Glucocorticoid‐induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2

Lin L, Dai S‐Dong, Fan G‐Yu. Glucocorticoid‐induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2. APMIS 2010; 118: 595–605. Long‐term clinical use of glucocorticoids often causes the serious side effect of non‐traumatic avascular osteonecrosis. The aim of this study was to examine the effects and mechanisms of a glucocorticoid, dexamethasone (Dex), on differentiation of primary cultured rat bone marrow mesenchymal cells (BMCs). We also tried to block the inhibitory effects of Dex on osteoblast differentiation. Adipocyte markers (peroxisome proliferator‐activated receptorγ‐2 and aP2) were increased in response to Dex treatment in a dose‐ and time‐dependent manner, while osteoblastic markers [Runx2, COL 1, osterix, alkaline phosphatase (ALP) and OC] were down‐regulated, consistent with ALP and osteocalcin promoter activity. To validate the effects of Runx2 on the expression of osteogenesis and adipocyte genes, pCMV/Flag‐Runx2 was transfected into BMCs, and relevant markers were detected after 10^?7 M Dex treatment for 48 h. The results indicated that Dex treatment induced adipogenic differentiation and suppressed proliferation. No significant difference was detected in expressions of these genes between Runx2‐transfected cells and Dex‐treated BMCs. These data suggest that Dex primarily induced adipocyte differentiation of BMCs. Exogenous Runx2 can antagonize the effect of Dex on osteoblast differentiation.     

Growth and adipogenic differentiation of mesenchymal stromal bone marrow cells during culturing in 3D macroporous agarose cryogel sponges

We studied the possibility of population of macroporous agarose cryogel sponges by mesenchymal stromal bone marrow cells with their subsequent adipogenic differentiation. After 7-day culturing of mesenchymal stromal cells in agarose cryogel, the level of cell proliferation was 35%. After 3-week culturing in a medium inducing adipogenesis we observed accumulation of intracellular neutral lipids positively stained with Oil Red O. These findings can be used for the development of bioengineering constructions of the adipose tissue on the basis of spongy carriers. Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 3, pp. 141–144, August, 2008     

miR-34b对骨髓间充质干细胞成骨分化的影响及相关机制

目的：探讨miRNA-34b在骨髓间充质干细胞成骨分化过程中的表达及其可能的作用靶点和作用机制。方法：采用密度梯度离心和全骨髓贴壁相结合的方法分离培养人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs),并在体外诱导成骨分化。采用实时荧光定量PCR技术,检测hBMSCs成骨分化过程中的miR-34b的表达水平;然后过表达miR-34b,进一步观察其对hBMSCs成骨分化的影响。同时检测过表达miR-34b对成骨分化的关键信号通路之一Notch信号通路的活性影响,初步探讨其可能涉及的作用机制。结果：成功分离出hBMSCs,并构建了hBMSCs体外诱导成骨分化模型;且随着成骨诱导培养时间的延长,miRNA-34b表达水平逐渐降低。ALP活性检测、茜素红染色检测结果显示过表达miRNA-34b后,ALP活性显著降低,且茜素红染色的钙盐结节明显减少;同时Western blot实验结果显示过表达miRNA-34b后,成骨特异性标记分子Runx2的蛋白表达水平显著下降(P<0.05)。此外,过表达miRNA-34b后,Notch信号通路的活性显著降低。结论：miRNA-34b能够负向调控人骨髓间充质干细胞成骨分化;其作用机制可能与抑制Notch信号通路的活性有关,提示miRNA-34b可以作为诊断和靶向治疗慢性炎症性骨疾病的潜在作用靶点。     

Amyloid‐Aβ Peptide in Olfactory Mucosa and Mesenchymal Stromal Cells of Mild Cognitive Impairment and Alzheimer's Disease Patients

