Peripheral Blood Of Healthy People Research Articles

MiR-21-5p protects against ischemic stroke by targeting IL-6R.

Ischemic stroke is a brain dysfunction disease caused by vascular obstruction. The expression of many kinds of microRNAs (miRNAs) is related to ischemic stroke. MiRNA has the ability to reduce or save ischemic injury. Therefore, we aimed to explore the protective miRNA in the ischemia-reperfusion process. The Gene Expression Omnibus (GEO) peripheral RNA sequencing (RNA-seq) datasets of ischemic stroke patients were analyzed to search for differentially expressed miRNAs in the ischemia-reperfusion process. The expression level of miRNA in 60 patients with ischemic stroke and 23 age-matched healthy control inpatients was tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The significantly changed miRNAs were verified through comparison of the peripheral blood of healthy people and patients of the hospital. The in-vitro ischemia-reperfusion model was established through oxygen-glucose deprivation (OGD) treated HEMC-1 cells. The cell viabilities and cell apoptosis are detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. Apoptosis-related proteins including Bcl-2, Bax, caspase-3, and caspase-9 expression levels were verified by western blot. Predict the combination of hsa-miR-21-5p and interleukin-6 receptor (IL-6R) through TargetScan database, clone the 2964-2961 site of IL-6R-3'-untranslated region (3'-UTR), establish IL-6R-3'-UTR and IL-6R-3'-UTR mutant plasmids, copy and clone wild type and mutant IL-6R-3'-UTR into luciferase report vector pGL3 respectively, and detect the activity of luciferase. The expression of hsa-miR-21-5p was regulated by using hsa-miR-21-5p mimic and hsa-miR-21-5p inhibitor. Through RNA-seq analysis, it was revealed that "hsa-miR-548ar-3p", "hsa-miR-651-5p", "hsa-miR-142-3p", "hsa-miR-21-5p", and "hsa-miR-30e-5p" were notably lower in ischemia patients, and that "hsa-miR-21-5p" was significantly decreased in the peripheral blood of hospital patients. Luciferase assay showed that hsa-miR-21-5p could directly bind to the 3'-UTR of the IL-6R gene and inhibit IL-6R translation; the level of IL-6R was also elevated in patients. In the OGD-treated HMEC-1 cells, overexpressed hsa-miR-21-5p mimic could enhance cell viabilities and decrease cell apoptosis. Moreover, IL-6R overexpression could reduce the protective effects of hsa-miR-21-5p. In the peripheral blood of ischemia patients, hsa-miR-21-5p is significantly decreased and IL-6R is elevated. The "hsa-miR-21-5p" could bind to the IL-6R gene and suppress IL-6R expression, thus alleviating the damage of OGD treatment in HMEC-1 cells.

Enhancement of Allo-NK Cell Activity in Myeloid Leukemia Cell Line KG-1a after Combined with Aclacinomycin

Abstract Objective: To investigate whether aclacinomycin can enhance the killing effects of allogeneic NK cells on acute myeloid leukemia and the molecular mechanisms, thus providing an experimental basis and a new thinking for the clinical treatment of acute leukemia. Methods: NK cells were isolated from the peripheral blood of healthy people by antibody-labeled immunomagnetic beads, the percentage of NK cells was detected by flow cytometry, and the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were detected by CCK-8 assay. In vitro killing assay was employed to detect the in vitro killing effects on NK-1a cells by NK cells with different effector/target ratios after treatment. Clone formation assay was used to detect the inhibitory effects of aclacinomycin in combination with allogeneic NK cells on the proliferation of KG-1a cells. Furthermore, the effects of aclacinomycin in combination with allogeneic NK cells in inducing the apoptosis of KG-1a cells was detected by flow cytometry, and changes in the expressions of IAP protein families and proteins associated with both intracellular and extracellular apoptosis signaling pathways after treatment of aclacinomycin in combination with allogeneic NK cells were analyzed by immunoblotting. Meanwhile, the differences in the expression levels of calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were detected. Results: The percentages of NK cells in the peripheral blood before and after isolation with anti-CD3-CD56+ antibody-labeled immunomagnetic beads were (15.2 ± 3.7)% and (84.9 ± 5.5)%, respectively. The results of CCK-8 assay showed that the inhibitory effects of aclacinomycin on the proliferation of KG-1a cells were increased in a time-dependent manner. At the same time, this study showed that after the co-culture of NK cells and KG-1a cells at the effector/target ratios of 10: 1 and 20: 1, the killing effects on KG-1a cells were not significant (P <0.05). After 72 hours of treatment at the ratio of 20: 1, aclacinomycin could significantly increase the killing effects of NK cells on KG-1a cells. The results of clone formation assay showed that at 72 hours after the treatment of acycloemycin in combination with allogeneic NK cells, the number of KG-1a cell clones was (11 ± 3), significantly lower than that in the aclacinomycin-alone group (68 ± 7) and the NK cell group (121 ± 12, P <0.05), the differences were statistically significant (P <0.001). The results of flow cytometry showed that the total apoptotic rate of KG-1a cells was increased significantly at 72 hours after the treatment of aclacinomycin in combination with allogeneic NK cells, and the results of immunoblotting showed that the expression of cIAP, XIAP and Bcl-2 were significantly down-regulated after the treatment of aclacinomycin in combination with NK cells, while the expressions of pro-apoptosis proteins Smac and caspase-3 were significantly up-regulated. Meanwhile, the expressions of immunogenic death-associated calreticulin, heat shock protein, adenosine triphosphate and high mobility protein-1 were significantly up-regulated. Conclusions: Aclacinomycin can significantly enhance the killing effects of allogeneic NK cells on the KG-1a cell line in vitro through such a molecular mechanism in which the expression levels of IAP protein family-related proteins are inhibited. Moreover, the mechanisms are associated closely with the activation of both intracellular and extracellular apoptosis signaling pathways and the activation of immunogenic death-associated proteins. DisclosuresNo relevant conflicts of interest to declare.

