Peripheral Blood Mononuclear Cells Of Group Research Articles

Cytokine changes and embryo attachment in mouse endometrial cells following treated with peripheral blood mononuclear cells (PBMCs) expressing ectopic hCG, and hCG–activated PBMCs

Objective: To compare the effect of human chorionic gonadotropin (hCG)-producing peripheral blood mononuclear cells (PBMCs) and PBMCs activated by hCG in vitro and expressions of related immune genes in mouse implantation. Methods: hCG-producing PBMCs (transfected PBMC) and PBMCs activated by hCG in vitro were introduced into isolated mouse endometrial cells, while cell cultures were divided into four groups: the control, PBMC, transfected, and activated PBMC groups. The expression of studied genes (IL-lβ, IL-6, Lif, and Vegf) was evaluated and blastocyst attachment on the cocultured cells (isolated endometrial cells and PBMC cells) was monitored in all four groups. Results: Data showed that expression decreased in the PBMC group compared to the treated PBMC (transfected and activated PBMCs) and increased in transfected PBMC compared to the activated PBMC. Attachment and migration of blastocysts were dramatically enhanced in the transfected PBMC group compared to the activated PBMC group (P<0.05). Conclusions: Use of hCG-producing PBMCs (transfected PBMC) has more influence on endometrial receptivity.

Intrauterine Administration of Activated Peripheral Blood Mononuclear Cells in Intrauterine Insemination: A Prospective Double-Blind Randomized Clinical Trial.

To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n= 108) underwent in utero administration of 1.106 cells on D2, while patients in the control group (n= 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n= 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated. Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0. Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.

Preventive cancer vaccination with P5 HER-2/neo-derived peptide-pulsed peripheral blood mononuclear cells in a mouse model of breast cancer.

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+ IFN-γ+, CD8+ IFN-γ+, and CD8+ granzyme B+ T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

Autologous peripheral blood-derived stem cells transplantation for treatment of no-option angiitis-induced critical limb ischemia: 10-year management experience

BackgroundPrevious studies have demonstrated that no-option angiitis-induced critical limb ischemia (NO-AICLI) could be significantly improved by transplantation of peripheral blood-derived stem cells (PBDSCs). Additionally, a randomized controlled trial (RCT) recently conducted by us suggested that peripheral blood-derived purified CD34+ cells (PCCs) were not inferior to non-purified peripheral blood mononuclear cells (PBMNCs) at limb salvage in treatment of NO-AICLI. However, most of these clinical trials whether RCT or single-arm studies were characterized with a small sample size and absence of long-term outcomes.MethodsTo analyze long-term clinical outcomes of PBDSCs transplantation for NO-AICLI, we reviewed clinical data of patients with NO-AICLI receiving PBDSCs transplantation at our center during the past decade. Meanwhile, we first compared the long-term safety and efficacy of intramuscular transplantation of PCCs versus PBMNCs in a sizable number of patients with NO-AICLI.ResultsFrom May 2009 to December 2019, a total of 160 patients with NO-AICLI patients were treated by PBDSCs transplantation (82 with PCCs, 78 with PBMNCs) at our center. Baseline characteristics between two groups were similar. Up to June 2020, the mean follow-up period was 46.6 ± 35.3 months. No critical adverse events were observed in either group. There was one death during the follow-up period. A total of eight major amputations occurred. The cumulative major amputation-free survival (MAFS) rate at 5 years after PBDSCs transplantation was 94.4%, without difference between two groups (P = .855). Wound healing, rest pain, pain-free walking time, ankle-brachial index, transcutaneous oxygen pressure, and quality of life (QoL) also significantly improved after PBDSCs transplantation.ConclusionsAutologous PBDSCs intramuscular transplantation could significantly decrease the major amputation rates and improve the QoL in patients with NO-AICLI. Long-term observation of a large sample of patients confirmed that the clinical benefits of PBDSCs transplantation were durable, without difference between the PCCs and PBMNCs groups.