Patients with mild cognitive impairment (MCI) or Alzheimer's disease (AD) might develop olfactory dysfunction that correlates with progression of disease. Alteration of olfactory neuroepithelium associated with MCI may be useful as predictor of cognitive decline. Biomarkers with higher sensitivity and specificity would allow to understand the biological progression of the pathology in association with the clinical course of the disease. In this study, magnetic resonance images, apolipoprotein E (ApoE) load, Olfactory Connecticut test and Montreal Cognitive Assessment (MoCA) indices were obtained from noncognitive impaired (NCI), MCI and AD patients. We established a culture of patient‐derived olfactory stromal cells from biopsies of olfactory mucosa (OM) to test whether biological properties of mesenchymal stromal cells (MSC) are concurrent with MCI and AD psychophysical pathology. We determined the expression of amyloid Aβ peptides in the neuroepithelium of tissue sections from MCI and AD, as well as in cultured cells of OM. Reduced migration and proliferation of stromal (CD90⁺) cells in MCI and AD with respect to NCI patients was determined. A higher proportion of anosmic MCI and AD cases were concurrent with the ApoE ε4 allele. In summary, dysmetabolism of amyloid was concurrent with migration and proliferation impairment of patient‐derived stem cells.     

TGF‐β affects development and differentiation of human natural killer cell subsets

Human peripheral blood NK cells may be divided into two main subsets: CD56^brightCD16^? and CD56^dimCD16⁺. Since TGF‐β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34⁺Lin^? hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF‐β represses development of NK cells from CD34⁺ progenitors and inhibits differentiation of CD16⁺ NK cells. Moreover, TGF‐β also results in conversion of a minor fraction of CD56^brightCD16⁺ cells found in peripheral blood into CD56^brightCD16^? cells, highlighting a possible role of the former as a developmental intermediate and of TGF‐β in influencing the genesis of NK subsets found in blood.     

Changes in thymic export of γδ and αβ T cells during fetal and postnatal development

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5–1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both αβ and γδ T cells, our results demonstrate that the proportion of thymic γδ T cells that are exported per day is much higher than that of thymic αβ T cells. Moreover, the export rate of γδ T cells increased from approximately 1 in every 60 γδ thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5⁺CD4^?CD8^?γδ^? T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both γδ and αβ T cells.     

β2 -Microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition,but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking β2 -microglobulin (β₂ m− mice) express greatly reduced levels of MHC class I molecules, and cells from β₂ m− mice are therefore highly sensitive NK cells. However, NK cells from β₂ m− mice fail to kill β₂ m− normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of β₂ m− NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to β₂ m− NK cells, we made the following observations: First, β₂ m− NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to β₂ m− concanavalin A (Con A)-activated T cell blasts. Secondly, β₂ m− NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than β₂ m+ NK cells. Third β₂ m− NK cells were not only tolerant to β₂ m− Con A blasts but also to Con A blasts from H-2Kb − /Db − double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of β₂ m− NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in β₂ m− mice, which may instead operate through mechanisms involving triggering pathways.     

Changes in thymic export of L-selectin+ γδ and αβ T cells during fetal and postnatal development

We have used the technique of in situ intrathymic injection of fluorescein isothiocyanate to examine L-selectin expression on γδ and αβ T cells immediately after emigrating from the thymus of fetal and postnatal animals. We found that the percentage of L-selectin⁺ thymocytes exported per day decreased by half after birth and that the export of T cells from the thymus does not rely on expression of the peripheral lymph node homing receptor, L-selectin. Analysis of L-selectin on emigrant and mature T cell subsets revealed a remarkable heterogeneity of expression, both in terms of the numbers of cells expressing this molecule as well as the level of expression. γδ T cells, reportedly not having a propensity for homing to lymph nodes, not only contained the highest proportion of L-selectin⁺ cells, but also expressed far more of this molecule than either CD4⁺CD8^? or CD4^?CD8⁺ αβ T cells. Furthermore, those emigrant T cells expressing L-selectin are somewhat immature in their expression of this molecule. Subsequent maturation resulted in up-regulation of L-selectin on mature peripheral blood T cells, maturation that was clearly independent of extrinsic antigen. This antigen-independent post-thymic maturation appeared to occur as part of the normal progression from immature thymocyte to mature peripheral T cell in both fetal and postnatal animals.     

