[LIM kinases and their roles in the nervous system].

LIM kinase-1 (LIMK1) and LIM kinase-2 (LIMK2) are kinases that have serine/threonine and tyrosine dual specificity. Although they show significant structural similarity, LIMK1 and LIMK2 have different expression patterns, subcellular localization, and functions. Activation of LIM kinases regulates the downstream of Rho GTPases, and influences the architecture of the actin cytoskeleton by regulating the activity of cofilin. Recent studies have shown that LIM kinases play important roles in the nervous system. For example, development of the central nervous system is reliant upon the presence of LIMK1, and deletion of Limk1 gene is involved in the development of the human genetic disorder Williams syndrome. Therefore, it is of vital physiological significance to investigate the neuronal function of LIM kinases. In this review, we outline the structure, phosphorylation regulation and neuronal function of LIM kinases, so as to provide new ideas for the treatment of these neurological diseases.

[Corrigendum] Enhanced antitumor activity by the combination of dasatinib and combretastatin A­4 in vitro and in vivo.

Following the publication of this article, an interested reader drew to the authors' attention that the flow cytometric (FCM) plots in Fig. 2A on p. 2278 showing the 'Dasatinib' and 'CA­4' experiments were duplicates of each other. After having re­examined their original data, and due to the overall similarity of the data, the authors have realized that these data were inadvertently assembled incorrectly in the figure. They realize that they also made a further mistake regarding the writing of the ratios of mitochondrial membrane­depolarized HO­8910 cells for these FCM plots (essentially, these were written the wrong way around): The percentage of mitochondrial membrane­depolarized HO­8910 cells should have been written as 22.50% for the dasatinib­treated cells (the centre­left FCM plot) and 15.71% for the CA­4­treated cells (centre­right plot). A revised version of Fig. 2 now showing alternative data for the FCM experiments shown in Fig. 2A, is shown on the next page. Note that the errors made in terms of assembling the data in Fig. 2A did not greatly affect either the results or the conclusions reported in this paper, and all the authors agree with the publication of this corrigendum. The authors regret that these errors went unnoticed prior to the publication of their article, and are grateful to the Editor of Oncology Reports for granting them this opportunity to publish a corrigendum. Furthermore, they apologize to the readership for any inconvenience caused. [Oncology Reports 29: 2275­2282, 2013; DOI: 10.3892/or.2013.2405].

B7-H4 enhances oncogenicity and inhibits apoptosis in pancreatic cancer cells.

B7-H4 is expressed in a variety of tumor cells and functions as a negative regulator of T cells. However, clarification is needed as to whether B7-H4 mediates tumorigenesis through mechanisms, such as apoptosis, in addition to mediating tumor immune escape. We investigate the mechanisms involved in enhanced oncogenicity and the inhibition of apoptosis by B7-H4 in pancreatic cancer cells. Short interfering RNAs (siRNAs) specific for B7-H4 were evaluated for their ability to knockdown B7-H4 mRNA and protein expression in pancreatic cancer cells and the most effective siRNA was selected for investigating the effect of B7-H4 gene silencing in a number of functional assays. The inhibition of B7-H4 increased cell-cell adhesion and decreased the formation of pseudopodia. It also increased the expression of E-cadherin and decreased the expression of vimentin and CD44. B7-H4 siRNA inhibited cell proliferation, colony formation and migration of pancreatic cancer cells. Moreover, increased apoptosis in pancreatic cancer cells following B7-H4 silencing was demonstrated in vitro by using flow cytometry and in a xenograft tumor model and was associated with increased caspase activity and decreased Erk1/2 phosphorylation both in vitro and in vivo. Loss of B7-H4 function thus prevents tumor growth through many processes, including the induction of apoptosis and inhibition of the Erk1/2 signaling pathway indicating that B7-H4 is a cancer promoter and a potentially important therapeutic target. B7-H4 inhibition might offer an exciting opportunity to inhibit the progression of human pancreatic cancers.

Wnt/ß-catenin signaling induces the aging of mesenchymal stem cells through promoting the ROS production.

