To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8 Chlamydia-triggered reactive arthritis (Ct-ReA) is strongly associated with HLA-B27 like other spondylarthropathies, and especially ankylosing spondylitis [Chlamydia trachomatis or gastroenteral infection with enterobacteria such as Yersinia enterocolitica [+-reactive arthritis patients, 20–40% move on to ankylosing spondylitis after 10–20 years, suggesting that the ReA-associated bacteria can cause ankylosing spondylitis [+ T cells [Chlamydia-specific CD8+ T cells capable of lysing target cells primed with Chlamydia antigens [+ T cell responses in spondylarthropathies other than Ct-ReA have also been described [ndylitis ,2. ReA ocolitica . After acolitica . Of HLA-ndylitis  and that T cells -9. Recenantigens . CD8+ T escribed -15.in situ [Recently a new method for antigen-specific T cell recognition has been established by using multimerized MHC/peptide molecules . These min situ .+ T cell response to Chlamydia-derived peptides in patients with Ct-ReA.In humans, MHC class I tetramers are widely used, and HLA-A2 tetramers in particular are an important tool in tumour immunology . HoweverThis is the first report of a systematic use of HLA-B27 tetramers in humans in an HLA-B27-related disease.+ and three HLA-B27- patients with ReA after infection with Chlamydia trachomatis .The quantification of HLA-B27 binding affinity was conducted with two different programs that analyse HLA-peptide binding motifs, one called SYFPEITHI described by Rammensee and colleagues  and the Nonamer peptides were synthesized by standard 9-fluorenyl-methyloxy-carbonyl solid-phase synthesis methods on a Syro-Synthesizer , purified by high-performance liquid chromatography  and identified by mass spectroscopy . The purity of the peptides was more than 95%. Peptides were dissolved in dimethyl sulphoxide. For T cell stimulation and fluorescence-activated cell sorting (FACS) analysis of intracellular cytokine staining, the peptides were further diluted with serum-free medium at a concentration of 5 mg/ml and frozen at -80°C.g; Haereus Biofuge Pico; Kendro Laboratories, Langenselbold, Germany) followed by biotinylation and gel filtration with a Superose 12 column (Pharmacia) on an Äkta Basic system (Pharmacia). Correct folding and biotinylation were analysed by gel filtration  and gel electrophoresis (Bio-Rad). Tetramers were generated by adding phycoerythrin (PE)-labelled streptavidin (Molecular Probes) at a ratio of 1.5:1. We generated HLA-B27 tetramers with the EBV EBNA peptide (residues 258–266) [Chlamydia-peptide-specific CD8+ T cells we used the previously described immunodominant peptides 8, 68, 80, 131, 133, 138, 144, 145, 146, 194, 195 and 196 [HLA-B27 tetramers were generated as described previously , with so258–266) . For the and 196   from synovial fluid or peripheral blood were incubated with tetramer and PerCP-labelled anti-human CD8 antibody  in parallel for 30 min at room temperature (20°C) followed by washing twice with phosphate-buffered saline (PBS)/2% bovine serum albumin (BSA) and incubation with Cy5-labelled anti-human CD3 antibody for 30 min at room temperature. Cells were washed twice in PBS/2% BSA and resuspended in Annexin V buffer (Molecular Probes) and 2.5 μl of Alexa 488-labelled Annexin V (Molecular Probes) was added. CD8+ T cells were further separated by FACS sorting. Sorted cells (1000) were incubated with 500,000 autologous antigen-presenting cells in the presence of 20 U/ml interleukin (IL)-2, 10 ng/ml IL-7 and 10 ng/ml IL-15 added every 3–4 days.Peripheral MNCs were incubated for 30 min with Cy5-labelled anti-CD8 antibody (BD) and 5 μg/ml PE-labelled HLA-B27/EBV EBNA (258–266) tetramer at room temperature. Cells were washed twice and incubated for 15 min at 4°C with anti-PE-labelled MACS beads (Miltenyi) at a ratio of 20 μl of beads to 80 μl of cell suspension. Labelled cells were loaded on an LS MACS column (Miltenyi) and eluted after the column had been washed three times with washing buffer including PBS, EDTA and BSA. MACS-sorted tetramer-positive and CD8The refolding rate of recombinant HLA-B27 monomer was analysed by gel filtration and by determining the relative amount of soluble HLA-B27 monomer eluted at 13.7 ml in comparison with precipitated protein eluted earlier in a Superose 12 column (Pharmacia). An Akta basic system (Pharmacia) was used. The elution profile was analysed by using Unicorn (version 4) software (Pharmacia). Refolding was defined as ++ when more than 75% of proteins loaded on the gel filtration column after refolding, biotinylation and sharp centrifugation was soluble HLA-B27 monomer molecule; + for more than 50% soluble HLA-B27 monomer, (+) for more than 10% soluble HLA-B27 monomer, and - for less than 10% soluble HLA-B27 monomer  plus single peptides (10 μg/ml) or without antigenic peptide as a negative control. Brefeldin A was added after 2 hours to stop the stimulation, and cells were harvested after a further 4 hours and then stained with 5 μg/ml anti-CD69-PE antibody (BD Pharmingen) and 1 μg/ml anti-CD8-PerCP (BD Pharmingen). Cells were then fixed in 2% formalin and resuspended in saponin buffer, followed by incubation with 1 μg/ml Cy5-conjugated anti-human interferon-γ antibody . Gated CD8+ cells from peripheral blood were incubated for 1 hour with anti-CD14-conjugated magnetic beads (Miltenyi) and sorted by MACS. The purity of separated cells was confirmed by FACS analysis. Cells  were cultured for 7 days in 24-well plates at 37°C at 5% CO2 in 1 ml of RPMI culture medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and 50 ng/ml granulocyte/macrophage colony-stimulating factor and 10 ng/ml IL-4 to induce transformation to dendritic cells (DCs). Cells were washed and harvested and incubated for 24 hours with infectious elementary bodies of Chlamydia trachomatis at a ratio of 1:50. DCs were analysed by FACS with the use of anti-CD80, anti-CD86, anti-HLA-DR, anti-CD14 (BD Pharmingen) and anti-Chlamydia trachomatis lipopolysaccharide antibodies (Dako) before and after infection with viable Chlamydia trachomatis.CD14+ T cells from peripheral blood with Chlamydia trachomatis-infected peripheral-blood-derived DCs at a ratio of 50:1 in RPMI culture medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Recombinant IL-7 (10 ng/ml) and IL-15 (10 ng/ml) were added on both days 2 and 7. T cells were analysed by FACS on day 14.We stimulated CD8Chlamydia peptide tetramers.To determine optimal conditions for the refolding procedure of soluble HLA-B27 monomers we used HLA-A2 tetramers and HLA-B27 tetramers with well-described HLA-B27 and HLA-A2 restricted viral epitopes from EBV. The binding scores of the two immunodominant EBV peptides to the HLA-A2  score 2, althoug+ T cells could be sorted and further cultured we stained peripheral MNCs with HLA-B27/EBV EBNA (258–266) tetramer. Before MACS, 0.22% of peripheral MNCs were CD8+ and tetramer-positive. After MACS, EBV EBNA (258–266)-specific T cells were enriched to 41.4%. MACS-sorted cells were further separated by FACS sorting and cultured for 4 weeks in the presence of IL-2. The purity of antigen-specific CD8+ was increased to 95.0%, as shown by tetramer staining  with Ct-ReA. Four of the six HLA-B27+ patients had a specific T cell binding to at least one HLA-B27/Chlamydia peptide tetramer.On the basis of our recently identified n Ct-ReA  we succeChlamydia peptide 195 tetramer bound to the synovial T cells of three  of these four patients. Two patients (nos 5 and 6) showed a T cell response to Chlamydia peptide 133 as detected by tetramer staining, and one (patient no. 3) had a T cell response to Chlamydia peptide 68.The results of tetramer staining in all patients are summarized in Table Chlamydia peptides 195 and 68 were detected with HLA-B27/Chlamydia peptide tetramers in patient no. 3: 0.09% of CD8+ T cells were positive for peptide 195 and 0.06% were positive for peptide 68. All other HLA-B27 tetramers with Chlamydia-derived peptides such as peptide 138 were negative (data not shown). In patient no. 2 we detected 0.06% HLA-B27/Chlamydia peptide 195 tetramer-positive T cells . We did not analyse the cytokine secretion profile of CD8+ T cells in response to chlamydial peptides in these two patients. The example of patient no. 6 is shown in Fig. Chlamydia peptide 133 tetramer binding to CD8+ T cells but no binding to any of the other HLA-B27 Chlamydia peptide tetramers. In patient no. 6 we were able to repeat this experiment and obtained a similar result, with 0.02% of tetramer binding to CD8+ T cells.The results of three patients are illustrated in Figs lls Fig. . All othChlamydia peptide 133-specific T cell stimulation for 1 week ex vivo, which revealed 0.22% tetramer-positive CD8+ T cells . The HLA-B27- patients with Ct-ReA and all six HLA-B27+ healthy controls had no HLA-B27-restricted, Chlamydia-peptide-specific T cell response (data not shown). Tetramer staining of synovial T cells from three HLA-B27+ patients with enterobacteria-triggered ReA and from three patients with rheumatoid arthritis revealed no specific staining of CD8+ T cells with 0–0.01% tetramer binding to CD8+ T cells.We also analysed CD8Chlamydia-specific CD8+ T cells in these patients is low in synovial fluid and absent in peripheral blood with both methods (tetramer staining and intracellular cytokine staining), we investigated whether enrichment of these cells could be achieved by short-term stimulation with autologous Chlamydia-infected DCs. By doing this we intended to obtain a higher frequency of tetramer-positive CD8+ T cells, to underline the specificity of tetramer staining.Because the frequency of + monocytes from peripheral blood of patient no. 5; they were separated by MACS first. After 7 days of cultivation in vitro, the cells turned into DCs, as indicated by the loss of CD14 receptors and the upregulation of HLA-DR, CD80 and CD86 receptors (data not shown). We infected these DCs with viable Chlamydia trachomatis and confirmed infection by using an anti-Chlamydia trachomatis lipopolysaccharide antibody and by quantification of Chlamydia-positive cells by FACS analysis (data not shown). We revealed at least 41.3% Chlamydia-infected DCs.We generated DCs from CD14Chlamydia-infected DCs for 2 weeks in the presence of IL-7 and IL-15. Subsequently, FACS analysis for intracellular cytokine staining for IFN-γ performed after restimulation of this cell line with Chlamydia-infected DCs revealed 0.11% IFN-γ-secreting CD8+ T cells, and stimulation with different peptide pools including the nine relevant peptides revealed between 0.07% and 0.21% antigen-specific IFN-γ-secreting CD8+ T cells (data not shown). When the cell line was analysed with HLA-B27/Chlamydia peptide tetramers we found a similar quantity of expanded CD8+ T cells with significant tetramer staining of CD8+ T cells specific for Chlamydia peptides 8 (0.09%), 68 (0.10%), 133 (0.17%), 138 (0.08%), 195 (0.23%) and 196 (0.06%)  tetramer and might have been caused by aggregated tetramers. Staining of untreated peripheral MNCs from the same patient did not reveal any tetramer binding (data not shown); staining with an HLA-B27/EBV peptide tetramer was performed as a positive control. We repeated this procedure in an HLA-B27- patient with Ct-ReA . In neither case could