极小胚胎样干细胞的生物学特性

近年来,研究者从小鼠骨髓和其他组织脏器中分离并纯化了一类数量极其稀少的极小胚胎样干细胞(very small embryonic-like stem cells,VSELs).VSELs不仅表达多能干细胞的表面分子标记,并能向3个胚层方向分化.有学者推测,VSELs可能是在哺乳动物组织/器官的发育早期迁移并定居下来的,且能在特定情况下向组织特异的单潜能干细胞方向分化.据此,VSELs可能在成体组织的更新和损伤组织的再生修复过程中发挥重要作用.     

Very Small Embryonic-Like (VSEL) Stem Cells: Purification from Adult Organs,Characterization, and Biological Significance

In this review, we discuss current views of the bone marrow (BM) stem cell (SC) compartment and present data showing that BM contains heterogeneous populations of hematopoietic (H)SCs and non-HSCs. These cells are variously described in the literature as: endothelial progenitor cells (EPCs); mesenchymal (M)SCs; multipotent adult progenitor cells (MAPCs); marrow-isolated adult multilineage inducible (MIAMI) cells; and multipotent adult (MA)SCs. In some cases, it is likely that similar or overlapping populations of primitive SCs in the BM detected using various experimental strategies were assigned different names. Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4(+)) small SCs from the murine BM that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs. Our data indicate that VSELs may differentiate into cells from all three germ layers.     

Proinflammatory CD14+CD16+ Monocytes are Associated with Microinflammation in Patients with Type 2 Diabetes Mellitus and Diabetic Nephropathy Uremia

Diabetic nephropathy (DN) is a major cause of type 2 diabetes mellitus (T2DM) mortality. Innate immunity has been shown to be closely associated with the occurrence and progression of T2DM-associated complications. In this study, we investigated the expression of Toll-like receptor 4 (TLR4) and CD14⁺CD16⁺ monocytes in patients with T2DM and DN patients with uremia and TLR4 response to lipopolysaccharide (LPS), and to further explore the potential effects of inflammatory immune response in T2DM and DN uremia. Thirty DN patients with uremia, 28 T2DM patients, and 20 healthy volunteers were enrolled for the determination of CD14⁺CD16⁺ fluorescence intensity and TLR4 expression on monocytes by using peripheral blood flow cytometry. Serum C-reactive protein (CRP) level was determined by using the immunoturbidimetry. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with LPS for 24 h. monocytes were collected to detect NF-κB p65 and phosphorylated STAT5(p-STAT5) expressions by using Western blotting. Supernatants were sampled for the determination of interleukin-6 (IL-6) concentration by using ELISA. Compared to normal control, T2DM patients and DN uremic patients had a significantly higher CD14⁺CD16⁺ fluorescence intensity, TLR4 expression, serum IL-6 and CRP level, whilst these biomarkers were more upregulated in DN uremic patients than in T2DM patients. Following the exposure to LPS, PBMCs showed a significant upregulation in NF-κB-p65 and p-STAT5 expression and a remarked increase in Supernatants IL-6 level, in a positive correlation with disease severity. Our results suggest that the disturbance in proinflammatory CD14⁺CD16⁺ monocytes occurs in T2DM and DN uremic patients. Such immunological dysfunction may be related to the activation of TLR4/NF-κB and STAT5 signaling pathways underlying the immune abnormalities of CD14⁺CD16⁺ monocytes.     