A NKp80-Based Identification Strategy Reveals that CD56neg NK Cells Are Not Completely Dysfunctional in Health and Disease.

SummaryNatural killer (NK) cells are usually identified by the absence of other lineage markers, due to the lack of cell-surface-specific receptors. CD56neg NK cells, classically identified as CD56negCD16+, are very scarce in the peripheral blood of healthy people but they expand in some pathological conditions. However, studies on CD56neg NK cells had revealed different results regarding the phenotype and functionality. This could be due to, among others, the unstable expression of CD16, which hinders CD56neg NK cells’ proper identification. Hence, we aim to determine an alternative surface marker to CD16 to better identify CD56neg NK cells. We have found that NKp80 is superior to CD16. Furthermore, we found differences between the functionality of CD56negNKp80+ and CD56negCD16+, suggesting that the effector functions of CD56neg NK cells are not as diminished as previously thought. We proposed NKp80 as a noteworthy marker to identify and accurately re-characterize human CD56neg NK cells.

Reply to letter to the editor: "JC viremia and multiple sclerosis" by Focosi et al.

First of all, we would like to thank the authors for the interest and the opportunity to go into depth of our results concerning the possible role of beta interferon treatment on frequency of JC virus (JCV) DNA detection in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients. The authors are concerned because in their opinion DNA of JCV could not be found also in the peripheral blood of healthy people and, to this regard, they quote several scientific papers reporting negative results. However, the analysis of the literature performed by them is restricted because it ignores a large body of evidences reported by several authors, in more recent years, and showing the presence at a variable range of JCV DNA in the peripheral blood of both healthy subjects and patients with MS (Dorries et al, 1994, 2003; Azzi et al, 1996; Gu et al, 2003; Pietropaolo et al, 2005). At the end of the paragraph regarding this topic, the authors suspect that polymerase chain reaction (PCR) we used could be flawed by methodological errors. The only possible answer to these comments is that our experience in the field of JCV dates from a long time and, more important, we participated to the recently settled European Quality Control for JCV/BKV PCR methods validation with excellent results (www.qcmd.org, 2007). The authors also hypothesize that the presence of JCV in PBMCs of relapsing-remitting MS (RRMS) patients could be due to a misdiagnosis of progressive multifocal leukoencephalopathy (PML). We underline again that our MS patients were diagnosed according to the Poser et al (1983) criteria. Moreover, we believe that only inexpert neurologists could confuse PML with MS, mostly if we consider that our patients had MS diagnosed many years before the samples’ collection. We would also like to inform the authors and to highlight that the first scientist suggesting a possible etiological relation between MS and JCV was the late lamented Gerald Stoner (1991). On these themes Focosi and colleagues recall also that Du Pasquier et al showed a strong cytotoxic lymphocyte (CTL) response against JCV VP1 in MS patients (Du Pasquier et al, 2006). In our opinion, this is an indirect evidence of the fact that JCV circulates in the blood of MS patients, stimulating immune competent cells against viral antigens. Regarding our previous data (Ferrante et al, 1998) showing the presence of JCV DNA in cerebrospinal fluid (CSF), Focosi et al stated that other authors did not confirm this report. Recently, we confirmed these results (Mancuso et al, 2007) and we would like to point out again that MS is a disease that can last for more than 20 years and its diagnosis is usually made from 5 to 15 years after the real onset of the disease. In addition, MS is characterized by very high cellular trafficking from periphery to central nervous system (CNS), an aspect that, in our opinion, supports the transport of viruses, and not only JCV, to the CNS and to the CSF of MS patients. We could also speculate about the preanalytical phase of the study of viruses’ research in CSF (time of collection, length and methods of storage, etc.), but due to space restriction, we will not discuss this issue further. Again regarding the possible relation between JCV and MS, Focosi et al quoted, without any apparent reason, the paper of Agostini et al (2000), showing the high frequency of JCV genotype 2b in PML patients, a finding that was also shown by our group (Ferrante et al, 2001). Finally the authors underlined the differences between our data and those by Alvarez-Lafuente et al (2007) who did not find any statistically significant differences between treated and untreated MS patients. We would like to suggest that not only methodological aspects could be different but also the geographical provenience, clinical history, and perhaps genetic background of our patients and Alvarez-Lafuente’s patients could be different. However, this is only the beginning of a new era of interest in JCV and MS relationship and we believe that we must be open to different possible results during the coming years each time a new aspect of a very complex disease, such as MS, is studied.