we observe staining with any of the HLA-B27/Chlamydia peptide tetramers even after stimulation with Chlamydia-infected DCs .Here we have also demonstrated another useful property of HLA-B27 tetramers as a 'proof of principle'. We sorted antigen-specific tetramer-positive CD8Chlamydia peptides with higher binding scores, previously defined as immunodominant in Ct-ReA [Chlamydia-peptide-specific T cell responses in four of six patients with Ct-ReA. These experiments suggest that Chlamydia peptides 195 and 133 are immunologically important epitopes, because we could detect CD8+ T cells with such specificity in three and two, respectively, out of six HLA-B27+ patients. We identified these antigen-specific T cells at a frequency of 0.02–0.09% in the synovial fluid of these patients, which is concordant with previous results [Chlamydia-peptide-specific CD8+ T cells detected with HLA-B27 tetramers sometimes makes discrimination from non-specific staining difficult. We confirmed our tetramer staining result in one patient by expansion of peptide-specific CD8+ T cells followed by tetramer staining with increased amounts of tetramer-binding CD8+ T cells; even more importantly, peptide-specific CD8+ T cells were also detected by intracellular IFN-γ staining after peptide-specific stimulation. However, for future experiments it would be useful to confirm such findings with antigen-specific T cell expansion as shown here  without prior stimulation, we assume that the frequency of CD8+ T cells with such specificity in the peripheral blood is below the sensitivity of both methods. In contrast, low frequencies of Chlamydia-derived peptide-specific CD8+ T cells in the peripheral blood were observed by another group by using HLA-A2 tetramers [Chlamydia trachomatis major outer membrane protein (MOMP) 258 peptide-specific and MOMP 249 peptide-specific CD8+ T cells in patients with acute urogenital tract infection. They found 0.01–0.2% MOMP-specific CD8+ T cells in the peripheral blood of these individuals with acute infection, who had no clinical symptoms of Ct-ReA.Because we could not detect any etramers . These rChlamydia-specific HLA-B27-restricted CD8 T cells were detected in the synovial fluid of four of six HLA-B27+ patients with Chlamydia-induced reactive arthritis, their frequency was low, arguing against a major role in fighting Chlamydia and in the pathogenesis of arthritis. However, these antigens might be able to induce a cross-reactive T cell response to self-antigens, implying that the 'arthritogenic peptide' is not necessarily identical with the immunodominant peptide that is capable of inducing the T cell response to eliminate the microbe. Nevertheless, their frequency could be significantly expanded after stimulation in vitro with Chlamydia-infected autologous DCs, suggesting that these cells have full replicative capacity.We conclude that HLA-B27 tetramers are useful tools for the study of HLA-B27/peptide-specific T cells in HLA-B27-associated diseases. Although None declared.Chlamydia-triggered reactive arthritis; DC = dendritic cell; EBV = Epstein–Barr virus; FACS = fluorescence-activated cell sorting; IFN-γ = interferon-γ ; IL = interleukin; MACS = magnetic activated cell sorting; MNC = mononuclear cell; MOMP = major outer membrane protein; PBS = phosphate-buffered saline; PE = phycoerythrin.BIMAS = BioInformatics and Molecular Analysis Section; BSA = bovine serum albumin; Ct-ReA =


Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways.We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals.The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. The phosphoinositide-3-kinases (PI3Ks) constitute an enzyme family of p85/p110 heterodimers responsible for regulating numerous biological processes in diverse cell types . A key tPten in T-cells leads to autoimmunity and development of lymphoma [PI3K is activated in T-cells both in response to T-cell receptor (TCR) stimulation, and by engagement of the co-stimulatory receptor CD28. Gene targeting studies have emphasised the essential role PI3K plays in these responses: whereas PI3K-deficient mice are immunodeficient ,5, constlymphoma . These sin vitro [The Notch signalling pathway plays a critical role in a wide range of developmental processes in the embryo, including binary lineage decisions and boundary formation . Bindingin vitro -13 suggeMice expressing catalytically inactive PI3K subunit p110δ  or a delTo address whether the PI3K pathway is a regulator of Notch-responsiveness, we generated a Jurkat E6.1 T-cell line which stably expresses full-length human Notch2 cDNA (J-N2 cells). J-N2 cells were treated with plate-bound recombinant human Delta1 protein (hereafter referred to as Delta1-Fc) fusion protein, and Notch signalling was measured using a transiently-transfected CBF1-driven luciferase reporter construct (p10xCBF1-luc). Plate-bound recombinant Delta1-Fc efficiently transactivated CBF1-luc, driving around a 10-fold increase in activity Fig. . A similPten in J-N2 cells led to a decrease in both basal and Notch ligand-induced activation of CBF1-dependent gene expression , demonstrating that the effect of PI3K on Notch is exerted downstream of Notch cleavage. Thus, TCR activation dramatically increases the cellular response to Notch signals in primary human T cells via a mechanism requiring the PI3K-Akt pathway.We next investigated whether PI3K activity also regulates Notch-responsiveness in primary human CD4els Fig. . In the We then tested whether Akt activation is sufficient to boost Notch-responsiveness in human T cells. Over-expression of a constitutively-active Akt construct (gagAkt ) was suf+ T-cells treated with the GSK3β inhibitor lithium chloride. Increasing concentrations of LiCl induced a dramatic increase in Notch signalling  and kinase dead (Akt-KD) Akt expression constructs were a kind gift from Dr. Julian Downward [SH-6, lithium chloride, LY294002, Wortmannin and BDNF were from Calbiochem. Human IgG4 was from Sigma. Dual-Glo Luciferase assay system was from Promega. pN1-IC was constructed by inserting nucleotides coding for the intracellular domain of human Notch1 (amino acids 1758 to 2557) preceded by an initiator ATG codon into the BamHI and EcoRI sites of pcDNA3.1 (Invitrogen). pAdTATA-luc was constructed by inserting the Adenoviral major late promoter TATA sequence immediately 5' of the luciferase gene in plasmid pGL3Basic (Promega). p10xCBF1-luc was constructed by inserting 10 copies of the CBF1 binding site 5' of the AdTATA box in pAdTATA-luc. pTK-Renilla was from Promega. Human Downward . All pla+ T cells were isolated from freshly-collected donor blood as follows. Peripheral blood mononuclear cells were prepared by centrifugation over Ficoll-Paque, incubated with anti-CD4-coated magnetic beads (Miltenyi Biotec) and selected on LS positive selection columns (Miltenyi Biotec). After purification, primary CD4+ T-cells were cultured in RPMI1640 . CHO-N2 and CHO-Delta1 cells have been described previously [Jurkat E6.1 cells and Jurkat-N2 cells were grown in RPMI1640 . Primary CD4eviously  and wereeviously , except 5 cells/well into flat-bottomed 96-well plates which had been (where relevant) pre-coated o/n at 4°C with hDelta1-Fc. Cells were stimulated, incubated o/n and luciferase activity measured using the Dual-Glo system (Promega). Firefly luciferase signal from the Notch-dependent p10xCBF1-luc was normalised using the Renilla luciferase signal driven by the pTK-Renilla.Jurkat E6.1 cells and Jurkat-N2 cells were transfected using Lipofectamine 2000 (Invitrogen). Cells were left 24 h post-transfection, washed, counted and re-plated in triplicate at 2 × 10+ T cells were transfected using the Amaxa 'nucleofection' system according to the manufacturer's instructions. Briefly, 5 × 106 purified CD4+ T-cells were resuspended in 100 μl of the appropriate Amaxa solution and transfected with 5 μg plasmid DNA using Protocol U-14. Cells were immediately transferred to 1 ml of pre-warmed media, counted and plated as required. After stimulation luciferase activity was detected using the Dual-glo assay system (Promega).Primary CD4For neuronal experiments, rat primary hippocampal neurons were transfected after 9 days in culture using Lipofectamine 2000 and stimulated 36-48 hours later. Dual-Glo Luciferase assays were performed following o/n stimulation.© (Lonza Biologics). This plasmid was expressed in CHO cells, purified by affinity chromatography on ProteinA-Sepharose (Amersham Biosciences) before concentration and buffer exchange into PBS. Batch activity of this protein was confirmed as previously described [A fusion protein comprising the extracellular domain of human Delta1 fused to the Fc domain of human IgG4 was prepared by inserting a nucleotide sequence coding for the extracellular domain of human Delta1 (amino acids 1 to 537) into the expression vector pCONescribed .To assess the effect of isolated Delta1-Fc signalling on Jurkat-N2 and CD4+ T-cells, 96- or 24-well plates were coated overnight at 4°C with anti-hIgG4 . Plates were washed with PBS and incubated for 2 hours at 37°C with 20 μg/ml Delta1-Fc or hIgG4 control, washed once again with PBS, before cells were plated for stimulation.+ T-cells, 96- or 24-well plates were first coated overnight at 4°C with a mixture of capture antibodies consisting of anti-mouse IgG2a  and anti-hIgG4 . Plates were washed with PBS and incubated 2 hours at 37°C with a mixture of anti-CD3  and 20 μg/ml Delta1-Fc or hIgG4 control (Sigma). Plates were again washed with PBS and purified CD4+ T cells were seeded at 2 × 105/well in the presence of soluble anti-CD28 .To examine the effect of coincident Delta1-Fc and T-cell receptor signalling on primary CD44 CHO-N2 cells were plated per well into 96-well plates, and left to adhere for 2 hours. Notch signalling was activated by plating an additional CHO line which stably expresses full-length human Delta1 (CHO-hDelta1) onto the CHO-N2 cells. Cells were co-cultured in this manner overnight, and luciferase activity determined as described.This assay has been described previously . BrieflyRNA was isolated from cell samples using Rneasy kits (Qiagen). First strand cDNA was synthesised with SuperScriptII (Invitrogen) oligo-d(T) and random decamers (Invitrogen). Q-PCR was performed using SYBR green detection chemistry on the Roche Lightcycler system. Gene-specific quantification was achieved by comparing each cDNA to a serially-diluted standard plasmid containing the relevant amplicon. Relative Hes-1 expression levels were normalised to 18S rRNA housekeeping gene. Primer sequences used were as follows (5' to 3'): 18S rRNA, GTAACCCGTTGAACCCCATT and CCATCCAATCGGTAGTAGCG; Hes-1, CTCTCTTCCCTCCGGACTCT and GGCGCAATCCAATATGAAC.GM and GH conceived this study and wrote the manuscript. GM was experimentally involved in CHO, Jurkat and primary T-cell assays. GH was experimentally involved in the neuronal studies. GW carried out all the CHO and Jurkat assays and the primary T-cell experiments involving N1-IC transfection. YS and EB carried out the primary T-cell experiments involving Delta1-Fc stimulation. SP was experimentally involved in the neuronal studies. AL, BC and MT provided susbstantial intellectual input into all aspects of this study. All authors read and approved the final manuscript.


