Involvement of ganglioside GT1b in glutamate release from neuroblastoma cells

Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.     

Indoxyl 3-sulfate inhibits maturation and activation of human monocyte-derived dendritic cells

Indole is produced from l-tryptophan by commensal bacteria and further metabolized to indoxyl 3-sulfate (I3S) in the liver. Physiologic concentrations of I3S are related to a lower risk to develop graft versus host disease in allogeneic stem cell transplanted patients pointing towards an immunoregulatory function of I3S. Here we investigated the impact of I3S on the maturation of human monocyte-derived dendritic cells (DCs). Even pathophysiologic concentrations of I3S did not affect viability of mature DCs, but I3S decreased the expression of co-stimulatory molecules such as CD80 and CD86 on mature DCs. Furthermore, I3S inhibited IL-12 and IL-6 secretion by mature DCs while IL-10 was significantly upregulated. Co-culture of I3S-treated mature DCs with allogeneic T cells revealed no alteration in T cell proliferation. However, interferon gamma and TNF production of T cells was suppressed. As I3S exerted no direct effect on T cells, the defect in T cell activation was mediated by I3S-treated mature DCs. Our study suggests an anti-inflammatory and tolerizing effect of I3S on human DCs.     

Intestine-derived Clostridium leptum induces murine tolerogenic dendritic cells and regulatory T cells in vitro

Patients with autoimmune and allergic diseases frequently present with reduced numbers and functionally impaired regulatory T cells (Tregs) and/or tolerogenic dendritic cells (tDCs). tDC-mediated regulation of Treg proliferation (numbers) and activation is crucial to establishing and maintaining an appropriate level of immune tolerance. Colonic colonization of Clostridium spp. is associated with accumulation of Tregs, which inhibits development of inflammatory lesions. To investigate whether infection with the Clostridium leptum sp. can specifically induce Tregs and/or tDCs bone marrow-derived dendritic cells were cultured in the presence or absence of C. leptum then co-cultured with CD4⁺CD25⁻ T cells or not. Changes in tDC numbers, Treg numbers, percentages of T cell subsets, and expression of cytokines related to Tregs (IL-10 and transforming growth factor-beta (TGF-β1)), DCs (IL-12p40 and IL-6) and effector T cells (IFN-γ, IL-4, IL-5, IL-13, and IL-17A) were measured. In the co-culture system, C. leptum-stimulated tDCs were able to increase the percentage and total number of Tregs attenuate activation of T helper cells (Th1, Th2, and Th17), and decrease the amount of secreted IL-4, IL-5, IL-13, IFN-γ and IL-17A. Thus, C. leptum exposure can induce the tDC-mediated stimulation of Tregs while disrupting the immune inflammatory response mediated by Th1, Th2 and Th17 cells.     

MiR-9 promotes the neural differentiation of mouse bone marrow mesenchymal stem cells via targeting zinc finger protein 521

MicroRNAs (miRNAs) are a class of non-coding RNAs that function as endogenous triggers of the RNA interference pathway. Recent studies have shown that microRNA-9 (miR-9) plays a regulatory role in the development and differentiation of stem cells and neural precursor cells. We have found that miR-9 is able to promote the differentiation of bone marrow mesenchymal stem cells (MSCs), but the mechanisms of miR-9 in this process remains poorly understood. An increasing number of studies have found that zinc-finger protein 521 (Zfp521) expression is high in most immature cells and decreases with differentiation. Zfp521 could induce neural conversion of embryonic stem cells. However, little is known about the expression of Zfp521 and its relationship with miR-9 with respect to the neural differentiation of MSCs. In this study, we found the expression of Zfp521 declined with the neural differentiation of MSCs, and miR-9 could promote the neural differentiation via targeting Zfp521.     

Effects of valproic acid on the expression of trophic factors in human bone marrow mesenchymal stromal cells

The potential of human bone marrow-mesenchymal stromal cells (hBM-MSCs) to differentiate into diverse cell types and secrete a variety of trophic factors makes them an excellent cell therapy tool for intractable diseases. However, their therapeutic efficacy has not yet been satisfied in preclinical and/or clinical trials with autologous or allogenic stem cells. To improve the efficacy of stem cell therapy, optimized conditions for stem cells need to be defined. In this study, we evaluated the effects of valproic acid (VPA), an HDAC inhibitor, in human BM-MSCs and assessed the expression of trophic factors (ANG, BDNF, ECGF1, bFGF-2, GDNF, HGF, IGF-1, PIGF, TGF-β1, and β-Pix) in MSCs treated with 200μg/ml VPA for 12h. Under these conditions the features of MSCs were not changed. The VPA-treated MSCs also showed an increased cell protective effect against oxidative injuries in MTT assays and improved migratory ability when examined by the Boyden chamber assay. This suggests that MSCs may be improved by treatment with an optimal VPA dose and incubation time, which may increase the efficacy of stem cell therapy.     