Recent studies have demonstrated that the Wnt/ß-catenin signaling plays an important role in stem cell aging. However, the mechanisms of cell senescence induced by Wnt/ß-catenin signaling are still poorly understood. Our preliminary study has indicated that activated Wnt/ß-catenin signaling can induce MSC aging. In this study, we reported that the Wnt/ß-catenin signaling was a potent activator of reactive oxygen species (ROS) generation in MSCs. After scavenging ROS with N-acetylcysteine, Wnt/ß-catenin signaling-induced MSC aging was significantly attenuated and the DNA damage and the expression of p16(INK4A), p53, and p21 were reduced in MSCs. These results indicated that the Wnt/ß-catenin signaling could induce MSC aging through promoting the intracellular production of ROS, and ROS may be the main mediators of MSC aging induced by excessive activation of Wnt/ß-catenin signaling.

In vitro anti-cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L.

AIM: To examine the anti-cancer effects of chamaejasmenin B and neochamaejasmin C, two biflavonones isolated from the root of Stellera chamaejasme L (known as the traditional Chinese herb Rui Xiang Lang Du) in vitro. METHODS: Human liver carcinoma cell lines (HepG2 and SMMC-7721), a human non-small cell lung cancer cell line (A549), human osteosarcoma cell lines (MG63, U2OS, and KHOS), a human colon cancer cell line (HCT-116) and a human cervical cancer cell line (HeLa) were used. The anti-proliferative effects of the compounds were measured using SRB cytotoxicity assay. DNA damage was detected by immunofluorescence and Western blotting. Apoptosis and cell cycle distribution were assessed using flow cytometry analysis. The expression of the related proteins was examined with Western blotting analysis. RESULTS: Both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in the 8 human solid tumor cell lines. Chamaejasmenin B (the IC(50) values ranged from 1.08 to 10.8 µmol/L) was slightly more potent than neochamaejasmin C (the IC(50) values ranged from 3.07 to 15.97 µmol/L). In the most sensitive A549 and KHOS cells, the mechanisms underlying the anti-proliferative effects were characterized. The two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis. Furthermore, treatment of the cells with the two compounds caused prominent G(0)/G(1) phase arrest. CONCLUSION: Chamaejasmenin B and neochamaejasmin C are potential anti-proliferative agents in 8 human solid tumor cell lines in vitro via inducing cell cycle arrest, apoptosis and DNA damage.

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism.

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4(+)Foxp3(+) Treg cells and Gr1(+)CD11b(+) MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8(+) T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.

[Expression of high mobility group box chromosomal protein 1 in mice with lupus nephritis].

OBJECTIVE: To determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis. METHODS: Serum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR. RESULT: MRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA. CONCLUSION: The results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.

Enhanced effect of soluble transforming growth factor-beta receptor II and IFN-gamma fusion protein in reversing hepatic fibrosis.

OBJECTIVE: To examine the in vivo anti-fibrotic effect of rat soluble transforming growth factor beta receptor II (RsTbetaRII) and IFN-gamma fusion protein (RsTbRII-IFN-gamma) in rat hepatic fibrosis model. METHODS: Model rats were divided into five groups and treated i.m. for 8 weeks: 1) fibrotic model group (each rat, 100 microl of 0.9% NaCl day superset-1); 2) RsTbetaRII-IFN-gamma treatment group (each rat, 0.136 mg x day(-1); 3) IFN-gamma treatment group (each rat, 7.5 MU x day(-1); 4) RsTbetaRII treatment group (each rat, 0.048 mg x day(-1); and 5) mixture of IFN-gamma and RsTbetaRII treatment group (each rat, IFN-gamma 7.5 MU x day(-1)+ RsTbetaRII 0.048 mg x day(-1). After treatment, hepatic fibrogenesis was evaluated by histopathological analysis and measurement of collagen III, alpha-smooth muscle actin (alpha-SMA), TGF-beta1, TGF-betaRII and their mRNA. RESULTS: Immunohistochemistry, Western blot and real-time RT-PCR showed that RsTbetaRII-IFN-gamma treatment significantly inhibited liver expression of collagen III, alpha-SMA, TGF-beta1 and TGF-betaRII at both protein and mRNA levels. Histopathological analysis also showed that the enhanced anti-fibrotic effects were achieved in model rats treated with RsTbetaRII-IFN-gamma. CONCLUSION: Our results confirmed that RsTbetaRII-IFN-gamma has the enhanced effects in reversing hepatic fibrosis.

[Expression of B7-H4 in prostate cancer and its clinical significance].

