, 15.8±8.6 years and age-matched 20 healthy controls, 14.7±6.7 years, (p&amp;gt;0.05) was evaluated with the Whole Human Genome Microarray Agilent and analyzed by GeneSpring with a fold change cutoff of 1.5 and adjusted P value &amp;lt;0.05; pathways analysis was performed with the software IPA. Results: 326 genes were differentially expressed between T1D patients and controls, being 204 (63%) upregulated, and 122 (37%) downregulated in T1D. Interactions between the differentially expressed genes evidenced expression patterns of 24 networks. The most significantly enriched canonical pathways were namely ErbB (erythroblastic leukemia viral oncogene) signaling, micropinocytosis signaling, savage pathways of pyrimidine ribonucleotides and hepathocyte growth factor in descending order of significance. The up-regulated protein targets within these signaling pathways were NEK2, PLK1, ATM, PRKCD, CCND1, NRG2 and those down regulated were AREG, CDC42 and CDADC1. Conclusion: The majority of upregulated genes of patients with T1D was related to cell cycle control, mitotic progression, proliferation and DNA repair, probably related to changes in PBMC metabolism and function due to DNA damage associated with hyperglycemia. The lower expression of AREG and CDC42 can be related to reduce immunosuppressive activity of regulatory T cells and B cell functions. Disclosure A. Santos: None. C. Chevillard: None. N. Gonfinetti: None. F. Bertonha: None. C. Moreira-Filho: None. J. Kalil: None. E. Cunha-Neto: None. M. Silva: None. Funding European Foundation for the Study of Diabetes; São Paulo Research Foundation

Endogenous DNA damage levels in peripheral blood mononuclear cells in patients with muscle-infiltrating urothelial bladder carcinoma.

e16020 Background: Cisplatin-based combination chemotherapy is used as a standard neoadjuvant treatment of patients with muscle-infiltrating urothelial bladder carcinoma (MIUBC). DNA damage represents one of the most important factors contributing to its toxicity. The objective of this prospective study is to evaluate the prognostic value of the endogenous DNA damage level in peripheral blood mononuclear cells (PBMCs) from MIUBC patients before treatment with neoadjuvant chemotherapy. Methods: PBMCs isolated from 25 consecutive MIUBC patients (16 men, 64%) were included into this study. Karnofsky index &lt; 80% was present in 1 patient (4%). DNA damage levels in PBMCs were evaluated by the Comet assay and scored as percentage of DNA in tail by the Metafer-MetaCyte analyzing software. Cut-off of 5.25 was used to dichotomize DNA damage level as “high” or “low” based on the previous study. Results: At the median follow-up 12.1 months, 13 patients progressed (52%) and 8 patients (32%) died. The median and IQR (interquartile range) of endogenous DNA damage level was 7.52 (4.07-27.9). Patients with low DNA damage levels had non-significantly better progression free survival (HR = 0.33, 95% CI: 0.09-1.29) and overall survival (HR = 0.64, 95% CI: 0.11-3.87) compared to patients with high DNA damage levels. Conclusions: These data suggest that endogenous DNA damage levels in PBMCs from MIUBC patients may serve as a prognostic marker early identifying patients with poor outcome. This study is supported by VEGA 2/0053/19 and APVV-17-0384.

Superoxide Dismutase Isoenzymes Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Coronary Artery Disease.

SummaryBackgroundCoronary artery disease (CAD) is one of the most important causes of mortality and morbidity in wide world population. Dyslipidemia, inflammation and oxidative stress may contribute to disruption of endothelium structure and function, atherosclerosis and CAD. Our study was aimed to determine whether Cu/Zn superoxide dismutase (Cu/Zn SOD) and Mn superoxide dismutase (Mn SOD) gene expression could be modulated by oxidative stress in CAD patients.MethodsThis study included 77 CAD patients and 31 apparently healthy persons. Serum lipid levels, high sensitivity C-reactive protein (hsCRP), total antioxidant status (TAS) and thiobarbituric acid-reacting substances (TBARS) were measured. SOD isoenzymes gene expression was determined in peripheral blood mononuclear cells using quantitative polymerase chain reaction.ResultsMn SOD messenger ribonucleic acid (mRNA) levels were significantly lower in CAD patients than in controls (p=0.011), while Cu/Zn SOD mRNA levels did not change significantly between tested groups (p=0.091). We found significantly lower high-density lipoprotein-cholesterol (HDL-c) (p<0.001) and TAS (p<0.001) levels and significantly higher hsCRP (p=0.002) and TBARS (p<0.001) in CAD patients than in controls. There were significant positive correlations between TAS and Mn SOD mRNA (ρ=0.243, p=0.020) and TAS and Cu/Zn SOD mRNA (r=0.359, p<0.001). TBARS negatively correlated only with Cu/Zn SOD mRNA (ρ=-0.215, p=0.040). TAS levels remained independent predictor for Mn SOD mRNA levels (OR=2.995, p=0.034).ConclusionsResults of this study showed that Mn SOD gene expression were decreased in CAD patients compared to controls and can be modulated by non-enzymatic antioxidant status in blood.