Expansion of CD14+CD16+Monocytes in Critically Ill Cardiac Surgery Patients

We have asked whether critically ill cardiac valve surgery patients identified by a high APACHE II score exhibit an increase in the number of proin-flammatory CD14⁺ CD16⁺ monocytes. A group of 12 patients was studied over a period of 5 days post cardiac valve surgery for changes in blood monocyte populations. Patients were selected on day 1 post surgery to either be in good clinical condition (APACHE II Score of 14; N = 9) or to be critically ill (APACHE II score of 24; N = 3). The 14 patients had an uneventful course and could leave the ICU after 2–3 days. Among the 24 patients two showed a decrease of the score to 14 within the 5 days of observation and they could leave the ICU thereafter. One 24 patient (patient #2) had a persistently high score and finally died on day 28. Analysis of blood monocytes on day 1 post surgery revealed that the 14 patients had normal values of CD14⁺CD16⁺ monocytes (44 ± 9/l). By contrast the 24 patients had increased values of these cells with 243 ± 106 cells per 1 on day 1. The numbers of CD14⁺CD16⁺ monocytes returned to the control range over the 5 days of observation in 2 of the 24 patients concomitant with the improvement of the APACHE II score. CD14⁺CD16⁺ monocytes remained, however, at a high level in patient #2, the patient with persistently high APACHE II score.     

Stem Cells, Including a Population of Very Small Embryonic-Like Stem Cells, are Mobilized Into Peripheral Blood in Patients After Skin Burn Injury

Background  

Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), as well as very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue and organ injury (e.g., heart infarct or stroke).     

Haemodialysis monocytopenia: differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets

In peripheral blood the majority of circulating monocytes present a CD14highCD16- (CD14++) phenotype, while a subpopulation shows a CD14lowCD16+ (CD14+CD16+) surface expression. During haemodialysis (HD) using cellulosic membranes transient leukopenia occurs. In contrast, synthetic biocompatible membranes do not induce this effect. We compared the sequestration kinetics for the CD14+CD16+ and CD14++ monocyte subsets during haemodialysis using biocompatible dialysers. Significant monocytopenia, as measured by the leucocyte count, occurred only during the first 30 min. However, remarkable differences were observed between the different monocyte subsets. CD14++ monocyte numbers dropped to 77 +/- 13% of the predialysis level after 15 min, increasing to > or = 93% after 60 min. In contrast, the CD14+CD16+ subset decreased to 33 +/- 15% at 30 min and remained suppressed for the course of dialysis (67 +/- 11% at 240 min). Approximately 6 h after the end of HD the CD14+CD16+ cells returned to basal levels. Interestingly, the CD14+CD16+ monocytes did not show rebound monocytosis while a slight monocytosis of CD14++ monocytes was occasionally observed during HD. A decline in CD11c surface density paralleled the sequestration of CD14+CD16+ monocytes. Basal surface densities of important adhesion receptors differed significantly between the CD14+CD16+ and CD14++ subsets. In conclusion, during HD the CD14+CD16+ subset revealed different sequestration kinetics, with a more pronounced and longer disappearance from the blood circulation, compared with CD14++ monocytes. This sequestration kinetics may be due to a distinct surface expression of major adhesion receptors which facilitate leucocyte-leucocyte, as well as leucocyte-endothelial, interactions.     

Elevated blood levels of inflammatory monocytes (CD14+ CD16+ ) in patients with complex regional pain syndrome

Complex regional pain syndrome (CRPS) is a chronic pain disorder. Although its pathophysiology is not completely understood, neurogenic inflammation is thought to play a significant role. Microglia and astrocytes are activated following tissue injury or inflammation and have been reported to be both necessary and sufficient for enhanced nociception. Blood-borne monocytes/macrophages can infiltrate the central nervous system (CNS) and differentiate into microglia resulting in hypersensitivity and chronic pain. The primary aim of this study was to evaluate the proportion of the proinflammatory CD14(+) CD16(+) monocytes as well as plasma cytokine levels in blood from CRPS patients compared to age- and gender-matched healthy control individuals. Forty-six subjects (25 CRPS, 21 controls) were recruited for this study. The percentage of monocytes, T, B or natural killer (NK) cells did not differ between CRPS and controls. However, the percentage of the CD14(+) CD16(+) monocyte/macrophage subgroup was elevated significantly (P<0·01) in CRPS compared to controls. Individuals with high percentage of CD14(+) CD16(+) demonstrated significantly lower (P<0·05) plasma levels on the anti-inflammatory cytokine interleukin (IL)-10. Our data cannot determine whether CD14(+) CD16(+) monocytes became elevated prior to or after developing CRPS. In either case, the elevation of blood proinflammatoty monocytes prior to the initiating event may predispose individuals for developing the syndrome whereas the elevation of blood proinflammatory monocytes following the development of CRPS may be relevant for its maintenance. Further evaluation of the role the immune system plays in the pathogenesis of CRPS may aid in elucidating disease mechanisms as well as the development of novel therapies for its treatment.     

