Immunomodulatory and anti-proliferative effects of canine adipose-derived stromal vascular fraction on peripheral blood mononuclear cells: An in vitro model for therapeutic applications

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly defined immune cell subtypes, we quantify the natural variation in mitochondrial DNA copy number (mtDNAcn), citrate synthase, and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning four decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte subpopulations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

We examined expression and function of TLRs in enthesitis-related arthritis (ERA) patients. RNA levels of TLR1, TLR3, and TLRs 5–8 were measured in 24 ERA peripheral blood mononuclear cells (PBMC), 18 synovial fluid mononuclear cells (SFMC), and IRAK1, IRAK4, TRIF, TRAF3, and TRAF6 in 18 PBMC and 10 SFMC. IL-6 and IL-8 were measured in supernatants from ERA PBMC (n=7), SFMC (n=3), and healthy PBMC (n=5) cultured with ligands for TLR1/2 (Pam 3-cys), TLR3 (polyI:C), TLR5 (flagellin), and TLR2/6 (zymosan). TLRs 1, 3, 5, and 6 were measured in whole blood (n=20 ERA, seven healthy) and SFMC (n=2) by flow cytometry. ERA PBMC compared to healthy PBMC and SFMC compared to ERA PBMC had higher RNA expression of TLR1, TLR3, TLR5, TLR6, IRAK1, IRAK4, TRIF, TRAF3, and TRAF6. TLR7 and TLR8 RNA expression was similar in all study groups. IL-6 and IL-8 levels were higher in stimulated ERA SFMC compared to ERA PBMC and in ERA PBMC compared to control PBMC. TLRs 1, 3, and 6 were also overexpressed at the protein level.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

The aim of the present study was to investigate the expression levels of heat shock proteins (HSP) mRNA in the peripheral blood mononuclear cells (PBMC) of patients with asthma and chronic bronchitis to elucidate the role of HSP in the pathogenesis of asthma and chronic bronchitis. Using reverse transcription-DNA polymerase chain reaction, the expression levels of HSP70, HSP90alpha and HSP90beta genes in PBMC in normal state and after heat shock were investigated. No HSP70 gene but HSP90alpha and HSP90beta expressions were found in non-heat-shocked PBMC of normal controls; HSP90alpha and HSP90beta genes may be expressed in PBMC of all patients, independently of acute episodes. Expression of HSP70 was found in PBMC of asthmatic patients in acute episodes and three symptom-free patients with Aas 3, step 2. Among patients with chronic bronchitis, no HSP70 gene expression was found in PBMC of patients in convalescent period but in PBMC of patients in acute episode. HSP90alpha and HSP90beta genes were expressed in PBMC of both patient groups. After heat shock, expressions of the three genes increased significantly in PBMC of both normal controls and patients. Expression of HSP70 gene in PBMC of asthmatic and chronic bronchitis patients was different, indicating that HSP, especially HSP70, might be involved in the pathogenesis of asthma.

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.

Comparing the efficacy of γ- and electron-irradiation of PBMCs to promote secretion of paracrine, regenerative factors

The expression pattern of VISTA in the PBMCs of relapsing-remitting multiple sclerosis patients: A single-cell RNA sequencing-based study

Hormonal regulation of peripheral blood mononuclear cells in sheep

Objective To investigate whether human bone marrow mesenchymal stem cells (hBC-MSCs) have the immune-suppression ability on the proliferation of peripheral blood mononuclear cells (PBMCs) and their pro-inflammatory cytokine production in rats, to explore what kinds of human cytokines are required for the induction of hBM-MSCs to become immune-suppressive, and to observe the effect of intravenous delivery of hBM-MSCs on tumor necrosis factor (TNF)-α transcription and expression in the core infarct areas of rats after cerebral ischemia. Methods The fetal-originated hBC-MSCs and rat PBMCs were extracted; the rat PBMCs were activated by adding 10 μg/mL concanavalin A (ConA). (1) The first experiment was divided into hBM-MSCs+PBMCs group, hBM-MSCs+PBMCs+ConA group, PBMCs group and hBM-MSCs group; CCK-8 assay was employed to detect the proliferation of these cells. (2) The second experiment was divided into hBM-MSCs+PBMCs+ConA group, PBMCs+ConA group; ELISA was used to detect the TNF-α, interferon-γ (IFN-γ) and interleukin (IL)-10 expressions. (3) The third experiment was divided into hBM-MSCs+PBMCs (IFN-γ+IL-1α) group, hBM-MSCs+PBMCs (IFN-γ+IL-1β) group, hBM-MSCs+PBMCs (IFN-γ+TNF-α) group and hBM-MSCs+PBMCs group; CCK-8 assay was used to detect the proliferation of these cells. (4) Thirty SD rats were randomly divided sham-operated group, control group (giving normal saline after ischemia) and hBM-MSCs group (giving hBM-MSCs after ischemia, n=10); on the third d of ischemia, the TNF-α mRNA and protein expressions at the infarct areas was detected by real time PCR and Western blotting, respectively. Results (1) The optical density (OD) in the hBM-MSCs+PBMCs group was significantly increased as compared with that in the PBMCs group and hBM-MSCs group (P<0.05); OD in the hBM-MSCs+PBMCs group was significantly increased as compared with that in the hBM-MSCs+PBMCs+ConA group (P<0.05). (2) The TNF-α, IFN-γ and IL-10 levels in the PBMCs+ConA group were (1030±196) pg/mL, (2880±250) pg/mL and (330±45) pg/mL; the TNF-α and IFN-γ levels in hBM-MSCs+PBMCs+ConA group were (160±10) pg/mL and (240±55) pg/mL, which were significantly lower than those in the PBMCs+ConA group (P<0.05); the IL-10 level in hBM-MSCs+PBMCs+ConA group was (750±110) pg/mL, which was significantly higher than that in the PBMCs+ConA group (P<0.05). (3) The OD in the hBM-MSCs+PBMCs(IFN-γ+IL-1α) group, hBM-MSCs+PBMCs(IFN-γ+IL-1β) group and hBM-MSCs+PBMCs(IFN-γ+TNF-α) group was significantly decreased as compared with that in the hBM-MSCs+PBMCs group (P<0.05). (4) The TNF-α mRNA expression in the sham-operated group, control group and hBM-MSCs group was 0.490±0.128, 2.369±0.788 and 1.002±0.408; the TNF-α protein expression in the sham-operated group, control group and hBM-MSCs group was 0.144±0.028, 0.314±0.029, 0.240±0.029; the TNF-α protein and mRNA expressions in the hBM-MSCs group were significantly decreased as compared with those in the control group (P<0.05). Conclusions The allogeneic transplantation of hBC-MSCs is competent in suppressing the inflammation of rats in vitro and in vivo. Furthermore, this immune-suppression ability is not innate, but cytokine stimulation dependent. The immune-suppression ability of hBM-MSCs on rat PBMCs are at least partly responsible for the therapeutic effect of hBM-MSCs transplantation into the rat models, such as ischemia stroke. Key words: Cerebral ischemia; Human bone marrow mesenchymal stem cell; Mononuclear cell; Inflammation factor; Immune-suppression