Establishment and Preservation of Lymphoblastoid Cell Lines from Fresh and Frozen Whole Blood and Mononuclear Cells.

Although blood cells are interesting sources for genome investigations, one of the main problems in obtaining genomic DNA from blood is the restricted amount of DNA. This obstacle can be avoided by generating Epstein-Barr virus (EBV)-induced B cell lines. This study investigates the efficiency of four different methods to generate lymphoblastoid cell lines (LCLs). Blood samples (n = 120) were obtained from donors and categorized into four groups: fresh whole blood, frozen whole blood, fresh peripheral blood mononuclear cells (PBMCs), and frozen PBMCs. The samples were followed by EBV transformation to generate LCLs. Quality control and authentication of the cells were performed using multiplex PCR and short tandem repeat (STR) analyses. Finally, we assessed the success rate and amount of time to establish the cell lines in each group. The results showed that the cells were not contaminated nor were they misidentified or cross-contaminated with other cells. The success rate of LCLs generated from the whole blood groups was lower than the PBMC groups. The freezing procedures did not have any considerable effect on the establishment of lymphoblastoid cells. These established cells have been preserved in the human and animal cell bank of the Iranian Biological Resource Center (IBRC) and are available for researchers. Due to the management and transformation of a substantial number of blood samples, we recommend that researchers freeze PBMCs for further use with high efficiency and time-saving. We suggest that whole fresh blood should be directly transformed when the volume of the blood sample is less than 0.5ml.

Phosphorylation of Protein Kinase Akt by Mtorc2 in Peripheral Blood Mononuclear Cells of Patients with Cancer and Diabetes

Akt/mTOR/p70S6K1 signaling pathway plays an important role in the pathogenesis of cancer and diabetes. Macrophages and lymphocytes are involved in the pathogenesis of diabetes, diabetic atherosclerosis, formation of insulin resistance as well as immune response to cancer and tumor maintenance. The aim of the study was to determine the Akt activation by mTORC2 in peripheral blood mononuclear cell (PBMC) of patients with type 2 diabetes and cancer.The following groups were studied: control group, patients with type 2 diabetes, cancer patients and patients with both cancer and diabetes. The amounts of phospho-Akt (р-S473) and phospho-p70S6K1 (p-T389) were determined using ELISA kits.The amount of phosphorylated Akt significantly increases in PBMC of patients with cancer. There was no effect in PBMC from patients with type 2 diabetes and significant decrease in the amount of phospho-Akt in PBMC of the patients group both with cancer and diabetes. p70S6K1 activation was observed in PBMC of the groups 2 and 3 patients.Thus, chronic diseases such as type 2 diabetes and cancer can affect the signaling mechanisms in blood cells. The state of Akt phosphorylation in leukocytes can indicate the activity of mTORC1 and its substrates, which may be important for the evaluation of the pathological process and the efficacy of the drugs.

AIM2 levels and DNA-triggered inflammasome response are increased in peripheral leukocytes of patients with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is heavily infiltrated with leukocytes, expressing the DNA sensor absent in melanoma 2 (AIM2) and other inflammasome components. Using multicolour flow cytometry, we here compared the expression of the inflammasome components AIM2, NLRP3, and ASC in different peripheral immune cells derived from AAA patients with those from non-AAA patients in a case-control study. In parallel, peripheral blood mononuclear cells (PBMC) of AAA patients and controls were stimulated in vitro with poly-dA:dT or lipopolysaccharide (LPS) to analyze inflammasome activation. AIM2 expression was significantly increased in peripheral granulocytes (P = 0.026), monocytes (P = 0.007), B lymphocytes (P < 0.0001), and T lymphocytes (P = 0.004) of AAA patients. Expression of other inflammasome components did not differ between the groups. Following in vitro stimulation with foreign DNA, PBMC derived from AAA patients released significantly more IL-1β (P = 0.022) into the supernatant than PBMC from control patients. In contrast, IL-1β release upon LPS stimulation did not differ between the PBMC groups. The data indicate the increased activation of an AIM2 inflammasome in peripheral immune cells of AAA patients and point to a systemic AIM2-associated immune response to AAA.