OBJECTIVE: To investigate the expression of B7-H4 in prostate cancer tissue and the relationship between the expression and the clinicopathological features. METHODS: Immunohistochemical staining was used to detect the expression of B7-H4 in prostate cancer tissue. And the relationship between the expressions and pathology was evaluated. RESULTS: The B7-H4 was diffusely expressed in cytoplasm and/or membrane of the prostate cancer tissue; the expression was much higher than that in normal prostate tissue (P<0.05). The expression of B7-H4 in the prostate cancer tissue was higher in patients with higher tumor grade. CONCLUSION: B7-H4 may be used as an new indicator for the diagnosis and prognosis of prostate cancer and a novel target for immunotherapy.

[Effects of Sema3A derived from tumor cells on functions of dendritic cells].

OBJECTIVE: To investigate the effects of tumor cell-derived Sema3A on the immunological functions of murine dendritic cells (DCs). METHODS: Lung adenocarcinoma A549 cells were transfected with small interference RNA, Si-Sema and Si-mut, and the interference efficiency was determined by real-time PCR and Western-blot. The concentrated supernatants from cultured tumor cells, Si-Sema and Si-mut-infected tumor cells were subjected to DCs respectively. The immunophenotypes of DCs were analyzed by flow cytometry, the production of IL-12P70 and the ability of DCs to stimulate DO11. 10 T cells secreting IFN-gamma and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Knockdown with Si-Sema3A significantly decreased the secretion of Sema3A by A549 cells in comparison with the Si-mut cells. DCs exposed to supernatants from Si-Sema cells showed elevated levels of MHC, CD40 and CD80, more production of IL-12P70, and enhanced capability of activating antigen-specific T cells, as evidenced by the remarkably increased levels of IFN-gamma and IL-2. CONCLUSION: A549 cells secrete Sema3A to inhibit the maturation and functions of DCs, which might be associated with the unidentified mechanism of immune evasion by tumor cells.

[Expression and identification of recombinant mouse gene B7-H4].

OBJECTIVE: To construct a eukaryotic expression vector encoding the gene of extracellular region of mouse B7-H4, to express it in yeast cell line and to determine its biological activity. METHODS: The extracellular region of the mouse B7-H4 gene was amplified with Xho I and EcoR I by PCR from a mouse B7-H4 chimeric plasmid. Digested with Xho I and EcoR I, the mB7-H4 gene was inserted into the yeast expression plasmid Ppic9. The DNA sequence was confirmed by double digestion and the Ppic9-mB7-H4 plasmid was transfected into the yeast cells. The expression of mB7-H4 was confirmed by PCR, Western Blot and ELISA analysis, and its biological function was determined. RESULT: Ppic9-mB7-H4 transfectants expressed mB7-H4 in yeast cells, and mB7-H4 effectively inhibited the proliferation of T lymphocytes with a fractional inhibition rate of 28.3 % and inhibited IL-2, IL-4, IL-10 and IFN-gamma production with fractional inhibition rates of 68.8%, 78.8%, 67.6% and 77.7%, respectively. CONCLUSION: The eukaryotic expression plasmid mouse B7-H4 has been successfully constructed and the expressed products of B7-H4 possess biological activity.

Local blockade of TSLP receptor alleviated allergic disease by regulating airway dendritic cells.

Thymic stromal lymphopoietin (TSLP) emerges as a central mediator of T helper cell (Th)2-dominant allergic diseases. However, the role of TSLP receptor (TSLPR) in allergen-induced Th2 priming, and the effects of TSLP signaling blocking on the development of asthma remain unclear. Here we showed that allergen challenge caused a rapid accumulation of TSLP in the airways of asthmatic mice, correlating well with eosinophils counts and interleukin (IL)-5 productions. When TSLP signaling was blocked by intratracheal administration of anti-TSLPR antibody before sensitization, eosinophilic airway inflammation, goblet cell hyperplasia and Th2 cytokines productions were significantly reduced. The alleviating effects of TSLPR blocking were achieved by inhibition of maturation and migration of airway dendritic cells (DCs), as well as their abilities of initiating CD4+T cell responses. Thus, local application of anti-TSLPR prevented Th2-mediated airway inflammation, at least partly, by regulating DCs function, which might be exploited to develop novel treatments for asthma.

[Construction and identification of recombinant adenoviral vector encoding B7-H4 gene].