A proliferation-inducing ligand (APRIL) induced hyper-production of IgA from tonsillar mononuclear cells in patients with IgA nephropathy

IgA nephropathy (IgAN) is a tonsil-related disease. We previously showed that oligodeoxynucleotides with CpG (CpG-ODN) and B-cell activation factor (BAFF) are involved in hyperproduction of IgA from tonsillar mononuclear cells of patients with IgAN (IgAN-TMCs). In this study, we focused on a proliferation-inducing ligand (APRIL), homologous to BAFF. IgAN-TMCs produced more APRIL than non IgAN-TMCs in the presence of both CpG-ODN and control-ODN. TLR9 expression was higher in B-cells of IgAN-TMCs, and treatment with CpG-ODN enhanced transmembrane activator and CAML interactor (TACI) expression. IgA production from IgAN-TMCs was inhibited by APRIL neutralization antibody or TACI blocking antibody, and enhanced by co-treatment of APRIL and CpG-ODN. Serum APRIL levels were higher in patients with IgAN, and decreased after tonsillectomy. These findings suggest that APRIL is involved in the hyperproduction of IgA from IgAN-TMCs, and that CpG-ODN enhanced APRIL-induced IgA production by increasing TACI expression on B-cells of IgAN-TMCs.

Characterisation of peripheral blood mononuclear cells in patients with combination ipilimumab and nivolumab therapy-related colitis

Characterisation of peripheral blood mononuclear cells in patients with combination ipilimumab and nivolumab therapy-related colitis

Overexpression of Tumor Necrosis Factor-Like Ligand 1 A in Myeloid Cells Aggravates Liver Fibrosis in Mice.

Macrophages are the master regulator of the dynamic fibrogenesis–fibrosis resolution paradigm. TNF-like ligand 1 aberrance (TL1A) was found to be able to induce intestinal inflammation and fibrosis. Furthermore, significantly increased TL1A had been detected in liver tissues and mononuclear cells of patients with primary biliary cirrhosis (PBC). This study was to investigate the effect of myeloid cells with constitutive TL1A expression on liver fibrogenesis. We found that TL1A expressions in liver tissues and macrophages were significantly increased in mice with liver fibrosis induced by injection of carbon tetrachloride (CCl4). TL1A overexpression in myeloid cells induced liver function injury, accelerated the necrosis and apoptosis of hepatocytes, recruited macrophages, and promoted activation of hepatic stellate cells (HSCs) and fibrosis. In vitro results of our study showed that TL1A overexpression in macrophages promoted secretion of platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Culturing macrophages with TL1A overexpression could accelerate the activation and proliferation of primary HSCs. These results indicated that constitutive TL1A expression in myeloid cells exacerbated liver fibrosis, probably through macrophage recruitment and secretion of proinflammatory and profibrotic cytokines.

Lymphocyte Phenotypic Analysis of Peripheral Blood Mononuclear Cells in Patients with Systemic Mastocytosis

Lymphocyte Phenotypic Analysis of Peripheral Blood Mononuclear Cells in Patients with Systemic Mastocytosis

CXCL8 derived from mesenchymal stromal cells supports survival and proliferation of acute myeloid leukemia cells through the PI3K/AKT pathway.

The role of proinflammatory cytokines secreted by the bone marrow mesenchymal stromal cells (BM-MSCs) in the progression of acute myeloid leukemia (AML) is poorly understood. We compared C-X-C motif chemokine ligand (CXCL)8 expression levels in the BM-MSCs of patients with AML and normal control subjects and detected significantly higher levels in the former. Furthermore, CXCL8 was up-regulated in cocultures of BM-MSCs and leukemic cell lines compared with either monoculture. CXCL8 expression was significantly higher in MSCs compared with mononuclear cells in patients with de novo AML. To elucidate the function of paracrine CXCL8 in AML, we blocked CXCL8 binding to the C-X-C motif chemokine receptor (CXCR)2 in the AML cells using SB225002. Inhibition of CXCL8/CXCR2 binding decreased proliferation in the AML cells by inducing cell cycle arrest at the G0/G1 phase and apoptosis via decreased AKT phosphorylation. Blocking the PI3K/AKT signaling pathway by a specific inhibitor induced similar apoptosis induction and lower proliferation, suggesting that the PI3K/AKT signaling pathway was also involved in CXCL8 action. Taken together, our findings demonstrate that BM-MSCs are the main source of CXCL8 in the AML bone marrow microenvironment and promote leukemogenesis via the PI3K/AKT signaling pathway, indicating a novel therapeutic target.-Cheng, J., Li, Y., Liu, S., Jiang, Y., Ma, J., Wan, L., Li, Q., Pang, T. CXCL8 derived from mesenchymal stromal cells supports survival and proliferation of acute myeloid leukemia cells through the PI3K/AKT pathway.