Circulating CD14+CD16+ monocyte levels predict tissue invasive character of cholangiocarcinoma

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro‐tumorigenic activity. In this study we identified elevation in CD14⁺CD16⁺, a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14⁺CD16⁺ monocytes in CCA patient blood correlated with degree of MAC387‐positive (recent blood‐derived macrophage migrant‐specific marker) tumour‐associated macrophage infiltration as determined by immunohistochemistry. These CD14⁺CD16⁺ monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor‐related genes (epiregulin, VEGF‐A and CXCL3). Elevation of peripheral CD14⁺CD16⁺ monocyte levels was associated with features associated with poor prognosis CCA parameters (non‐papillary type and high number of tissue macrophages). These data indicate that the CD14⁺CD16⁺ monocytes from CCA patients with pro‐tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14⁺CD16⁺ monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.     

Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients

CD16+ monocytes are expanded in various inflammatory conditions. Recently it was reported that CD16+ monocytes can be divided into two subsets with contrasting potential of modulating inflammatory responses, namely CD14++CD16+ and CD14+CD16+ monocytes. Here, we characterized and quantified CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatic patients in the context of severity of disease and different treatment options. Subjects included seventeen severe asthmatics and eighteen moderate asthmatics treated with moderate-to-high doses of inhaled glucocorticosteroids (GCS), twenty nine steroid-naive mild asthmatics and fifteen healthy controls.First, we demonstrated that CD14++CD16+ monocytes, in contrast to CD14+CD16+ monocytes, present significantly higher expression of anti-inflammatory molecule CD163. The frequency of CD14++CD16+, but not CD14+CD16+ monocytes, was significantly higher in patients with severe asthma as compared to mild and moderate asthmatics. However, the frequency of both CD16+ monocyte subsets did not correlate directly with exhaled nitric oxide levels. Short-term administration of oral GCS in patients with exacerbations resulted in a preferential decrease of CD14+CD16+ monocytes. Our study indicates that CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatics are differentially modulated by both the inflammatory process and GCS treatment.     

Pro-angiogenic CD14++ CD16+ CD163+ monocytes accelerate the in vitro endothelialization of soft hydrophobic poly(n-butyl acrylate) networks

As the majority of the polymers used as cardiovascular grafts so far do not match the elasticity of human arteries (100–1000 kPa) and the required endothelialization, a multifunctional material approach is needed to allow the adjustment of the mechanical properties while at the same time exhibiting a haemocompatible surface. Recently soft poly(n-butyl acrylate) networks (cPnBA) with adjustable mechanical properties were introduced as candidate materials with a surface that can be endothelialized. In this study, angiogenically stimulated intermediate CD163⁺ monocytes/macrophages (aMO2) were utilized as a cellular cytokine release system to realize the functional endothelialization of the hydrophobic cPnBA surface. We investigated the influence of co-cultured aMO2 on the morphology, density and cytokine secretion of human umbilical venous endothelial cells (HUVEC) seeded on cPnBA with an elastic modulus of around 250 kPa (cPnBA0250). A functional confluent HUVEC monolayer could be developed in the co-culture within 3 days. In contrast, the HUVEC in the monoculture exhibited stress fibres, broadened marginal filament bands and significantly more and larger cell-free areas in the monolayer, indicating incomplete cell–substrate binding. Remarkably, a functional confluent monolayer formation could only be achieved in co-cultures; it did not develop with the sole supplementation of recombinant VEGF-A₁₆₅ to the HUVEC monocultures (unpublished data). The study demonstrated the multifunctional potential of cPnBA in combination with aMO2 as a cellular cytokine release system, adapting their secretion to the demand of HUVEC. In this way, a functional confluent monolayer could be generated within 3 days.     

Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3-10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7-14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16(-) and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16(-), the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.     

Plasmodium falciparum Infection of Human Volunteers Activates Monocytes and CD16+ Dendritic Cells and Induces Upregulation of CD16 and CD1c Expression

Antigen-presenting cells (APCs) are key players in the induction and regulation of immune responses. In Plasmodium falciparum malaria, determination of which cells and pathways are activated in the network of APCs remains elusive. We therefore investigated the effects of a controlled human malaria infection in healthy, malaria-naive volunteers on the subset composition and activation status of dendritic cells (DCs) and monocytes. While subsets of monocytes increased in frequency during blood-stage infection, DC frequencies remained largely stable. Activation markers classically associated with peptide presentation to and priming of αβT cells, HLA-DR and CD86, were upregulated in monocytes and inflammatory CD16 myeloid DCs (mDCs) but not in the classical CD1c, BDCA2, or BDCA3 DC subsets. In addition, these activated APC subsets showed increased expression of CD1c, which is involved in glycolipid antigen presentation, and of the immune complex binding Fcγ receptor III (CD16). Our data show that P. falciparum asexual parasites do not activate classical DC subsets but instead activate mainly monocytes and inflammatory CD16 mDCs and appear to prime alternative activation pathways via induction of CD16 and/or CD1c. Changes in expression of these surface molecules might increase antigen capture and enhance glycolipid antigen presentation in addition to the classical major histocompatibility complex class II (MHC-II) peptide presentation and thereby contribute to the initiation of T-cell responses in malaria. (This study has been registered at Clinicaltrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01086917","term_id":"NCT01086917"}}NCT01086917.)     

Preparations of intravenous immunoglobulins diminish the number and proinflammatory response of CD14+CD16++ monocytes in common variable immunodeficiency (CVID) patients

We have studied the effect of intravenous immunoglobulins (IVIG) on monocyte subpopulations and cytokine production in patients with CVID. The absolute number of CD14(+)CD16(++) monocytes decreased on average 2.5-fold 4h after IVIG and after 20h returned to the baseline. The cytokine level in the supernatants of peripheral blood mononuclear cells (PBMC) after ex vivo LPS stimulation demonstrated the >2-fold decrease in TNF production 4h after IVIG. The TNF expression, which is higher in the CD14(+)CD16(++) monocytes, was decreased in these cells by IVIG in 4/7 CVID cases. In vitro exposure of the healthy individuals' monocytes to the IVIG preparation resulted in reduced TNF production, which was overcome by blockade of the FcγRIIB in the CD14(+)CD16(++) CD32B(high) monocytes. Our data suggest that reduction in the number of CD14(+)CD16(++) monocytes and the blockade of their cytokine production via triggering CD32B can contribute to the anti-inflammatory action of IVIG.     

Comparative Analysis of the Morphological, Cytochemical, Immunophenotypical, and Functional Characteristics of Normal Human Peripheral Blood Lineage/CD16/HLA-DR/CD14 Cells, CD14 Monocytes, and CD16 Dendritic Cells