Bacterial trigger possibly causes disease exacerbation in enthesitis-related arthritis (ERA) patients. Microbes initiate immune responses through Toll-like receptors (TLRs). We studied TLR expression on blood and SF monocytes and the effect of TLR ligands on peripheral blood (PB) mononuclear cells (PBMCs) in ERA patients. PB from 26 ERA patients and 19 healthy subjects and paired SF from 13 patients were collected. Dual-colour flow cytometry was done for TLR and CD14 expression. Results are expressed as median fluorescence intensity (MFI). Real-time PCR was done for TLRs. PBMCs were stimulated with lipopolysaccharide (LPS) or peptidoglycan and levels of IL-6 and MMP-3 measured in the culture supernatants. PBMCs from ERA patients had higher expression of TLR-2 [MFI 295.5 (48.1-598) vs 179 (68.7-442); P < 0.05] and TLR-4 [MFI 448 (178-2581) vs 402 (229-569); P < 0.05] as compared with controls. TLR-9 expression showed no significant difference between the two groups. In paired samples, SF mononuclear cells (SFMCs) had higher expression of both TLR-2 [MFI 485 (141-1683) vs 353 (118-598); P < 0.05] and TLR-4 [MFI 1016 (42.4-3159) vs 513 (193-2581); P < 0.05] as compared with PBMCs. Difference in TLR-9 expression was not significant. TLR RNA expression data were similar. Patients' PBMCs produced more IL-6 (13.51 vs 6.54 ng/ml) and MMP-3 (61 vs 32.9 ng/ml) as compared with those of the controls, on stimulation by LPS. With peptidoglycan also, IL-6 (30.58 vs 10.84) and MMP-3 (102.54 vs 49.45) were higher than in controls. Increased TLR-2 and TLR-4 expression on PBMCs and SFMCs may recognize microbial/endogenous ligands and up-regulate IL-6 and MMP-3 leading to disease exacerbation.

IgE-Dependent Activation of T cells by Allergen in Atopic Dermatitis: Pathophysiologic Relevance

Exhaustion of CMV Specific T Cells with Enhanced PD-1 Expression In Persistent Cytomegalovirus Infection After Allogeneic Stem Cell Transplantation

We investigated the expression of miR-146a in peripheral blood mononuclear cell (PBMC) of patients with ankylosing spondylitis (AS) and its correlation with inflammatory factors to explore the clinical significance. In total 45 patients with AS were selected at the Weifang People's Hospital from June, 2014 to January, 2016. At the same time, 30 healthy volunteers were also selected to serve as control group. Expression level of miR-146a in PBMC cells of patients in each group was detected by quantitative real-time-polymerase chain reaction (qRT-PCR). Levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in serum and the supernatant of culture medium of PBMC derived from each group were detected by enzyme-linked immunosorbent assay (ELISA). Correlations between expression level of miR-146a and serum inflammatory factors, and clinical indicators were analyzed. Clinical indicators included bath ankylosing spondylitis disease activity index (BASDAI), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and duration of morning stiffness. Expression level of miR-146a in PBMC of AS patients was significantly higher than that of healthy control (P<0.01); levels of TNF-α, IL-1β and IL-6 in serum and the supernatant of culture medium of PBMC derived from AS patients were significant compared to those of control group (P<0.01); expression of miR-146a in PBMC of patients with AS was positively correlated with the levels of TNF-α, IL-1β and IL-6 in serum (r=0.632, P<0.01; r=0.574, P<0.01; r=0.483, P<0.01). In addition, expression level of miR-146a in PBMC of patients with AS was positively correlated with BASDAI, ESR, CRP and duration of morning stiffness (r=0.551, P<0.01; r=0.738, P<0.01; r=0.685, P<0.01; r=0.497, P<0.01). Expression level of miR-146a in PBMC of AS patients was significantly increased and the expression level was positively correlated with the levels of TNF-α, IL-1β and IL-6 in serum (P<0.05). In addition, expression level of miR-146a in PBMC of AS patients was also positively correlated with BASDAI, ESR, CRP and duration of morning stiffness. Those results suggest that miR-146a may be involved in the pathogenesis of AS, and the expression level of miR-146a in PBMC cells may be helpful for diagnosis of AS and judgment of disease activity.

Increased oxidative stress and apoptosis in peripheral blood mononuclear cells of fructose-fed rats