Elevated expression of pleiotrophin in pilocarpine-induced seizures of immature rats and in pentylenetetrazole-induced hippocampal astrocytes in vitro

Pleiotrophin (PTN) is a secreted extracellular matrix (ECM)-associated cytokine that has emerged as an important neuromodulator with multiple neuronal functions. In the present study, we detected and compared the dynamic expression of PTN in the hippocampus and adjacent cortex of immature rats with pilocarpine-induced epilepsy. Moreover, we also confirmed the results by examining PTN expression in hippocampal astrocytes cultured in the presence of pentylenetetrazole (PTZ). Immunohistochemistry showed faint immunostaining of PTN in the control hippocampus and adjacent cortex. Notably, PTN immunoreactivity began to increase in relatively small cells in the hippocampus and adjacent cortex at 2 h and 3 weeks after seizures, and the labeling intensity reached the maximum level in the hippocampus and adjacent cortex at 8 weeks after seizures. Furthermore, we also found that PTZ treatment significantly reduced astrocytic viability in a dose-dependent manner and time-dependently increased expression levels of PTN in hippocampal astrocytes. In conclusion, our data suggest that increased expression of PTN in the brain tissues may be involved in epileptogenesis.     

The pharmacological efficacy of mycophenolic acid before and after renal transplantation as estimated by the lymphocyte immunosuppressant sensitivity test (LIST)

The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of immunosuppressive agents. We herein estimated the mycophenolic acid efficacy using LIST before and 1, 3, and 12 months after the operation in 15 renal transplant recipients. The pharmacological efficacy of mycophenolic acid as assessed before transplantation showed small individual variations, whereas the variability greatly increased at 1 and 3 months after transplantation. Furthermore, the individual IC50 variation among these subjects at 1-year after operation was closely similar to that observed before surgery. No significant implications of these individual differences in the mycophenolic acid sensitivity were noted in regard to the clinical courses of these recipients.     

Deficiency of monoclonal antibody (Leu 7) defined NK cells in newly diagnosed insulin-dependent diabetes mellitus

Peripheral blood from 11 newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM) was studied for the proportion of monoclonal antibody (HNK 1, Leu 7) defined natural killer (NK) cells using a fluorescence-activated cell sorter analyzer. The proportion of Leu 7+ cells in patients with IDDM (7.0 +/- 4.0) was significantly (P less than 0.001) lower than in simultaneously studied healthy controls (16.8 +/- 7.0). A 2-yr-old boy with recent onset IDDM had a deficiency of Leu 7+ NK cells (6.1%), while his healthy identical twin had normal proportions of Leu 7+ cells (22.2%), when compared to a simultaneously studied healthy control. Two patients reexamined in remission and one other studied in remission alone, showed deficiency of Leu 7+ NK cells. This study demonstrates a quantitative deficiency of monoclonal antibody (Leu 7+) defined NK cells in newly diagnosed patients with IDDM that persists during remission of the disease and therefore appears to be independent of metabolic abnormality. The deficiency of NK cells may predispose genetically susceptible individuals to viral-induced islet cell injury, contributing to the pathogenesis of IDDM.     

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.     

p62 protects SH-SY5Y neuroblastoma cells against H2O2-induced injury through the PDK1/Akt pathway

The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H₂O₂ treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H₂O₂-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H₂O₂-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3θ protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.     

Curcumin induces permanent growth arrest of human colon cancer cells: link between senescence and autophagy

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53−/− cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53−/− HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.     

Artificial antigen-presenting cells expressing CD80, CD70, and 4-1BB ligand efficiently expand functional T cells specific to tumor-associated antigens