OBJECTIVE: To construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities. METHODS: The full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol I and EcoR V the B7-H4 gene was inserted into pshuttle-CMW(PSC). Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac I linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested. RESULTS: The full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination, and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production. CONCLUSION: The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells.

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.

Activated NKT cells facilitated functional switch of myeloid-derived suppressor cells at inflammation sites in fulminant hepatitis mice.

Myeloid-derived suppressor cells (MDSCs) confer immunosuppressive properties, but their roles in fulminant hepatitis have not been well defined. In this study, we systematically examined the distribution of MDSCs in bone marrow (BM), liver and spleen, and their functional and differentiation status in an acute fulminant hepatitis mouse model induced by lipopolysaccharide and D-galactosamine (LPS-GalN). Moreover, the interaction between NKT cells and MDSCs was determined. Our study revealed that BM contained the largest pool of MDSCs during pathogenesis of fulminant hepatitis compared with liver and spleen. MDSCs in liver/spleen expressed higher levels of chemokine receptors such as CCR2, CX3CR1 and CXCR2. At inflamed tissues such as liver or spleen, activated NKT cells induced differentiation of MDSCs through cell-cell interaction, which markedly dampened the immunosuppressive effects and promoted MDSCs to produce pro-inflammatory cytokines and activate inflammatory cells. Our findings thus demonstrated an unexpected pro-inflammatory state for MDSCs, which was mediated by the activated NKT cells that precipitated the differentiation and functional evolution of these MDSCs at sites of inflammation.

Heat shock up-regulates TLR9 expression in human B cells through activation of ERK and NF-kappaB signal pathways.

Toll-like receptors (TLRs) play a critical role in innate immunity and TLR9 is essential for CpG ODN signaling. As "dangerous signal", heat shock may regulate immune response. However, little is known about TLRs expression and signaling after heat shock. In this study, we investigated regulation of TLR9 expression and function in human B cell line RPMI8226 by heat shock. We demonstrated that TLR9 expression was up-regulated remarkably following heat shock. Coincidently, CpG ODN stimulation significantly increased IL-6 production and up-regulated expressions of MHC I, MHC II and CD86 by heat-shocked B cells. Heat shock activated ERK and NF-kappaB signal pathways, and pretreatment of B cells with specific inhibitors of ERK or NF-kappaB signal pathways inhibited heat shock-induced up-regulation of TLR9 expression. These results demonstrated that heat shock promotes TLR9 expression and signaling through activation of ERK and NF-kappaB signal pathways in B cells, suggesting that heat shock might modulate host immune response by regulating TLR expression.

Intratumoral injection of interferon-gamma gene-modified dendritic cells elicits potent antitumor effects: effective induction of tumor-specific CD8+ CTL response.

PURPOSE: To examine the antitumor efficacy of intratumoral injection of interferon-gamma gene-modified dendritic cells (DC-IFN-gamma) in a B16 melanoma model and to investigate its related immunological mechanisms. METHODS: C57BL/6 mice-derived DC were transfected with adenovirus encoding IFN-gamma or beta-galactosidase (DC-LacZ). Secretion of IFN-gamma and TNF-alpha by DC was detected by ELISA. Nitric oxide (NO) production was measured by Griess reaction. Cytotoxicity of DC against tumor cell lines and activity of cytotoxic T lymphocytes (CTLs) were determined by 51Cr-release assay. TRP-2aa180-188-specific CD8+ CTLs in tumor-bearing mice with different treatment were determined by ELISPOT. RESULTS: DC-IFN-gamma could secrete high levels of IFN-gamma, NO and TNF-alpha. DC-IFN-gamma were cytolytic to B16 melanoma cells in vitro, but DC-LacZ and DC were not. Significant inhibition of tumor growth and prolonged survival were achieved in tumor-bearing mice intratumorally injected with DC-IFN-gamma when compared with those in tumor-bearing mice intratumorally injected with DC, DC-LacZ, fibroblasts, IFN-gamma gene-modified fibroblasts or PBS. After treatment with DC-IFN-gamma, enhanced Th1 and decreased Th2 responses were observed, and B16 melanoma antigen TRP-2aa180-188-specific CD8+ CTLs were induced significantly in the tumor-bearing mice. CONCLUSIONS: Intratumorally injected DC-IFN-gamma can uptake tumor antigens in situ and cross-present tumor antigens to specific CD8+ T cells, hereby eliciting effective antitumor effects in murine model with preestablished B16 melanoma.