The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines  derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [bis-acetatoammine dichloro (cyclohexylamine) platinum (IV)] and JM221 [ammine dibutyrato dichloro (cyclohexylamine) platinum (IV)]. The range in IC50 values across the five lines was approximately 2.5-fold for cisplatin, carboplatin and iproplatin, 13-fold for tetraplatin and JM216, and 25-fold for JM221. No significant correlation (P > 0.05) was observed between platinum drug chemosensitivity and either glutathione levels or cadmium chloride sensitivity, an indicator of metallothionein levels. In addition, there was no significant correlation (P > 0.05) between cisplatin cytotoxicity and intracellular cisplatin accumulation or JM216 cytotoxicity and intracellular JM216 accumulation over the dose range 5-100 microM (2 h exposure). The exceptional sensitivity of HX/156 to JM216 appears, at least partially, to be a result of enhanced accumulation of JM216. An 8.6-fold acquired cisplatin resistant stable variant of HX/155 has been generated in vitro. Intracellular cisplatin accumulation was reduced by 2.4 +/- 0.3-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). Glutathione levels in HX/155cisR were elevated by 1.3-fold in terms of protein content and by 1.6-fold in terms of cell number. HX/155cisR was 1.9-fold resistant to cadmium chloride. Total platinum bound to DNA after cisplatin exposure  was 3.6 +/- 0.6-fold (mean +/- s.d.) lower in HX/155cisR. Hence the mechanism of acquired cisplatin resistance in HX/155cisR appears to be multifocal, with reduced intracellular drug accumulation and elevated glutathione and metallothionein levels combining to reduce DNA platination levels. While HX/155cisR was cross-resistant to tetraplatin and carboplatin, novel platinum (II) and (IV) ammine/amine complexes, including JM216 and JM221, partially circumvented resistance (resistance factors of 1.5-2). Non cross-resistance was observed to iproplatin and nine non-platinum anticancer agents. Intracellular tetraplatin accumulation was reduced by 1.8 +/- 0.1-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). In contrast, after JM216 exposure (1-100 microM for 2 h), no significant difference in intracellular platinum levels between HX/155 and HX/155cisR was observed.(ABSTRACT TRUNCATED AT 400 WORDS)


Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells. One of the best-studied cellular signaling relays is the mitogen-activated protein kinase (MAPK) signaling cascade, the central mechanism by which growth factors steer cellular key decisions such as proliferation and differentiation . It has The dephosphorylation of ERK1/2 regulatory tyrosine and threonine residues can be achieved by three different phosphatase types: protein serine/threonine phosphatases, dual specificity (serine/threonine/tyrosine) phosphatases (MKPs/DUSPs) and classical phosphotyrosine-specific protein tyrosine phosphatases (PTPs) . The latMammals contain three classical PTP families that may regulate ERK activity by KIM-dependent binding and dephosphorylation: HePTP/LC-PTP, STEP and PTPRR Fig. . The haeIn the current study we assessed the contribution of the KIM-domain-containing classical PTPs to the regulation of ERK activity in rat PC12 cells. HePTP and STEP mRNAs were not detectable in PC12 cells. For the PTPRR gene a single transcript type, encoding the transmembrane isoform PCPTP1, was encountered. We exploited retroviral transduction to increase or decrease PTPRR protein levels and monitored growth factor-induced ERK activation using phosphospecific antibodies. Our results demonstrate that KIM-domain-containing tyrosine-specific PTPs are dispensable for proper ERK-dependent signaling in PC12 cells and put forward the dual-specificity MAPK phosphatases as the physiological regulators of ERK activity in these cells.To determine which of the three KIM-containing phosphotyrosine-specific PTPs are endogenously expressed in rat PC12 cells, an RT-PCR analysis on total RNA was performed. Expected amplicons for STEP and HePTP were obtained from mouse cerebrum and mouse spleen RNA, respectively, but STEP and HePTP messengers remained undetectable in PC12 material Fig. , in concWe next investigated PTPRR isoform expression in PC12 cells at the protein level. Both Western blot analysis of PC12 cell lysates and immunoprecipitation experiments revealed multiple immunoreactive bands  in PC12 cells has been reported previously to result in reduced phospho-ERK5 and phospho-ERK1/2 levels following EGF administration . Those dDrosophila, for example, redundancy regarding ERK inactivation was encountered for DMKP3, a homolog of mammalian MKP-3/MKP-4, and PTP-ER, the Drosophila homolog of PTPRR [PCPTP1, the rat homologue of mouse PTPBR7, is abundantly expressed in several rat brain regions, including the cerebellum, cerebral cortex and the hippocampus . Intriguof PTPRR . Potentiof PTPRR . We detein vitro studies might be hampered by the fact that potential ligands for the PCPTP1/PTPBR7 extracellular part, which may critically control the activity of the phosphatase, have not been identified yet. Also in view of the restricted expression pattern of the Ptprr gene [in vivo.What other substrate targets for PCPTP1 may exist in PC12 cells to explain its endogenous presence? Besides ERK1/2, PTPRR proteins were shown to bind the MAPK family members p38α and ERK5 ,13,27,29prr gene ,24,30 anprr gene ,20,30 itThe PTPRR isoform PCPTP1 is the sole tyrosine-specific PTP in PC12 cells that is able to bind ERK MAPKs through its KIM-domain and it is readily up-regulated upon growth factor-induced differentiation of these cells. Still, the physiological regulation of ERK1/2 signaling in PC12 cells could not be ascribed to this PTP. We conclude that other, dual-specificity MAPK phosphatases may act as major regulators of EGF- and NGF-induced signaling pathways in these cells.- Reverse Transcriptase  and used for PCR. Amplified products were analyzed on agarose gels. Primers used are: PCPTP1-forward 5'-CCTCAATGCACACACTATGAGG-3'; PCPTP1-Ce-forward 5'-CTTCAGCTCGCAGGC TTTC-3'; PTPPBSγ (rat)-forward 5'-GTGCAGGACGTGGAGGAAG-3'; PTPRR-reverse 5'-TCCTTCTTTGCTCCAGAT-3'; STEP-forward 5'-GAGGACTACCGGCTGCGAC-3'; STEP-reverse 5'-GGCTCATGGCGTGGTGCAC-3'; HePTP-forward 5'-CATCTGCTCAGT GAACACACC-3'; HePTP-reverse 5'-GTCTGCTACAGTGTTGGGC-3'; β-actin-forward 5'-GCTAGAGCTGCCTGACGG-3' and β-actin-reverse 5'-GAGGCCAGGATGGAGCC-3'.Total RNA from exponentially growing PC12 cells, rat cerebellum, mouse cerebrum or spleen was purified using RNazol B . RT-PCR analyses were performed essentially as described . BrieflyXhoI-NotI fragment from pIRES2-eGFP (Clontech) into XhoI-NotI digested retroviral plasmid pLXSN (Clontech) generated pLXSN-IRES2-eGFP. The BamHIsite in pLXSN-IRES2-eGFP was subsequently used to insert the BglII insert from pSG5-PTPPBSγ-FL [EcoRI fragment from PTPBR7-eGFP [EcoRI-digested pLXSN-PTPPBSγ-IRES2-eGFP we generated pLXSN-PTPBR7-IRES2-eGFP. Mutant pLXSN-PTPPBSγ constructs were generated as follows. First mutant PTP-SL open reading frames were excised from pRK5-PTP-SL expression vectors  were grown on collagen-coated dishes in DMEM supplemented with 10% FCS and 5% horse serum (HS) at 37°C. Ecotropic Phoenix packaging cells  were cul6 cells/well. The next day medium was replaced by DMEM without serum. After o/n serum deprivation, cells were treated with 0.075–100 ng/ml EGF  or 50 ng/ml NGF  for different time periods. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) before being scraped with a rubber policeman in 75μl lysis buffer: 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% triton X-100; 100 mM NaF; 2 mM Na3VO4; 20 mM Na4P2O7; 1 mM PMSF; protease inhibitor cocktail . Lysis was continued on ice for 30 min and insoluble components were subsequently pelleted by centrifugation for 25 min at 14.000 rpm and 4°C. Supernatants were stored at -20°C until further use. Protein concentrations were determined spectrophotometrically [Cells were seeded in six-well plates at a density of 1*10trically .Protein samples (8 μg) were subjected to SDS-PAGE on 10% PA gels and blotted onto polyvinylidene fluoride (PVDF) membranes. Blots were blocked for 30 min using 3% non-fat dry milk in TBS-T  and incubated overnight at 4°C with a 1:2000 dilution of a phospho-ERK1/2 monoclonal antibody . Following three washes with TBS-T, blots were incubated for 1 h with a 1:10,000 dilution of peroxidase-conjugated goat anti-mouse IgG  in 3% non-fat dry milk in TBS-T. Immunoreactive bands were detected with Lumi-LightPLUS Western Blotting Substrate (Roche Diagnostics GmbH) using a BioChemi imaging system and obtained signals were quantified with the Labworks 4.0 program . Blots were then stripped for 40 min at 60°C , washed with TBS-T and reprobed for 1 h with rabbit polyclonal ERK1 (C-16) antibody  at a dilution of 1:8000 in TBS-T containing 3% non-fat dry milk. Peroxidase-conjugated goat anti-rabbit IgG (Pierce Biotechnology Inc.) was used for detection and quantitation as described above. Ratios of the phospho-ERK1/2/total ERK1 signals were determined for every sample and, to be able to compare signal ratios originating from different blots, a reference standard sample (corresponding to a PC12 cell lysate following 5 min treatment with EGF) was included on each blot.PTPRR expression levels were determined by subjecting 50 μg of total protein to SDS-PAGE and immunoblot analysis as described above, using monoclonal antibody 6A6 . Phospho2) containing 4% paraformaldehyde, and subsequently washed with PBS and permeabilized for 5 min using 0.2% Triton X-100 in PBS. Aspecific sites were blocked by incubating for 30 min with 4% BSA in PBS before cells were incubated for 1 h with a 1:500 dilution of rabbit polyclonal α-SL antiserum [Cells, cultured on collagen-coated coverslips, were fixed at room temperature for 30 min in PHEM buffer  declare that they have no competing interests.YN carried out the experiments and helped to draft the manuscript. PJ assisted in RNAi experiments. WH conceived and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.