Professional antigen-presenting cells (APCs), notably dendritic cells (DCs), are the most potent for expanding antigen-specific T cells ex vivo. However, the labor-intensive and expensive procedure for customized preparation of autologous APCs has hampered their broad clinical application. Artificial APC (aAPC) systems, which can be readily prepared from off-the-shelf components, have been proposed as a promising alternative to custom-made professional APCs. Here, in order to develop a novel aAPC system, we established K562 erythroleukemia cells expressing different combinations of co-stimulatory molecule ligands, CD80, CD70, and/or 4-1BB ligand (4-1BBL). When nucleofected with in vitro-generated mRNA encoding a tumor-associated antigen, MART-1, the K562 cells expressing all of CD80, CD70, and 4-1BBL were the most efficient for expansion of functional T cells specific to an HLA-A2-restricted immunodominant epitope, MART-1_26–35. In addition, only the K562 cells expressing all three of these co-stimulatory molecule ligands could clearly expand T cells specific to other less immunogenic antigen epitopes, gp100_154–162 and Cyp1B1_239–247, through transfection with in vitro generated gp100 and Cyp1B1 mRNA, respectively. These results indicated that non-redundant and synergistic effects of co-stimulation via CD28/CD80, CD27/CD70, and 4-1BB/4-1BBL might be critical for eliciting efficient expansion of T cells; co-stimulation via the 4-1BB/4-1BBL interaction might expand antigen-specific T cells by preventing apoptotic cell death triggered by specific antigens in the presence of the CD28/CD80 and CD27/CD70 signaling. Taken together, our findings suggested that this K562-based aAPC system expressing CD80, CD70, and 4-1BBL would be useful for efficiently stimulating functional antigen-specific T cells ex vivo, in particular when detailed information on the epitope specificities is unavailable.     

The effect of azithromycin on the maturation and function of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells capable of initiating primary/adaptive immune responses and tolerance. DC functions are regulated by their state of maturation. However, the molecular pathways leading to DC development and maturation remain poorly understood. We attempted to determine whether inhibition of nuclear factor kappa B (NF-κB), which is one of the pivotal pathways underlying these processes, could induce immunophenotypic and functional changes in lipopolysaccharide-induced mature DCs derived from murine bone marrow. A comparative in vitro study of five clinically used drugs that are known to inhibit NF-κB demonstrated that azithromycin, a macrolide antibiotic, significantly inhibited expression of co-stimulatory molecules (CD40 and CD86) and major histocompatibility complex (MHC) class II by DCs. It also reduced Toll-like receptor 4 expression, interleukin-12 production and the allostimulatory capacity of DCs. These data suggest that azithromycin, as not only an NF-κB inhibitor but also an antibiotic, has potential as a novel drug for manipulation of allogeneic responses.     

Purified high molecular weight synthetic Aβ(1–42) and biological Aβ oligomers are equipotent in rapidly inducing MTT formazan exocytosis

Synthetic soluble Aβ oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of Aβ oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10–100 nM). Purified oligomers, but not monomers or fibrils, elicited an increase of a detergent-insoluble form of MTT formazan within 2 h as opposed to a control toxin (H₂O₂). This effect was comparable for biological and synthetic peptide in both cell types. Monomeric Aβ attenuated the effect of soluble oligomers. This study suggests that the activities of biological and synthetic oligomers are indistinguishable during early stages of Aβ oligomer–cell interaction.     

The effect of human immunodeficiency virus-1 on monocyte-derived dendritic cell maturation and function

Dendritic cells (DC) are mediators of the adaptive immune response responsible for antigen presentation to naive T cells in secondary lymph organs. Human immunodeficiency virus (HIV-1) has been reported to inhibit the maturation of DC, but a clear link between maturation and function has not been elucidated. To understand further the effects of HIV-1 on DC maturation and function, we expanded upon previous investigations and assessed the effects of HIV-1 infection on the expression of surface molecules, carbohydrate endocytosis, antigen presentation and lipopolysaccharide (LPS) responsiveness over the course of maturation. In vitro infection with HIV-1 resulted in an increase in the expression of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as well as decreases in maturation-induced CCR7 and major histocompatibility complex (MHC)-II expression. Retention of endocytosis that normally occurs with DC maturation as well as inhibition of antigen presentation to CD8(+) T cells was also observed. Mitogen-activated protein kinase (MAPK) responsiveness to LPS as measured by phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK)1/2 was not affected by HIV-1 infection. In summary, in-vitro HIV-1 impairs DC maturation, as defined by cell surface protein expression, with selective alterations in mature DC function. Understanding the mechanisms of DC dysfunction in HIV infection will provide further insight into HIV immune pathogenesis.     

The neuroprotective role of tissue inhibitor of metalloproteinase-2 in MPP- or 6-OHDA-treated SK-N-BE(2)C and SH-SY5Y human neuroblastoma cells

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases (MMPs), and the aberrant expressions of MMPs are strongly associated with neuroinflammation and neuronal cell death. In the present study, we found that two well-known dopaminergic neurotoxins, MPP⁺ and 6-OHDA, reduced TIMP-2 expression at the mRNA and protein levels in two human neuroblastoma cell lines (SK-N-BE(2)C and SH-SY5Y). To investigate the role of TIMP-2, these cells were transfected with TIMP-2 expression plasmid and viabilities were compared after treating cells with MPP⁺ or 6-OHDA. It was found that TIMP-2 overexpression attenuated the cell deaths induced by MPP⁺ or 6-OHDA, and that the degree of protection conferred was greater for MPP⁺-treated cells. Furthermore, the introduction of TIMP-2 siRNA into SK-N-BE(2)C cells aggravated the cell deaths induced by MPP⁺ or 6-OHDA. These findings collectively show that endogenously expressed TIMP-2 has a neuroprotective role, and they imply that the inhibition of TIMP-2 expression by MPP⁺ or 6-OHDA may contribute, in part, to neuronal cell death. These findings suggest that TIMP-2 expressional enhancement provides a potential therapeutic strategy for the treatment of neurodegenerative diseases such as Parkinson's disease.     

