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Objective To determine the involvement of Toll-like receptor (TLR) 2, TLR9, and TLR2+9 in renal ischemia/reperfusion (I/R) injury. Methods Mice with gene knock-out of TLR2, TLR9, and TLR2+9 underwent renal I/R injury. Tubular damage, expression of proinflammatory cytokines and chemokines, and kidney dysfunction were evaluated on different days after reperfusion. Results Mice deficient in TLR2, but not TLR9, were protected from renal I/R injury with less tubular damage, decreased cytokine production, and lower serum creatinine and urea levels than wild-type mice. TLR2+9 double-deficiency did not have an additional effect on tissue injury compared with TLR2 deficiency alone. Conclusion These results suggest that activation of TLR2 signaling contributes to the pathogenesis of renal I/R injury, whereas TLR9 may be redundant for the development of this injury.

Distance-based indexing is a widely used technique for general purpose search. Pivot selection is the most crucial step of bulkloading a metric-space indexing tree. Current pivot selection methods are mainly based on linear methods. A non-linear method based on Locally Linear Embedding is proposed. Empirical results demonstrate that the performance of new method is superior to existing methods. Keywords-similarity search; metric-space indexing; pivot selection; locally linear embedding; dimensional reduction

Metric-space indexing is a general method for similarity queries of complex data. The quality of the index tree is a critical factor of the query performance. Bulkloading a metric- space indexing tree can be represented by two recursive steps, pivot selection and data partition, while pivot selection dominants the quality of the index tree. Two heuristics, based on covariance and correlation, for pivot selection are proposed. Empirical results show that their performance is superior or comparable to existing methods. Keywords-similarity query; metric-space indexing; pivot space model; pivot selection; I. INT RODUCT ION

Hormesis induced by low-dose ionizing radiation (LDIR) is often mirrored by its stimulation of cell proliferation. The mitogen-activated protein kinases (MAPK)/ extracellular-signal- regulated kinases (ERK) pathway is known to play important roles in cell growth. Therefore, this study was to examine the effects of LDIR on rat mesenchymal stem cell (MSC) proliferation and MAPK/ERK signaling pathway. Rat MSCs were isolated from the bone marrow from 6 to 8-week-old male Wistar rats and cultured in vitro. Exponentially growing cells within 4-5 passages were irradiated with low doses of X-rays at 20, 50, 75 and 100 mGy with a dose rate of 100 mGy/min. Cell proliferation was evaluated by counting total viable cell number with trypan-blue staining and MTT assay. Cell cycle changes were also evaluated by flow cytometry and the activation of MAPK/ERK signaling pathway was assayed by Western blotting. Results showed that LDIR at 50 and 75 mGy significantly stimulated the proliferation of rat MSCs with the most stimulating effect at 75 mGy. There was a significant increase in the proportion of S phase cells in MSCs in response to 75 mGy X-rays. Activation of several members in the MAPK/ERK signaling pathway, including c-Raf, MEK and ERK were observed in the cells exposed to 75 mGy X-rays. To define the role of ERK activation in LDIR-stimulated cell proliferation, LDIR-treated MSCs were pre-incubated with MEK specific inhibitor U0126, which completely abolished LDIR-increased phosphorylation of ERK and cell proliferation. These results suggest that LDIR stimulates MSC proliferations in the in vitro condition via the activation of MAPK/ERK pathway.

Bmi-1 is a transcriptional repressor belonging to the Polycomb group and is associated with the cell proliferation and carcinogenesis of a variety of human cancers. The level of Bmi-1 expression correlates with the aggressiveness of many cancers, and is considered an important marker for cancer diagnosis. However, its role in gastric carcinoma is unknown.We used lentiviral mediated interfering short hairpin RNA to knockdown Bmi-1 expression in gastric carcinoma human gastric cancer cell line (AGS cells), then tested the cell proliferation by MTT assay, rate of colony formation by colony formation assay, cell cycle distribution by fluorescence-activated cell sorting and cell invasiveness by cell invasion assay. To analyze the expression and localization of Bmi-1 in gastric tumor tissues, we further performed the immunohistochemistry analysis on a gastric cancer tissue array.We found that knocking down Bmi-1 led to slower cell growth, lesser cell invasiveness, decelerated colony formation, and altered cell cycle progression. In addition, a positive relationship between nuclear expression of Bmi-1 and gastric cancer was observed, suggesting that nucleus localization of Bmi-1 in the cells may be a novel marker of gastric cancer.Our study highlights critical roles for Bmi-1 in gastric cancer, and suggests that Bmi-1 nuclear localization could be an important marker for the diagnosis of gastric cancer.

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.

Skeletal muscle stem cell–derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. We demonstrate that Janus kinase 1 (JAK1) is required for myoblast proliferation and that it also functions as a checkpoint to prevent myoblasts from premature differentiation. Deliberate knockdown of JAK1 in both primary and immortalized myoblasts induces precocious myogenic differentiation with a concomitant reduction in cell proliferation. This is caused, in part, by an accelerated induction of MyoD, myocyte enhancer–binding factor 2 (MEF2), p21Cip1, and p27Kip1, a faster down-regulation of Id1, and an increase in MEF2-dependent gene transcription. Downstream of JAK1, of all the signal transducer and activator of transcriptions (STATs) present in myoblasts, we find that only STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts. Leukemia inhibitory factor stimulates myoblast proliferation and represses differentiation via JAK1–STAT1–STAT3. Thus, JAK1–STAT1–STAT3 constitutes a signaling pathway that promotes myoblast proliferation and prevents premature myoblast differentiation.

Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5′-phosphatase SHIP1 is a normal physiologic counter-regulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP3). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway.

We used two inhibitors of the signaling enzyme phosphatidylinositol 3-kinase (PtdIns3K), wortmannin and LY294002, to evaluate the potential involvement of PtdIns3K in the activation of the MAP kinases (MAPK), Erk1 and Erk2. In dose-response studies carried out on six different cell lines and a primary cell culture, we analyzed the ability of the inhibitors to block phosphorylation of protein kinase B/akt (PKB/akt) at Ser473 as a measure of PtdIns3K activity, or the phosphorylation of Erk1/2 at activating Thr/Tyr sites as a measure of the extent of activation of MAPK/Erk kinase (MEK/Erk). In three different hemopoietic cell lines stimulated with cytokines, and in HEK293 cells, stimulated with serum, either wortmannin or LY294002, but never both, could partially block phosphorylation of Erks. The same observations were made in a B-cell line and in primary fibroblasts. In only one cell type, the A20 B cells, was there a closer correlation between the PtdIns3K inhibition by both inhibitors, and their corresponding effects on Erk phosphorylation. However, this stands out as an exception that gives clues to the mechanism by which cross-talk might occur. In all other cells, acute activation of the pathway leading to Erk phosphorylation could proceed independently of PtdIns3K activation. In a biological assay comparing these two pathways, the ability of LY294002 and the MEK inhibitor, U0126, to induce apoptosis were tested. Whereas LY294002 caused death of cytokine-dependent hemopoietic cells, U0126 had little effect, but both inhibitors together had a synergistic effect. The data show that these two pathways are regulating very different downstream events involved in cell survival.

Objective: The purpose of this experiment was to determine whether inflammatory cytokines or TLR ligands could be combined with influenza vaccine in older adult PBMC to enhance the response to live influenza virus challenge. Methods: PBMC from five older adults (>65 years old) and six young (20-38 years old) adults were obtained at six months post-vaccination and cultured for five days with split-virus vaccine (SVV) and the TLR3 ligand, poly I:C, or a cocktail of pro-inflammatory cytokines (TNF-α, IL-1, IL-6; T/1/6). Stimulated PBMC were then challenged with live influenza virus for 20 hours and supernatants and lysates prepared for assays of IL-1, IL-6, TNF-α in 5-day supernatants, and the IFN-γ:IL-10 ratio and granzyme B activity in 20-hour virus-challenged PBMC. Results: We observed an increased response to SVV in younger compared to older adults, but a dose-response relationship between poly I:C (added to a fixed dose of SVV) and the IFN-γ:IL-10 ratio and granzyme B levels improved the response in older adult PBMC. This response was associated with a dose-response increase in pro-inflammatory cytokine levels in SVV/poly I:C-stimulated PBMC. However, a cocktail of these inflammatory cytokines (T/1/6) added to the PBMC culture suppressed rather than enhanced the T cell response in older adults. Conclusion: Poly I:C combined with influenza vaccine was shown to enhance the T cell response to influenza in aged PBMC by mechanisms that cannot be replicated by the simple addition of inflammatory cytokines to the vaccine.

BackgroundSmall cell lung cancer (SCLC) is the most devastating type of human lung cancer. Patients usually present with disseminated disease to many organs (extensive stage). This study was to investigate the efficacy and safety of cellular immunotherapy (CIT) with autologous natural killer (NK), γδT, and cytokine-induced killer (CIK) cells as maintenance therapy for extensive-stage SCLC (ES-SCLC) patients.MethodsA pilot prospective cohort study was conducted with ES-SCLC patients who had responded to initial chemotherapy. Patients received either CIT as maintenance therapy (CIT group), or no treatment (control group). Progression-free survival (PFS), overall survival (OS), and adverse effects were compared.ResultsForty-nine patients were recruited in this study, with 19 patients in the CIT group and 30 patients in the control group. The patient characteristics of the 2 groups were comparable except for age, as patients in the CIT group were older than those in the control group (P

The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in B cell activation and differentiation. Recruitment of pleckstrin homology (PH) domain-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. We have found that the PH domains of Bam32 and tandem PH domain-containing protein 2 (TAPP2) specify a temporally distinct wave of membrane recruitment compared with that of Bruton’s tyrosine kinase (Btk), with recruitment of these two adaptors representing a later stage of the response. In this study we provide direct evidence that PH domain-dependent recruitment of Btk to the membrane is blocked by coligation of the inhibitory receptor FcγRII in human B lymphoma cells. In contrast, recruitment specified by the Bam32 or TAPP2 PH domains is completely insensitive to FcγRII inhibition. This differential regulation can be accounted for by Src homology 2-containing inositol polyphosphate 5-phosphatase (SHIP) activity alone, as expression of membrane-targeted SHIP completely abrogated Btk recruitment, but had no inhibitory effect on Bam32 or TAPP2 recruitment. Strikingly, kinetic analysis revealed that membrane recruitment of Bam32 and TAPP2 is actually more rapid under “inhibitory” signaling conditions. Analysis of 3-phosphoinositide generation under activating and inhibitory signaling conditions indicated that recruitment of Bam32 and TAPP2 is inversely correlated with the SHIP substrate/product ratio (phosphatidylinositol 3,4,5-trisphosphate/phosphatidylinositol 3,4-bisphosphate). Overexpression of TAPP2 in B cells led to an increase in the sustained phase of the calcium response and increased NF-AT-dependent transcriptional activation after B cell Ag receptor ligation. Together, these results suggest that Bam32 and TAPP2 adaptors define a novel group of SHIP-activated targets of PI3K that regulate B cell Ag receptor signaling.