Differential expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins PTEN and p63 in verrucous carcinoma of the head and neck

Odar K, Bo?tjan?i? E, Gale N, Glava? D & Zidar N
(2012) Histopathology  61, 257–265 Differential expression of microRNAs miR‐21, miR‐31, miR‐203, miR‐125a‐5p and miR‐125b and proteins PTEN and p63 in verrucous carcinoma of the head and neck Aims: To investigate the expression of microRNAs miR‐21, miR‐31, miR‐203, miR‐125a‐5p and miR‐125b and proteins phosphatase and tensin homologue (PTEN) and p63 in verrucous carcinoma (VC) of the head and neck. Methods and results: Thirty cases of VC, 50 cases of conventional squamous cell carcinoma (SCC) and 30 samples of normal epithelium of the head and neck were included. Real‐time polymerase chain reaction and immunohistochemistry were used to analyse the expression of microRNAs and proteins, respectively. In comparison to normal epithelium, miR‐21 was overexpressed in both VC and SCC and miR‐31 was overexpressed in VC and in well‐ and moderately differentiated SCC. Levels of miR‐203 were elevated in VC but unaltered or reduced in SCC, and levels of miR‐125a‐5p and miR‐125b were reduced in VC but unaltered in SCC. PTEN was down‐regulated in both VC and SCC, whereas p63 was down‐regulated in VC but up‐regulated in SCC. Differential expression of p63 in VC correlated inversely with the expression of miR‐21 and miR‐203. Conclusions: Differences between VC, SCC and normal epithelium in expression profiles of investigated molecules indicate their association with the pathogenesis and clinicopathological characteristics of VC. Our results suggest that some microRNAs and proteins, particularly miR‐125b, miR‐203 and p63, might be useful in the diagnosis of VC.     

Primary over‐expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK‐AβPP transgenic mouse

The aim of this study is to determine whether primary over‐expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK‐AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild‐type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over‐expression of full length AβPP in transgenic mouse muscles, deposition of amyloid‐β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase‐deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild‐type animals was the presence of tubular aggregates which was age‐related and largely restricted to male animals. Expression of full length AβPP in this MCK‐AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid‐β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re‐deriving transgenic mouse strains in different laboratories.     

Interferon-γ arrests proliferation and causes apoptosis in stromal cell/interleukin-7-dependent normal murine pre-B cell lines and clones in vitro,but does not induce differentiation to surface immunoglobulin-positive B cells

Normal pre-B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin-7 (IL-7). Their IgH loci are partly in germ-line, partly in D_HJ_H-rearranged configuration, while their light chain loci are in germ-line configuration. They express the pre-B cell-specific genes V_preB and λ₅. Proliferation of these pre-B cells is inhibited by interferon (IFN)-γ, with half-maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre-B cells exposed to IFN-γ die by apoptosis, as is evidenced by the disintegration of pre-B cell DNA into oligonucleosomal multimers of 180-200 bp. While the proliferation of pre-B cells from Eμ-bcl-2 transgenic (tg) mice is inhibited by IFN-γ, these cells do not die by apoptosis. IFN-γ does not induce differentiation to more mature B lineage cells. In the absence of IL-7 normal pre-B cells differentiate to V_HD_HJ_H/V_LJ_L-rearranged, surface immunoglobulin-positive B cells expressing the a chain of the IL-2 receptor. They also down-regulate the expression of V_preB and X.5, and lose the capacity to proliferate on stromal cells in the presence of IL-7. In contrast, both normal and Eμ-bcl-2 tg pre-B cells exposed to IFN-γ in the presence of stromal cells and IL-7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of V_preB and λ₅, do not express the α chain of the IL-2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL-7, once IFN-γ is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B-ALL) with IFN-γ is discussed.