All the compounds tested inhibited soybean lipoxygenase activity. 2-(4-Trimethylsilyloxypiperidin-1-yl)-N-[4-(p-methoxyphenyl)-thiazol-2-yl]-acetamide (19) was the most potent displaying inhibition against lipoxygenase (ID50 = 0.01 mmol). It also possessed moderate cytotoxic effect  concerning MG-22A cell lines.A number of trimethylsiloxyalkyl and trialkylsilylalkyl thiazole derivatives have been evaluated for their anti-inflammatory activity, lipoxygenase inhibiting properties, and cytotoxicity. The investigated compounds have been found to protect in vivo against carrageenin-induced edema, especially 3-(4-trimethylsiloxypiperidin-1-yl)-N-(thiazol-2-yl)-propionamide ( The aim of this investigation was to study anti-inflammatory as well as lipoxygenase inhibitory activities and cytotoxicity of a series oforganosilicon-containing thiazole derivatives.It is well known that thiazolyl derivatives possessanti-inflammatory activity –6. Todayin vivo.Organosilicon compounds attract scientific attention due to somedifferent reasons, especially due to a number of interestingresults in the field of their biological action. Modernorganosilicon chemistry coincided with the emergence ofbiomaterials and bioengineering fields fifty years ago. It hasbeen reported that some organosilicon compounds affect thecollagen biosynthesis in cartilagenous tissue . Newapp1H NMR spectra were recorded with a Mercury 200 (Varian) spectrometer using 3CDCl as solvent andhexamethyldisiloxane (HMDSO) as internal standard (forunsilylated compounds). Mass spectra under electron impactconditions were recorded on a Hewlett-Packard apparatus . Analytical thin-layer chromatography(TLC) was performed on Macherey-Nagel silica plastic plates, withvisualization under UV (254 mm). Column chromatography wasperformed using Merck silica gel (0.040–0.063 mm). Solventsand reagents were purchased from the following commercial sources:Fluka, Aldrich, Acros. Melting points were determined on a Boetiusmelting point apparatus and were uncorrected. Elemental analyseswere performed on Carlo Erba 1108 elemental analyzer. Elementalanalyses  for all compounds synthesized were within±0.4% of theoretical values. The following compounds weresynthesized according to literature procedures:2-chloro-N-(thiazol-2-yl)-acetamide (1) -acetamide(19), which contains bulky p-MeO-6H4C-group at 4C-position.The nature of substituent in 6, 10)or their silyl ethers  leads tocytotoxic effect noticeable increase for propionamides 10and 21 in comparison with the corresponding acetamides6 and 17. Trimethylsilyl ether 18 wasthe most active against mouse hepatoma among all the compoundsstudied and in comparison with its unsilylated precursor7.The distance elongation between thiazolyl and piperidylheterocycles either in parent compounds (22) or lipoxygenase inhibition activity display(thiazolyl acetamides 18 and 19). At the sametime, compound 4-methyl-5-(β-trimethylsiloxyethyl)-thiazole(12) was the least active concerning all the biologicalproperties studied.It can be noted that the data obtained do not allow to concludedefinitely the existence of relationship amonganti-inflammatory activity, lipoxygenase inhibition, andcytotoxicity. But in some cases, cytotoxic properties wereaccompanied by anti-inflammatory activity (organosilicon saltThe wide possibility for variation of substituents around thesilicon atom can promote finer selection of perspective compoundfor further investigations.


CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ . Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted “Swiss cheese-like” cribriform morphology (CM) comprising multiple abnormal “back to back” lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked 1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors.Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH) Oncogenic perturbation of cell-cell interactions and hierarchical, three-dimensional 3D) tissue organization characterizes cancer development  and progD tissue PTEN, CDC42, PRKC and PARD genes   . In accoy Figure  and disps Figure . Caco-2 cellular  with grecellular  than CacRC cells  also indRC cells , S1F andRC cells .2D3 treatment enhanced vitamin D receptor (VDR) expression, perinuclear and nuclear VDR localization in Caco-2 and Caco-2ShPTEN cells, indicating biological responsiveness of the model system  rescue of Caco-2 ShPTEN gland morphology m Figure , S2B. Trs Figure  and rests Figure , formatis Figure  and singelopment , S2D. Corphology , S2F whirphology , S2H. 1,lularity . Hence, 2D3 initiates rapid nongenomic biological responses, in addition to transcriptional effects on target genes  . For confocal microscopy, primary antibodies were used in conjunction with Alexa Fluor 568 (anti-rabbit) and Alexa Fluor 488 .Laboratory chemicals were purchased from Sigma-Aldrich, Dorset, England unless otherwise stated. Antibodies included mouse anti- LTVDCCs , anti-GAPTEN-deficient Caco-2 ShPTEN cells were generated by transfection of parental Caco-2 cells with replication-defective retroviral vectors encoding PTEN short hairpin RNA (shRNA), using the Phoenix™ retroviral expression system , as previously described [PTEN knockdown was conducted in SK-CO-15 colorectal cells  that have the capacity for 3D organotypic growth [PTEN+/+ and PTEN−/− HCT116 cells were used in signalling assays, as previously described [2. PTEN+/+ and PTEN−/− HCT116 cells were cultured in McCoys 5A media supplemented with 10% FCS, 1mM L-glutamine and 1mM sodium pyruvate, as previously described [Stable escribed , 36. Trac growth . PTEN+/escribed . Caco-2 escribed , 36.PTEN-expressing Caco-2, PTEN-deficient Caco-2 ShPTEN cells, parental PTEN-expressing SK-CO-15 cells and a subclone rendered PTEN-deficient by SiRNA knockdown, in organotypic cultures. Cells were cultured and embedded in Matrigel matrix , then imaged by confocal microscopy during progressive development of multicellular glandular architecture, as previously described [SLC9A3R1, PRKCZ or p-PRKCZ were undetected in these cells, in this study. Cribriform morphology (CM) was defined as multiple aberrant lumens surrounded by abnormal stratified epithelium in 3D multicellular structures in culture and in tumors [Development of multicellular architecture was assessed in escribed , 36. SK-escribed  and apicPTEN 3D cultures were transfected with mutant CDC42 constructs, as previously described [2 D3 (10−7M), inhibitors of L-type calcium channels (nifedipine) [PRKCZ pseudosubstrate peptide containing a membrane-targeting myristoylation tag that functions as an effective PRKCZ pseudosubstrate inhibitor (PRKCZI) [Caco-2 and/or Caco-2Shescribed , 36 and/edipine) , calciumedipine)  or a myr(PRKCZI) .T+/0;PtenF/F mice and 29 Ah::CreERTT+/0;Pten+/+ mice were enrolled into cohorts for prolonged follow up. Tissues were harvested, fixed, and processed according to standard protocols, as previously described. [PTEN-deficient murine intestinal epithelium was assessed by H&E histology. Cribriform morphology was assayed as previously defined [All animal procedures were conducted in accordance with local and national regulations. Mice were generated, housed, and genotyped, and Cre activity was induced as previously described . A totalscribed. . Animalsscribed. . The mor defined .KRAS-genotyped CRCs. We assessed CM at low power microscopy according to previously defined criteria [PTEN RNA expression by RNAscope in situ hybridization [SLC9A3R1 intensity by immunohistochemistry (IHC) as readout of apical PRKCZ activity as outlined previously [PTEN protein expression, PTEN IHC was also assessed in cohort (ii) TMAs. Samples used in this research were released from the Northern Ireland Biobank (NIB13-0090), approved by the Office of Research Ethics Committees Northern Ireland (Reference number 11/NI/0013/-/NIB13-0090).We conducted 2 separate studies of polarity signalling against cribriform morphology (CM) in human colorectal cancer (CRC). We used anonymised formalin fixed, paraffin embedded (FFPE) samples from (i) 35 patients with non-genotyped CRCs and (ii) 92 patients with criteria  in both eviously , in FFPEPTEN RNA values were log transformed to provide a normal distribution.Descriptive statistics were expressed as the mean ± sem. Statistical analyses were by one or two-way ANOVA or Student's t test using SPSS for Windows release 22.0  or Graphpad Prism software . Scatterplots and bar charts were used for display of quantitative numerical or categorical data.