Human Peripheral Blood Mononuclear Cells Incubated in Vasculogenic Conditioning Medium Dramatically Improve Ischemia/Reperfusion Acute Kidney Injury in Mice

Acute kidney injury (AKI) is a major clinical problem that still has no established treatment. We investigated the efficacy of cultured human peripheral blood mononuclear cells (PBMNCs) for AKI. Ischemia/reperfusion injury (IRI) was used to induce AKI in male nonobese diabetic (NOD/severe combined immunodeficiency) mice aged 7 to 8 wk. PBMNCs were isolated from healthy volunteers and were subjected to quality and quantity controlled (QQc) culture for 7 d in medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin 6. IRI-induced mice were divided into 3 groups and administered (1) 1 × 106 PBMNCs after QQc culture (QQc PBMNCs group), (2) 1 × 106 PBMNCs without QQc culture (non-QQc PBMNCs group), or (3) vehicle without PBMNCs (IRI control group). PBMNCs were injected via the tail vein 24 h after induction of IRI, followed by assessment of renal function, histological changes, and homing of injected cells. Blood urea nitrogen and serum creatinine (Cr) 72 h after induction of IRI in the QQc PBMNCs group dramatically improved compared with those in the IRI control and the non-QQc PBMNCs groups, accompanied by the improvement of tubular damages. Interstitial fibrosis 14 d after induction of IRI was also significantly improved in the QQc PBMNCs group compared with the other groups. The renoprotective effect noted in the QQc PBMNCs group was accompanied by reduction of peritubular capillary loss. The change of PBMNCs’ population (increase of CD34+ cells, CD133+ cells, and CD206+ cells) and increased endothelial progenitor cell colony-forming potential by QQc culture might be one of the beneficial mechanisms for restoring AKI. In conclusion, an injection of human QQc PBMNCs 24 h after induction of IRI dramatically improved AKI in mice.

Response of peripheral blood mononuclear cells to conditioned medium from cultured oral squamous cell carcinomas

The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50% conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.

Obesity alters the expression profile of clock genes in peripheral blood mononuclear cells. Preliminary results

IntroductionThe aim of this study was to investigate the association between the variation in expression profile of clock genes and obesity using peripheral blood mononuclear (PMN) cells.Material and methodsThe subjects comprised 10 obese patients and 10 healthy volunteers. Blood was collected at different time-points during the day and levels of blood sugar, IRI, adiponectin and leptin were determined. Peripheral blood mononuclear cells were sampled, and expression levels of brain and muscle Arnt-like protein-1 (BMAL1), Period (PER)1, PER2, Cryptochrome (CRY)1, CRY2, and REV-ERBα mRNA were quantified.ResultsDuring the day, the expression levels of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells of the obese group were all significantly higher compared to those in the non-obese group. In addition, expression of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells increased between 12:00 and 21:00 in the obese group. In PMN cells of both groups, PER1 gene expression showed a bimodal pattern, with high expression at 9:00 and 18:00.ConclusionsDifferences were observed in the expression profile variation of clock genes between the obese and non-obese groups. This study reveals the differences in clock gene expression profiles between obese and non-obese subjects, with evidence for two distinct chronotypes, and suggests a contribution of these chronotypes to fat accumulation in humans.