B7-H3单抗交联作用对Mo-DC体外生物学功能的影响

探讨B7-H3分子对人外周血单核细胞来源树突状细胞(Mo-DC)体外成熟和生物学功能的影响。采用常规方法从健康人外周血单核细胞诱导DC,在诱导过程中,加入B7-H3单抗21D4共培养,经流式细胞术检测Mo-DC上B7-H3分子和其他共刺激分子的表达,ELISA试剂盒检测培养上清中细胞因子IL-10和IFN-γ的分泌量,并采用3H-TdR掺入法测定T细胞的增殖。结果:B7-H3分子在未成熟和成熟Mo-DC上均有高水平表达,抗人B7-H3单抗21D4能上调Mo-DC表面CD80、CD86和CD83的表达,提高Mo-DC的共刺激能力,促进T细胞的体外增殖,并能显著促进T细胞分泌IL-10。由此表明,B7-H3单抗21D4交联作用可以促进Mo-DC体外成熟,上调其共刺激T细胞的能力。     

Chemosensitization of human leukemia K562 cells to taxol by a Vanadium-salen complex

Vanadium complexes are a heterogeneous class of compounds exhibiting interesting biological properties. Herein, we report the effect of a vanadium-salen complex (VO-salen) on proliferative behavior of K562 cell line. The results revealed that VO-salen at 6–32 μM inhibited K562 proliferation with no distinct alteration in cell morphology, extent of apoptosis and/or differentiation. Our results indicated that VO-salen complex has just a cytostatic effect and capable of arresting the affected cells in G2/M phase of cell cycle. In addition, we evaluated the combined effects of VO-salen complex and taxol. The cell cycle analyses showed that VO-salen complex enhanced taxol-induced G2/M arrest and also increased taxol-induced apoptosis through a decrease in the ratio of Bcl-2/Bax which might account for the decrease in the apoptosis threshold among the affected cells. These findings support that combination of VO-salen, as a chemosensitizer, and taxol might constitute an affective new strategy for leukemia therapy.     

Treatment with anti-CC chemokine receptor 3 monoclonal antibody or dexamethasone inhibits the migration and differentiation of bone marrow CD34 progenitor cells in an allergic mouse model

Background: The migration and in situ differentiation of CD34⁺ progenitors contribute to inflammatory eosinophilia in asthma and corticosteroids have been widely used in asthma. However, little is know about whether and how corticosteroids modulate the migration and differentiation of CD34⁺ progenitors. This study was aimed to investigate the impact of anti‐CC chemokine receptor 3 (CCR3) or dexamethasone on inflammatory eosinophilia in asthma and possible mechanism(s) underlying the action of dexamethasone or anti‐CCR3 on migration and differentiation of CD34⁺ progenitors in asthmatic context. Methods: Using an asthmatic mouse model, airway inflammation of anti‐CCR3‐ or dexamethasone‐treated mice and that of controls were characterized. And the migration and differentiation of CD34⁺ progenitor cells were analyzed in vivo, ex vivo or in vitro. Results: Treatment with anti‐CCR3 or dexamethasone significantly inhibited allergen‐induced eosinophilia and CD34⁺ progenitor cell infiltration in the lung, which was accompanied by lower levels of airway hyper‐responsiveness and mucus production. Moreover, anti‐CCR3 inhibited the eotaxin‐mediated migration and IL‐5/eotaxin‐induced differentiation of CD34⁺ progenitors in vitro. Dexamethasone was also shown to mitigate eotaxin‐mediated migration and IL‐5 or eotaxin‐promoted differentiation of CD34⁺ progenitor cells ex vivo, which were associated with the down‐regulation of CCR3 expression on bone marrow progenitor cells. Conclusions: Treatment with anti‐CCR3 or dexamethasone can inhibit the migration and differentiation of CD34⁺ progenitor cells by regulating the eotaxin/CCR3 axis in asthmatic mice. Our findings provide new insights into understanding the mechanism(s) underlying the action of dexamethasone and CCR3‐mediated signaling in allergic inflammation and aid in the design of new immunotherapy for intervention of human asthma.