Combination of alisertib with PI3Kα (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (∼9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-κB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) ∼30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate ∼30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4 Peripheral T-cell non-Hodgkin lymphomas (PTCL) belong to a rare ∼15% of NHL) and heterogeneous group of lymphoid malignancies, comprised of several subtypes: extra-nodal  and nodal PTCL (NOS) (not otherwise specified), anaplastic large cell lymphoma  and angioimmunoblastic T-cell lymphoma (AITL) 5% of NHL. PTCL’s PTCL’s are a heterogeneous collection of malignancies originating from CD4 and CD8 T-cells, however the role of immune checkpoints in the pathogenesis of PTCL is not well established. Targeting PD-1 (Program Death-1) in Hodgkin lymphoma and follicular lymphoma have shown sustainable responses , howeverhypothesize that immune checkpoint mediated PTCL proliferation is amenable to targeting by inhibiting AK and PI3K and that these targets can be developed as a novel therapeutic strategy.Immunohistochemistry (IHC) of tumor samples from patients treated with alisertib for relapsed and refractory PTCL (SWOG 1108) [In vitro, alisertib plus PI3Kα isoform and pan-inhibitors showed synergistic anti-PTCL activity in CRL-2396 cells that were synthetic lethal with vincristine (VCR). Syngeneic immune competent PTCL mouse xenograft models showed synthetic lethality of alisertib plus anti-PD-L1 therapy. This effect was further enhanced by pan-PI3K inhibition ± VCR in this mouse xenograft model. Together, our data suggests that targeting immune escape associated proliferation is a key concept to effective PTCL therapy.We OG 1108)  showed hTargeting PD-1 (Program Death-1) in Hodgkin lymphoma and follicular lymphoma have shown sustainable responses however the role of immune checkpoints in the pathogenesis of PTCL is not well established. Western blotting demonstrated that the PTCL cell lines CRL-2396, TIB-48 and SUP-T1 over-express PD-L1 relative to PD-1 Figure . SimilarREG) in the Tumor Microenvironment (TME). Constitutively active PI3K plays key roles in G2/M transition and leads to defects in the DNA damage checkpoint control [50 values for MLN1117 (a PI3Kα inhibitor) indicate a micro-molar differential activity: TIB-48 = 9.75μM, Jurkat = 36.45μM and CRL-2396 = 21.37μM respectively  or IPI-145 has shown durable responses , most li control . These oy Figure . In conty Figure , indicats Figure , similar50 ranging from 17nM – 10.0μM , 0.10nM (DERL-2), 0.64nM (TIB-48), 1.12nM (CRL-2396) and 1.52nM (Jurkat)  Figure . VCR plu) Figure . VCR plu) Figure .in vitro synergy studies prompted a mouse PTCL xenograft study to evaluate the above-described therapies for anti-tumor activity, tolerability and safety in vivo. The mouse PTCL CRL-2396 BALB/c xenograft model treated with alisertib  ± PI3Kα inhibitor MLN1117  yielded a tumor growth inhibition (TGI) of ∼30% with alisertib alone versus control . Treatment with alisertib + MLN1117 resulted in a TGI of ∼60% (mean decreases 1288 mm3 p<0.001). The addition of VCR (0.375 mg/kg IV Q1W x 3) to alisertib plus MLN1117 resulted in a TGI of > 90% with a p<0.001  into different groups based on their tumor volume but independent of body weight which results in low weight mice grouped in negative control group. All treatments were well tolerated with no body weight changes except on day-16 there was only significant changes between negative control group and alisertib as a single agent group with p=0.043 , likely due to inhibition of cell growth. Vincristine synergizes with alisertib  but triple therapy did not induce further synergism in CRL-2396 cells (p=0.004) and SUP-T1 cells (p=0.004) which may be due to growth inhibitory nature of PF04691502. However, this combination is more potent in in vivo mouse models  at different doses (50 μg and 100 μg IV Q1W x3) day 3 post-implantation of CRL-2396 cells were ineffective (data not shown). In a subsequent study in a ARK/J syngeneic mouse xenograft model, drug dosing was initiated at day 3 post-implantation and mice were pair-matched. Anti-PD-L1 Mab  given at 10 mg/kg, IP, every other day (QOD) x3 weeks was ineffective versus control  despite expression of PD-L1 on CRL-2396 cells. In contrast, alisertib  plus anti-PD-L1 Mab  led to a TGI of >90%. However, 40% of mice relapsed within 2 weeks after discontinuation of therapy  and T-cell proliferation  in PTCL is not well established. A pilot study showed that CRL-2396 cells when injected into the flank of BALB/c or ARK/J mice grew rapidly to >2500 mmy Figure . The addy Figure . Kaplan-y Figure . Westerny Figure . These rThe development of effective therapies for PTCL remain a serious challenge and the identification of novel targets to guide innovative treatment strategies should be a leading priority. The aurora kinase (AK) inhibitor alisertib is efficacious in rapidly proliferating tumors such as PTCL , 6, 8. AIHC of patients with relapsed and refractory PTCL (SWOG 1108)  showed ain vitro consistent with a prior report [The PI3K pathway is known to induce PD-L1 expression  and to pr report . The PI3in vitro and in vivo), AMG319 a PI3Kδ inhibitor plus VCR also demonstrated synergy with increased cytotoxicity [in vitro cell culture modeling for CRL-2396 cells.Vincristine sensitizes AK inhibition with alisertib in PTCL. The shift in the cytotoxicity curve to the left observed in PTCL for alisertib plus VCR was also observed in MCL  and DLBCtoxicity . Our mouREG) within the TME thereby altering the pro-inflammatory tumor promoting cytokine milieu. In CLL, lenalidomide (imid) causes a cytokine release syndrome due to up-regulation of PI3Kδ p110 with activation of AKT. However, Idelalisib plus lenalidomide dampens this immune response without compromising anti-tumor activity [The mechanistic link between immune suppression and proliferation in PTCL is not well established with respect to immune checkpoint regulators. Our syngeneic mouse PTCL xenograft model demonstrated that alisertib as a single agent had a TGI of ∼30% that significantly improved TGI to >90% when combined with an anti-PD-L1 monoclonal antibody which had no single agent activity. This exciting result indicates alisertib synergizes with anti-PD-L1 and supports the concept that immune escape by suppression is closely linked to mitotic proliferation that can be successfully co-targeted. However, 14 days after end of treatment 40% mice do relapse. An active PI3K pathway is known to induce the PD-1/PD-L1 axis promoting immune suppression . Since aactivity  providinIn conclusion, we demonstrate that the AK inhibitor alisertib synergizes with anti-PD-L1 treatment thus providing a novel backbone therapy for PTCL. In addition, alisertib also synergizes with pan-PI3K inhibitor PF-04691502 targeting both PTCL and immune suppressive T-cells in the TME providing a second approach to PTCL therapy. Thirdly, alisertib has exquisite synergy with VCR further augmenting anti-PD-L1 and PI3K inhibition in PTCL. Since alisertib shifts the anti-proliferative curve to the left significantly with either VCR or pan-PI3K inhibition, the addition of anti-PD-L1 completes the blocking of the immune inhibitory loop enhancing the anti-tumor response in PTCL. We thus propose a novel therapeutic approach to treating PTCL with alisertib based synergistic combinations that target immune suppression  and proliferation  that could be evaluated in early phase therapeutic trials in PTCL patients relapsing after CHOP-like therapies.2. Alisertib and MLN1117 were provided by Takeda pharmaceuticals . PF-04691502, idelalisib, duvelisib and vincristine were purchased from SelleckChem . Mouse Anti-PD-L1 (BE0101) antibodies were purchased from BioXCell .Peripheral T-cell non-Hodgkin lymphoma (PTCL) murine cell lines TIB-48 and CRL-2396 (TK1) and human leukemia cell line Jurkat clone E6-1 (ATCC) and SUP-T1, DERL-2 (DSMZ) were maintained in RPMI medium  supplemented with 10% fetal bovine serum, 2mM sodium pyruvate at 37°C in a humidified atmosphere containing 5% COParaffin-embedded sections were de-paraffinized and rehydrated with stepwise addition of distilled water. Antigen retrieval was carried out by bringing slides to a boil in 1 mM Sodium Citrate Buffer pH 6.0 followed by 20 min at a sub-boiling temperature. After washing in TBS three times, the slides were incubated in 3% hydrogen peroxide in TBS for 10 min and then blocked with blocking solution  for 2hr at room temperature. The slides were then immune-stained using 1:250 anti-PD-L1, 1:250 anti-PD-1, or 1:500 anti-Ki-67 antibodies in blocking solution. The reaction was incubated overnight at 4°C. After washing 3 times with TBS, the anti-rabbit or anti mouse HRP conjugated secondary antibodies  were incubated at a dilution of 1:500 in blocking solution for 2 hr at room temperature. The signal was checked using DAB staining (Sigma) following the manufacturer’s protocol. Primary or secondary antibody replacements with normal serum from the same animal species were used as negative controls. Analysis of samples was performed using IHC profiler on ImageJ to compare the staining density of DAB at 20x using spectral deconvolution with a pixel by pixel comparison . Compari50 values were estimated by Calcusyn software . For combination studies of alisertib and PI3K inhibitor or vincristine, an equipotent ratio was calculated to determine a combined graded combination treatment. A control group was established for each drug treatment in six replicates. The effects of the combined treatments were determined by the combination-index (CI) and isobologram methods derived from the median-effect principle of Chou and Talalay [PTCL cells were seeded at 8,000 per well in 96-well culture plates and allowed to grow for 24 hr followed by the desired treatment with increasing concentrations of the indicated agents for 4 days. Viable cell densities were determined using a CellTiter 96 Cell Proliferation Assay . The studies were performed in 4 independent sets of triplicates repeat and IC Talalay .Annexin V staining was used