Age-related susceptibility of naive and memory CD4 T cells to apoptosis induced by IL−2 deprivation or PHA addition

The increased age-associated incidence of infectious and cancer diseases has been related to the alteration of immune functioning found in the elderly (immunosenescence). The reduction of naive T cells, which determine an impaired ability to mount immune responses to new antigens, represents a hallmark of the aging process. The aim of this study was to evaluate the susceptibility to apoptosis of purified naive and memory CD4(+) T cells from peripheral blood of healthy people ranging in age from 20 to 98 years. Two mechanisms of T cell elimination by apoptosis have been evaluated: cytokine deprivation and activation-induced cell death. After Interleukin-2 deprivation, the percentage of naive and memory CD4(+) apoptotic cells significantly increased with donor age concomitantly with a reduction of Bcl-2 expression and an increase of intracellular content of reactive oxygen species. After phytohemagglutinin addition, the percentage of apoptotic cells, the expression of CD95, and the intracellular reactive oxygen species, were not significantly correlated with age both in naive and memory CD4(+) T cells. Our data demonstrate the existence of functional alterations of naive and memory T cell populations during ageing. These alterations are mainly related to the mechanism of the apoptotic event rather than to the type of cell population involved (naive or memory). The alterations of naive and memory T cells may have implications in the age-related susceptibility to diseases.

Activated T lymphocytes from patients with high risk of type I diabetes mellitus have different ability to produce interferon-γ, interleukin-6 and interleukin-10 and undergo anti-CD95 induced apoptosis after insulin stimulation

Type I Diabetes mellitus (DM1) is the effect of T cell dependent autoimmune destruction of insulin producing beta cells in the pancreas islet. T cells are activated in response to islet dominant autoantigens, the result being the development of DM1. Insulin is one of the islet autoantigens responsible for activation of T lymphocyte functions, inflammatory cytokine production and development of DM1. The experiments reported in this study have shown the spontaneous increase of CD95 molecule expression on lymphocytes of the first-degree relatives of DM1 patients. The autoantigen insulin is responsible for stimulation in vitro of potentially hazardous ‘memory’ lymphocytes to produce interleukin-6 (IL-6) and interleukin-10 (IL-10) interleukins. Insulin induced stimulation of lymphocytes in vitro was observed in patients at high risk of developing diabetes mellitus (prediabetics). Phytohaemagglutinin (PHA) stimulates lymphocytes of all groups in the same way. Stimulated lymphocytes in second cultures undergo apoptosis induced with anti-Fas specific antibodies. The deletion in vitro of resting peripheral lymphocytes is nonfunctional. Insulin activated T lymphocytes, which undergo apoptosis were not observed in peripheral blood of healthy people and in patients with DM1. This observation suggests that insulin is involved as autoantigen in DM1 progression in patients with high risk of diabetes type I. The autoreactive T lymphocytes may persist in peripheral blood of patients with high risk DM1. Defective elimination of autoreactive T cells may result in autodestructive damage of islets β cells in the prediabetic stage and disease progression to DM1.

Carotenoid levels in human lymphocytes, measured by Raman microspectroscopy

Abstract

T-CELL INDUCERS OF SUPPRESSOR LYMPHOCYTES CONTROL LIVER-DIRECTED AUTOREACTIVITY

The sensitisation of helper T cells of patients with autoimmune chronic active hepatitis to a liver-cell membrane-expressed asialoglycoprotein receptor protein is shown to be associated with a defect of T cells that specifically induce suppressor lymphocytes. These lymphocytes are found in an activated state in the peripheral blood of healthy people and may form part of an immunoregulatory network which actively prevents autoimmunity.

Simultaneous determination of T, B, and mixed rosette-forming lymphocytes in human peripheral blood

Preliminary treatment of erythrocyte suspension with trypan blue solution did not affect their capacity to participate in the reaction of spontaneous rosette formation, but permitted to differentiate them in a mixture with other erythrocytes after staining with methyl-green-pyronine on a fixed slide. By using a mixture of such marked erythrocytes sensitized with antibodies and a complement, the presence in the peripheral blood of healthy people of a small lymphocyte subpopulation connecting these two types of indicator particles was demonstrated.