Human peripheral blood (PB) CD14^lo/HLA-DR⁺ cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16⁺/HLA-DR⁺ cells compared to both PB CD14⁺ monocytes and CD16⁻ DC. In contrast to CD14⁺ monocytes, purified CD16⁺/HLA-DR⁺ cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and α-naphthyl acetate esterase. Normal human PB CD16⁺/HLA-DR⁺ cells also displayed phenotypic characteristics different from those of CD14⁺ monocytes: they lacked the CD64 Fcγ receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14⁺ monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16⁻ DC, CD16⁺/HLA-DR⁺ cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16⁺/HLA-DR⁺ cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16⁻ DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-γ-stimulated PB CD16⁺/HLA-DR⁺ cells produced significant amounts of IL1β, IL6, IL12, TNFα, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16⁺/HLA-DR⁺ cells than in CD14⁺ monocytes. In addition, upon comparing CD16⁺/HLA-DR⁺ cells with CD33⁺⁺⁺/CD16⁻ DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16⁺/HLA-DR⁺ cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16⁻ DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow.     

Elevated levels of peripheral blood CD14brightCD16+ and CD14dimCD16+ monocytes may contribute to the development of retinopathy in patients with juvenile onset type 1 diabetes

The study aimed to analyze the CD14^brightCD16⁺ and CD14^dimCD16⁺ monocyte subsets in juvenile‐onset complication‐free diabetes mellitus type 1 in the context of their association with microvascular complications. 61 children with type 1 diabetes and 30 healthy individuals were enrolled in a study. CD14^brightCD16⁺ and CD14^dimCD16⁺ monocytes were quantified in peripheral blood by means of flow cytometry. At the time of sampling blood glucose concentration was taken along with biochemical measurement of renal function, CRP and glycosylated hemoglobin. The Spearman's correlations were used to compare the relationship between CD16⁺ monocyte subsets and the clinical parameters that can predict the development of microangiopathies. The flow cytometric analysis of monocyte subsets in peripheral blood of analyzed subjects revealed that the numbers of CD14^brightCD16⁺ and CD14^dimCD16⁺ monocytes were significantly higher in patients with type 1 diabetes than in the healthy individuals. As to the relationship between CD16⁺ monocyte subsets and the clinical parameters that can predict development of microangiopathies, it was shown that both CD16⁺ subsets were associated with increased risk of retinopathy development, defined as retinopathy development value. Elevated levels of intermediate CD14^brightCD16⁺ and non‐classical CD14^dimCD16⁺ monocytes predict development of diabetic retinopathy in patients with type 1 diabetes.     

Small (CD14+/CD16+) monocytes and regular monocytes in human blood.

Compared to the obvious phenotypic and functional heterogeneity of tissue macrophages, little information is available on subsets of blood monocytes. We have employed two-color immunofluorescence and flow cytometry for the definition of regular and small monocytes, the latter characterized by the low-density expression of CD14 and the strong expression of the CD16 (Fcy-RIII) antigen. These cells comprise 15% of the blood monocytes and they appear to be similar in phenotype to the alveolar macrophage. The CD14+/CD16+ small monocytes can perform phagocytosis and they produce reactive oxygen, while their capacity for cytokine production is strongly reduced when compared to regular monocytes. At this point it is still unclear as to whether the CD14+/CD16+ small monocytes comprise a specific level of activation or differentiation or a distinct sublineage of human blood monocytes.     

CD14(dim)/CD16(bright) monocytes in hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an extremely rare and highly lethal chronic inflammatory disease, which is mediated by proinflammatory cytokines. In the peripheral blood of a boy suffering from HLH, a chronic expansion of CD14(dim)/CD16(bright) inflammatory monocytes was detected. Compared with CD14(bright) monocytes, their immunophenotype correlated with more mature monocytic cells differentiating to macrophages: they showed lower expression of CD11b, CD64 and CD35. Such CD14(dim)/CD16(bright) monocytes produce the inflammatory cytokines IL-1beta, IL-6 and TNF-alpha. They fit in well with the pathophysiological concept of HLH as an inflammatory state of lymphocytes and of the monocyte/macrophage system. In the presented patient the percentage of these circulating inflammatory monocytes decreased over time during clinical response to immunosuppressive therapy. This finding may indicate that CD14(dim)/CD16(bright) monocytes represented the degree of inflammation in this extremely rare and highly lethal disease.