to detect apoptosis. Treated cells were harvested and rinsed with cold PBS once. After centrifugation for 5 min, cells were suspended in 500 μl of 1X Annexin V binding buffer  and then 5μl of Annexin V-FITC and 5μl of propidium iodide  were added. After incubation for 5 min at room temperature in the dark, the samples were analyzed by flow cytometry. All studies are performed in three or more biological replicates and then quantitatively expressed as mean±S.E.Cells are lysed in 1X RIPA buffer and supplemented 1:100 Protease/ Phosphatase inhibitor cocktail . Protein concentrations are determined using the BioRad protein assay kit  and 30μg of protein resolved by electrophoresis on a 10% SDS-PAGE. The proteins are transferred onto a nitrocellulose membrane and nonspecific binding is blocked by incubating with 5% nonfat milk in TBS-T buffer  at room temperature for 1h. The membrane is subjected to the indicated antibodies and fluorescence detected using a LI-COR Odyssey Infrared Imaging System. Mouse Anti-PD-L1 (BE0101), mouse anti-PD-1(BE0146) and control rat IgG2A (BE0090) antibodies were purchased from BioXCell . Anti-Ki-67 (sc-15402) was obtained from Santa Cruz Biotechnology . Human Anti-PD-L1 (CST #13684), human anti-PD-1 (CST #86163), Anti-aurora A (CST #14475), Anti-phospho-aurora A (Thr288) (CST #3079), anti-Akt (CST #4691), anti-phospho-Akt (Ser473) (CST #4060), anti-ERK1/2 (CST #4695), anti-phospho-ERK1/2 (CST #4370), anti-PD-L1 (human) (CST #13684), anti-NFκβ (CST #8242) and anti-GAPDH (14C10) (CST #2118), anti-β-Actin (CST #3700) antibodies were purchased from Cell Signaling Technology .6 CRL-2396 cells subcutaneously into the right hind flank. In the second study AKR/Jmice were injected with 1×106 CRL-2396 cells subcutaneously into the right hind flank. Three days after the initial injection, mice were divided randomly (pair-matched) into different groups with 12 mice per cohort. The first study evaluated control (PBS) versus alisertib (30 mg/kg PO QD x 3 weeks) versus alisertib plus MLN1117 (30 mg/kg PO QD x 3 weeks) versus alisertib plus MLN1117 plus vincristine .Animal care and treatment were performed at Arizona Cancer Center’s experimental mouse shared services (EMSS) core facility. In the first study, BALB/c mice were injected with 1×10V) was calculated as: TV = (L×W2)/2. Mice were sacrificed at the end of treatment (3 mice per cohort), end of study or if they reached >2000 mm3 at any time during the study.The second study treated mice with intraperitoneal (IP) control antibody (IgG with DMSO) versus anti-PD-L1 antibody alone  versus anti-PD-L1 plus alisertib (30 mg/kg PO QD x 3 weeks) versus anti-PD-L1 plus alisertib plus PF-04691502 (10 mg/kg QD) versus anti-PD-L1 antibody plus alisertib plus PF-04691502 and vincristine . The length (L) and width (W) of the subcutaneous tumors were measured by calipers and the tumor volume  or end of study (censored). The Kaplan-Meier method was used to estimate survival. The log rank test was used to compare survival between the respective treatment groups. Statistical adjustments were made for multiple comparisons. Analysis was performed using Prism . All p-values ≤0.05 were considered statistically significant.


Background: We aimed to determine whether early or delayed wheel-running (W) after a cortical lesion in rats influences functional recovery and protein expression involving synaptic plasticity.Methods: 57 rats were arranged in 4 groups: (1) Sham, (2) NMDA, (3) W-24 h or (4) W-72 h (W-24 h and W-72 h means wheel-running for 14 days starting day 1 or day 3 after NMDA lesion). NMDA produced a standardized lesion in the unilateral sensorimotor cortex and detectable behavioral deficits. Synaptogenesis was measured by immunohistochemistry.Results: Wheel-running starting after 24 h had no detectable effect, but it significantly speeded functional recovery when delayed to after 72 h. These results were in accordance with a marker linked to synaptogenesis.Conclusion: Wheel-running starting 3 days, but not 1 day, after an NMDA lesion is associated with improved functional recovery.


Scientific Reports 10.1038/s41598-017-10782-4, published online 04 September 2017Correction to: This Article contains typographical errors in the Acknowledgements section.“National Natural Science Foundation of China (U13022081)”should read:“National Natural Science Foundation of China (U1302224)”.


Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG). Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG) generates reactive oxygen species (ROS) and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth.Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In  Caenorhabditiselegans, transgenesis can be quickly accomplished following microinjection of DNAs into the germline, which then form heritable extrachromosomal arrays  domain-derived Mini Singlet Oxygen Generator (miniSOG) generates reactive oxygen species (ROS) upon blue-light illumination  plates with OP50 bacteria as previously reported  + C. briggsaeminiSOG-3′ UTR( (Cbr)-unc-119(+)]unc-119(ed3) III III (knjSi1[Pmex-5-his-72::tbb-2)] IVminiSOG-3′UTR (pRF4) (ttx-3-RFP-3′UTR(unc-54) plasmids were used to examine the efficiency of optogenetic integration. For GFP::RPS-18 expression, Prps-18-GFP::rps-18 plasmid (pCZGY3162) (juIs531[GFP::rol-6(su1006dm)]rps-18 +  transgene was examined in the rps-18(ok3353)/nT1[qIs51 (Pmyo-2::GFP + Ppes-10::GFP + PF22B7.9::GFP)] background. Prgef-1-GFP::efa-6N-3′UTR(unc-54) (pLC665) was made by recombination between GFP::efa-6N entry vector and Prgef-1-GTW destination vector using Gateway system (Invitrogen). pLC665 contains exons corresponding to the first 150 amino acids of EFA-6, which was fused to GFP with a 22 amino acid linker. For overexpressing dlk-1, we used pCZ848 expressing Prgef-1-GFP::DLK-1L and Cbr-unc-119, which was generated for MosSCI insertion (Cbr-unc-119 overexpression does not cause uncoordinated behavior.Bristol ed3) III , which eCZ20310 as previously described . Among these F1 plates, we selected the plates with >50% F2 worms carrying transgenes and recovered single transgenic F2 onto five individual plates. A few days later, we examined for 100% transgene-positive F3 worms. The potential integrated strains were crossed to N2 males and confirmed for Mendelian segregation. We did not find any integrants having two integration sites based on the segregation. We note that integrants may not show 100% penetrance of phenotypes as described in Results and Discussion. To distinguish between true integrants and extrachromosomal array lines with high-transmission rate, we used a few ways in addition to outcross. First, if the transgenes have fluorescence, we examined them under an epifluorescence upright microscope instead of a fluorescent stereo scope. Integrants sometimes show 100% fluorescence of the transgene despite variable expression of a co-injection marker. Second, we picked animals without phenotypes of the co-injection marker and checked if they produced transgenic progeny. True integrants will produce marker-positive progeny from marker-negative parents.Plasmid DNAs for microinjection were purified with QIAprep Spin Miniprep Kit (Qiagen), mixed at 100 ng/µl in total unless otherwise noted, and centrifuged at 15,000 rpm for 10 min at room temperature to avoid clogging microinjection needles. The plasmid solution was injected into the gonad of escribed . These P2, 100 µg/mL Proteinase K). These worms were then lysed by incubating at 65° for 60 min and subsequently at 95° for 15 min for inactivation of Proteinase K. Two microliters of the worm lysis were used as the templates for quantitative PCR (qPCR) using iQ SYBR Green Supermix (Bio-Rad) and a C1000 thermal cycler (Bio-Rad) with CFX96 Real-Time system (Bio-Rad). The parameters for the qPCR were 95° for 3 min and 40 cycles of 95° for 10 sec and 55° for 30 sec. The following primers were used for detecting integrated transgenes as well as rps-25 for the internal control: rps-25:Twenty adults were picked and put into the lysis solution (10 mM Tris (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgClttx-3: YJ12212 (GAGCATACGCTTCGTCGGACT) and YJ12213 (GGAAAGTCATGTTGCGCGAGAG), rol-6: YJ12214 (AGGAAGGACCAGATGGGCAC) and YJ12215 (GGTGAAGCATCCTGTTGGTGG), efa-6: YJ12216 (GCATCGAGCATTCACCCACATC) and YJ12217 (CGATGGAAACGAAACTCCCGC). Copy number of integrated transgenes was calculated by normalizing to N2 and rps-25.YJ12209 (TCACACCATCCGTCGTCTCTG) and YJ12211 (GACCTGTCCGTGATGATGAACG), Bright field images and a movie were obtained from young adults using a Stemi508 stereo microscope (Zeiss) and a digital camera L-835 (Hozan). For fluorescence imaging, worms were immobilized with 25%(v/v) of 50 nm polystyrene beads  in M9 solution. Representative images were collected from young adults using a LSM710 confocal microscope (Zeiss) equipped with a 10x (NA = 0.3), 63x (NA = 1.4), or 100x (NA = 1.46) objective lens. The zoom function of ZEN software (Zeiss) was used for imaging cell bodies of mechanosensory ALM neurons. Single-plane images or maximum-intensity-projections obtained from several z-sections (0.5 or 1 µm/section) using ZEN software (Zeiss) were shown. Fluorescence intensities were analyzed after subtracting background signals using FIJI software .https://doi.org/10.25387/g3.6127709.The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. Supplemental material available at Figshare: rol-6(su1006dm), which produces dominant roller (Rol) phenotype , and the ratio of Rol F2 progeny was examined , ∼75% of its F2 worms should show Rol phenotypes. To avoid overlooking, we propagated any transgene-bearing F2 worms with >50% transmission rate (2). If singly propagated Rol F2 worms (F2 plates) produced 100% Rol F3 progeny, we treated them as potential integrants and proceeded to outcrossing of the F3 lines to wild type, followed with confirmation of the Mendelian segregation of Rol. In all integrants identified by Rol, we observed red fluorescence in AIY neurons. These results suggest that the rol-6(su1006dm) and Pttx-3-RFP transgenes were co-integrated in the genome. We were able to obtain such integrants among