Telomerase activity in peripheral blood mononuclear cells from senile patients with pneumonia

To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43 +/- 0.04; post-stimulation, 0.63 +/- 0. 03) were significantly lower than those in PBMCs from both group A patients (A values: pre-stimulation, 0.65 +/- 0.05; post-stimulation, 1.26 +/- 0.13; P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63 +/- 0.03; post-stimulation, 0.93 +/- 0.03; P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35 +/- 0.03) was also significantly lower than that in group A patients (A value: 0. 55 +/- 0.04; P<0.05) and group B patients (A value: 0.46 +/- 0.03; P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

Analysis of cytokine levels and NF-κB activation in peripheral blood mononuclear cells in influenza virus-associated encephalopathy

Background Influenza virus-associated encephalopathy (IE) is suggested to be a proinflammatory cytokine-related disease. Methods We measured the concentrations of interleukin-6 (IL-6), TNF-α, soluble TNF receptor 1 (sTNFR1), IL-10, interferon-γ, IL-2, IL-4, and soluble E-selectin (sE-selectin) in serum and CSF during the acute stage in 30 children with IE, 20 with influenza virus-associated febrile seizures (IFS), and 39 with influenza virus infection without complications (Flu). Moreover, the activation of transcription factor NF-κB in peripheral blood mononuclear cells (PBMC) of 5 children with IE was examined. Results The serum IL-6, sTNFR1, and IL-10 levels in the IE group with a poor prognosis (Group A) were significantly higher than those in the IE group without sequelae in IE (Group B), IFS, and Flu. In particular, the serum levels of IL-6, sTNFR1, and IL-10 in 5 deceased patients were markedly higher. The CSF IL-6 levels in Group A were significantly higher than those in Group B and IFS. Flow cytometric analysis revealed that NF-κB activation in PBMC in Group A was higher than that in Group B, IFS, and Flu. Conclusion We suggest that cytokines are produced by PBMC in IE, and that the levels of serum IL-6, sTNFR1, and IL-10, CSF IL-6, and NF-κB activation in PBMC are useful indicators of the severity of the disease.

Cell-associated magnesium and QT dispersion in hemodialysis patients

Background: Impaired magnesium (Mg) homeostasis has been implicated in a variety of cardiovascular disturbances, including ventricular arrhythmias and changes in the interval between the onset of wave Q to the end of wave T (QT interval) on electrocardiogram. Cardiac arrhythmias are common in patients on hemodialysis therapy. Methods: We investigated the relationship between QT interval corrected for heart rate (QTc) dispersion and Mg content in peripheral blood mononuclear cells (PBMC) of chronic hemodialysis patients treated with high-dose calcium carbonate providing Mg in excess (group I; n = 18) or low-dose calcium carbonate and smaller Mg load (group II; n = 13). Results: Mean Mg content in PBMC of group I patients (27.9 ± 4.2 [SD] μmol/L/mg protein) was significantly greater than in group II patients (10.4 ± 4.1 μmol/L/mg protein; P < 0.05) and greater in both groups than in healthy control subjects (2.75 ± 0.6 μmol/L/mg protein; P < 0.05). Mean QTc dispersion was significantly longer (74.6 ± 21.4 milliseconds) in group I than group II (37.8 ± 13.1 milliseconds; P < 0.02) and longer in both groups than in controls (27.3 ± 9.6 milliseconds; P < 0.05). After dialysis, in both groups of patients, cell-associated Mg (c-a Mg) levels and QTc dispersion were significantly greater (P < 0.05) than before dialysis started. One week after stopping calcium carbonate treatment, group 1 patients showed significant reductions in predialytic c-a Mg levels (to 19.5 ± 9.8 μmol/L/mg protein; P < 0.05) and QTc dispersions (to 48.9 ± 23.7 milliseconds; P < 0.05). Plasma Mg and other electrolyte concentrations prior to and during hemodialysis did not correlate with QTc dispersion. Conclusion: Prolongation of QTc dispersion in patients on chronic hemodialysis therapy could be, at least in part, a consequence of increased concentrations of c-a Mg resulting from excess daily Mg intake. Am J Kidney Dis 41:196-202. © 2003 by the National Kidney Foundation, Inc.