progeny of P0 animals that received blue light treatment 6, 12, or 18 hr after microinjection . Microinplasmids . Rol F1 examined  and 1B. ion rate , High F2njection . The higondition , light, 0 worms treated with blue light 12 hr after microinjection, we obtained 61.3 ± 11.5 transgenic F1 worms , and 12.2 ± 2.5 F2 animals carrying germline transmittable transgenes with >50% transmission rate. Among them, we obtained 1.6 ± 0.3 integrants. Statistical analysis showed that the optogenetic integration depends on both Histone-miniSOG transgene and light illumination  ranged from 31 to 144 when injected at 25 ng/µl . Extrachromosomal arrays are estimated to contain hundreds of copies of injected plasmids (25 ng/µl . In cont25 ng/µl . This tr18 (rps-18) (rps-18 with a co-injection marker rol-6(su1006dm) (juIs531 and juIs532[Prps-18-GFP::rol-6(su1006dm)]rps-18 + ). We detected consistent GFP::RPS-18 expression among different individuals from a single parent (juIs504 and juIs505[rol-6(su1006dm) + Pttx-3-RFP], showed stochastic expression of RFP in AIY, despite that Rol phenotype was 100% penetrant . This stochastic expression is probably not due to optogenetic integration protocol because variable expression is also reported in the integrants generated by traditional methods  in neurons inhibits axon regeneration and perturbs neuronal development  from 12 P0 animals, and these transgenic animals showed uncoordinated behavior . Thus, optogenetic integration is suitable for the integration of transgenes with toxicity to organism development and/or function.Traditional methods for transgene integration require establishment of transgenic lines before mutagenesis, which is not suitable for integrating transgenes that cause sickness of worms. For example, overexpression of a dominant-interfering N-terminus of Exchange Factor for promoter . We exampromoter . The coppromoter  and negapromoter . Thus, tpromoter , making C. elegans has a strong demand on fast and efficient integration for rapid expansion of newly available reporters and genetic tools. For example, cGAL4-UAS system has recently become available in C. elegans community (C. elegans as models for neurodegenerative diseases (In summary, we have established a one-step optogenetic integration method that bypasses the need to first establish transgenic extrachromosomal arrays lines. This technical improvement has two benefits. One is significant reduction of time and labor for integration. Research using ommunity . For exadiseases .


We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis.Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133 Mutaeceptors . Furthereceptors –9. There+ mononuclear cells isolated from adult peripheral blood are a functional angiogenetic modulator of postnatal neovascularization [+ and CD133+ cells [Endothelial progenitor cells (EPCs) identified as CD34rization . Periphe3+ cells . Cytokin3+ cells –14. In t3+ cells . Further3+ cells , 17. Howin vitro co-culture system similar to the bone marrow niche using HESS-5 bone marrow stromal cells to investigate the functional importance of Notch signals for human EPC-mediated neovascularization and the proliferation and differentiation of human CB-derived EPCs in vitro and in vivo.In this study, we established an + CB cells were separated from 1 × 108 mononuclear cells by a magnetic bead separation method . In brief, CD133+ cells were labeled with a hapten-conjugated monoclonal antibody (mAb) against human CD133 (clone AC133: Miltenyi Biotec), followed by microbeads coupled with an anti-hapten mAb. The bead-positive cells were enriched on positive selection columns twice in a magnetic field. Flow cytometric analysis of purified cells using a phycoerythrin (PE)-conjugated anti-CD133 mAb of a different clone  showed that 95% of the selected cells were positive for CD133.Human umbilical CB samples (50–100 mL) were collected in sterile blood packs  containing a citrate-dextrose solution as the anticoagulant. Written informed consent was obtained from all mothers before labor and delivery. Protocols for sampling human umbilical CB were approved by our Institutional Review Board, the Clinical Investigation Committee at Tokai University School of Medicine. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. The mononuclear cell layer was collected, washed twice with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS, and resuspended in degassed PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. CD1334 magnetically separated CD133+ CB cells or 1 × 105 cultured CB cells collected from each transfected HESS-5 stroma were washed twice with PBS containing 1% fraction V BSA , preincubated with 10 μL FcR blocking regent (Miltenyi Biotec) to block nonspecific binding for 30 minutes at 4°C, and then incubated with mAbs for 30 minutes at 4°C. For kinase-insert domain containing receptor (KDR) staining, biotinated anti-mouse IgG1  and allophycocyanin (APC)-conjugated streptavidin (BD Biosciences) were used as secondary and tertiary antibodies. The stained cells were washed three times with PBS containing 1% BSA, resuspended in 0.5 mL PBS/1% BSA/propidium iodide , and analyzed by the FACSCalibur flow cytometer. Dead cells were excluded from the plots based on positive PI staining. The following mAbs were used to characterize the cultured CD133+ CB cells: CD34-fluorescein-5-isothiocyanate (FITC) (clone 581), CD34-PE (clone 581), CD45-FITC (clone HI30), CD31-FITC (clone WM59) (BD Biosciences), AC133-APC (clone AC133) (Miltenyi Biotec), and KDR (Sigma). For negative isotype controls, mouse IgG1-FITC (clone MOPC-21), mouse IgG1-PE (clone MOPC-21), mouse IgG1-APC (clone MOPC-21), and unlabeled mouse IgG1 (clone MOPC-21) (BD Biosciences) were used.Flow cytometric profiles were obtained with a FACSCalibur flow cytometer and CellQuest software . Briefly, 1 × 10ex vivo-generated human hematopoietic stem cells and does not express Jagged-1 or Dll-1 [We used the murine bone marrow-derived stromal cell line HESS-5 , which maintains the reconstitution ability of or Dll-1 . OriginaJagged-1), and hDll-1(HESS-5 cells transfected with human Dll-1). Full-length cDNAs encoding human Jagged-1 and Dll-1  were cloned into the NotI and XhoI restriction sites of GCDNsamI/N retrovirus vectors. These vectors were transfected transiently into PLAT-E packaging cells  using Lipofectamine transfection reagent . The culture supernatants were harvested as the source of the retrovirus as described previously [3 HESS-5 cells were seeded in a 24-well plate. The cells were then incubated in 0.1 mL of retroviral supernatant containing either human Jagged-1, human Dll-1, or empty viruses in the presence of 8 μg/mL polybrene (Sigma) for 16 hours at 37°C with 5% CO2. The transfection mixture was removed, α-MEM with 10% horse serum was added, and the cells were cultured for 24 hours. Then, the cells were transferred to a 6-well plate for further culture in α-MEM with 10% horse serum and penicillin/streptomycin. At 48 hours after culture, the transfected HESS-5 cells were stained with human nerve growth factor receptor (NGFR)-PE (clone CD40-1457) (BD Biosciences) and analyzed by flow cytometry (+ cells.We established three types of feeder cells: control (HESS-5 cells transfected with an empty vector), hJagged-1 . The proteins were transferred to nitrocellulose membranes , and incubated overnight at 4°C with primary antibodies: goat polyclonal IgG against human Jagged-1 (C-20) , rabbit polyclonal IgG against human Dll-1 (H-265) (Santa Cruz Biotechnology), or rabbit polyclonal IgG against actin (Sigma). The membranes were washed three times and incubated with horseradish peroxidase-conjugated donkey anti-goat (Santa Cruz Biotechnology) or goat anti-rabbit  IgG for 2 hours at room temperature. Antibody-labeled proteins were detected using an enhanced chemiluminescence detection system . hJagged-1 or Dll-1 proteins was detected only in each transduced HESS-5 cell line, but not in the empty vector-transduced control .5 NIH/3T3 cells stably expressing Notch1 or 8 × 105 CHO cells stably expressing Notch2  were transfected with RBP-Jk-luc (five RBP-J-binding sites) and pTK-Renilla plasmids (constitutive expression of Renilla luciferase for transfection efficiency control) by Transfast Transfection Reagent . At 1 day after transfection, the cells were collected, and 5 × 104 of cells were co-cultured with 5 × 104 of each transduced HESS-5 stromal cell line for 48 hours. Then, luciferase activities were measured using a Dual Luciferase Kit (Promega) according to the manufacturer’s instructions. hJagged-1- and hDll-1-transduced HESS-5 cells were activated by RBP-Jk, a major Notch target. Control HESS-5 cells were not activated (n = 3) (Luciferase reporter assays were performed as described previously . In brie (n = 3) .+ CB cells, which mostly overlap with CD34+ cells, were plated onto the various stroma. Briefly, at 24 hours before co-culture experiments, hJagged-1, hDll-1, and control HESS-5 cells (1 × 104 in 0.5 mL medium) were plated in flat-bottomed, collagen-I-coated 24-well plates. CD133+ CB cells (1 × 104) were plated onto irradiated HESS-5 cell layers in 24-well plates containing Stem Span SFEM  supplemented with 5% fetal bovine serum (FBS)  and cytokines including human stem cell factor (SCF) (100 ng/mL), interleukin (IL)-6 (20 ng/mL), thrombopoietin (TPO) (20 ng/mL), Flt-3 ligand (100 ng/mL), and vascular endothelial growth factor (VEGF) (50 ng/mL). Human SCF, IL-6, and TPO were a generous gift from Kirin Brewery Co. Ltd. . Flt-3 ligand and VEGF were purchased from PeproTech . All cytokines were pure human recombinant molecules. At 7 days after incubation at 37°C with 5% CO2, the cells were harvested, counted, and used for subsequent analyses and experimental assays. In some cultures, 10 μg/mL γ-secretase inhibitor IX  was added to the culture medium to inhibit Notch signal transduction.To evaluate the effects of Notch ligand-expressing stromal cells on the progeny of EPCs, the human EPC fraction of CD133+ CB cells using an RNeasy Micro Kit  according to the manufacturer’s instructions. First-strand DNA was synthesized from 100 ng RNA with random primers by a First Strand cDNA Synthesis Kit (Invitrogen) and amplified with specific primer pairs by Taq DNA polymerase . The human specific primer pairs, PCR conditions, and products sizes are shown in + human CB cells, we measured the band intensities in gel images. After the images were recorded in a computer, the band intensities were processed with Image J software (http://imagej.en.softonic.com/). Specific mRNA expression levels were normalized to the intensities of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH).Total RNA was obtained from cultured CD1332 cultured CD133+ CB cells, Iscove’s modified Dulbecco’s medium (IMDM) (Gibco), Methocult H4236 (Stem Cell Technologies), 30% FBS (JRH Bioscience), penicillin/streptomycin, and cytokines, including human SCF (100 ng/mL), VEGF (50 ng/mL), basic fibroblast growth factor (FGF) (50 ng/mL), epidermal growth factor (EGF) (100 ng/mL), insulin-like growth factor (IGF)-1 (100 ng/mL), IL-3 (20 ng/mL), and heparin (2 IU/mL), were plated in 35-mm Primaria culture dishes . All cytokines were pure human recombinant molecules. Human IL-3 was a generous gift from Kirin Brewery Co. Ltd. Basic FGF, EGF, IGF-1 (PeproTech), and heparin  were purchased. Cultures were incubated at 37°C with 5% CO2 for 18 days, and then the number of cells was counted by microscopy. For flow cytometric analysis and the cell adhesion assay, EPC colonies were stained with EPC fluorescent chemical markers [Ulex europaeus lectin type 1 (UEA-1)  for 1 hour at 4°C, followed by flow cytometry. For the cell adhesion assay, stained and harvested cells from EPC colonies were counted, and then 2 × 104 cells were incubated at 37°C in 0.1% BSA/IMDM with 100 ng stromal derived factor-1 (PeproTech) on 0.1% gelatin-coated (Sigma) 24-well plates. After 20 minutes, non-adherent cells were removed by washing with PBS, and adherent cells were counted by fluorescence microscopy.Vasculogenic methylcellulose culture for the EPC colony-forming assay was performed in triplicate for each sample as described previously . Briefly markers . BrieflyAll experimental procedures were conducted in accordance with the national and institutional guidelines. The protocols were approved by the Institutional Animal Care and Use Committee of the Isehara Campus, Tokai University School of Medicine, based on the Guide for the Care and Use of Laboratory Animals . Sixteen male athymic nude mice , 8 to 9 weeks old and 17 to 20 grams in weight, were separated into four groups  and used. Unilateral hindlimb ischemia was induced in the mice by ligating and excising the left femoral artery, as described previously , 24. All+ cells were harvested and washed three times with a sufficient amount of IMDM to remove the culture medium completely. Then, 1 × 105 cells in 50 μL of fresh unused IMDM were injected into the ischemic limb muscle of the mice immediately after the ligation procedure. As additional control animals, mice with hindlimb ischemia were identically injected with only medium of IMDM.Cultured CB CD133Regional blood flow in ischemic hind limbs was recorded and analyzed by laser Doppler perfusion imaging (LDPI) at 4, 7, 14 and 28 days after transplantation as described previously . In the Statistical analysis was performed using StatView v5.0 . All values are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated by one-way analysis of variance. Differences of P < 0.05 were considered statistically significant.+ cells significantly increased to 8.76 ± 2.07 × 105 (P < 0.05) compared with control cells (6.09 ± 1.50 × 105), whereas the number of hDll-1-stimulated CD133+ cells was reduced to 3.54 ± 1.07 × 105 (P < 0.05)  . The exp < 0.05) . No prol+/CD38- hematopoietic progenitor cells and CD31+/KDR+ endothelial lineage cells were decreased among hDll-1-stimulated CD133+ cells  increased significantly among hJagged-1-stimulated CD133+ cells compared with the control , but were decreased significantly by 0.5-fold among hDll-1-transfected CD133+ cells (P < 0.01). Suppression of Notch signaling by GSI reversed these cellular changes compared with the controls  (The numbers of CD34 control . Furtherith GSI) .+ cells compared with Dll-1-stimulated CD133+ cells (both P < 0.05)  mRNAs by competitive RT-PCR analysis. All cell types examined expressed VEGF-A and eNOS mRNA. There was significantly higher VEGF-A and eNOS mRNA expression in hJagged-1-stimulated CD133 < 0.05) . HESS-5 + cells were stimulated with each stromal cell type at day 7 for EPC colony forming assays. EPC colonies expressed endothelial differentiation markers KDR, eNOS, and VE cadherin (data not shown). Compared with controls, the number of EPC colonies were significantly higher in hJagged-1-stimulated CD133+ cell cultures (P < 0.05) and significantly lower in hDll-1-stimulated CD133+ cell cultures (P < 0.05) (+ cells were enriched with Ac-LDL/UEA-1 double-positive cells and showed significantly increased adhesive activity (P < 0.05), whereas hDll-1-stimulated cells showed a lower adhesive activity (Isolated CD133 < 0.05) . Compareactivity .+ cells.Thus, HESS-5 bone marrow stromal cells expressing hJagged-1 enhanced EPC colony formation of CD133+ cells. Blood flow was significantly increased at 28 days after injection of hJagged-1-stimulated CD133+ cells compared with hDll-1-stimulated cells or control cells (P < 0.05)  . Capilla < 0.05) .+ cells co-cultured with Notch ligand-transduced stromal cells were identified in the vasculature by double staining of human HLA-ABC and human vWF. Human endothelial cells were identified in mice that received all three cell types, indicating that the transplanted human CD133+ cells had differentiated into endothelial cells in mouse ischemic muscles  have been identified in mammalian systems. Previously, we found that CD133eceptors . It has eceptors . Among teceptors . Choi eteceptors . Not onleceptors , while Jeceptors , 42. ThuVEGF is a potent promoter of angiogenesis and regulator of blood vessel homeostasis. Furthermore, VEGF contributes to postnatal neovascularization by mobilizing EPCs . In previn vivo in mouse ischemic hindlimbs. Immunohistochemical analysis demonstrated that transplanted CD133+ CB cell-derived EPCs differentiated into endothelial cells in ischemic limb muscles, but a significant difference in the numbers of human endothelial cells was not found among the groups. It has been reported that the angiogenic potential of transplanted EPCs in ischemia is due to endothelial differentiation and the local production of angiogenic cytokines[+ CB cells might be caused by an enhancement of EPC potential (colony formation and release of angiogenic factors including VEGF). Although the proliferation rate was different, we obtained the largest number of ex vivo-expanded EPCs from hJagged-1-stimulated CD133+ cells. Thus, hJagged-1 signaling in the bone marrow niche might be effective to enhance the therapeutic potential of CD133+ CB cell-derived EPCs.In this study, the transplantation of hJagged-1-stimulated EPCs recovered impaired regional blood flow and capillary density cytokines. Thus, e+ CB progenitors compared with hDll-1-expressing cells. Thus, hJagged-1 signaling in the bone marrow niche might aid the expansion of EPCs for therapeutic angiogenesis.In conclusion, this study is the first report regarding the effect of human Notch ligand signaling on EPC proliferation and differentiation. hJagged-1-expressing bone marrow-derived stromal cells induced the proliferation and differentiation of CD133S1 Fig+ CB cells (1 × 105) co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells for 7 days or fresh unused medium only (IMDM) were injected into mouse ischemic limbs. Representative double immunofluorescence staining of human vWF (red), HLA-ABC (green), and nuclear counterstaining with DAPI (blue) are shown for each group at 28 days after transplantation. All images show merged staining at ×400 magnification. Human endothelial cells were identified as vWF and HLA-ABC double-positive yellow cells (arrow). Scale bar represents 20 μm.CD133(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-13457-2, published online 12 October 2017Correction to: In the PDF version of this Article, Equation 6 is formatted incorrectly. The correct Equation 6 appears below.


T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease caused by the malignant transformation of immature progenitors primed towards T-cell development. Clinically, T-ALL patients present with diffuse infiltration of the bone marrow by immature T-cell blasts high blood cell counts, mediastinal involvement, and diffusion to the central nervous system. In the past decade, the genomic landscape of T-ALL has been the target of intense research. The identification of specific genomic alterations has contributed to identify strong oncogenic drivers and signaling pathways regulating leukemia growth. Notwithstanding, T-ALL patients are still treated with high-dose multiagent chemotherapy, potentially exposing these patients to considerable acute and long-term side effects. This review summarizes recent advances in our understanding of the signaling pathways relevant for the pathogenesis of T-ALL and the opportunities offered for targeted therapy. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor arising from the malignant transformation and subsequent clonal expansion of T-cell precursors expressing immature T-cell markers. Based on the maturational stage of normal thymic development at which transformation occurs, T-ALL can be classically sub-classified into early cortical, late cortical or mature . RecentlAt the molecular level T-ALL is a heterogeneous disease, where a wide spectrum of genetic lesions  and micrIn this review, we summarize and discuss recent advances in understanding the signal transduction pathways that are de-regulated in T-ALL, the emerging role in cancer development of leukemic adaptation to metabolic stresses encountered in the native environment and evaluate their potential as therapeutic targets.−CD25+) stage of development, the expression of a constitutively active pre-T cell receptor    ; LIM-onlnd LMO2) ,23; home1/HOX11) , TLX3 (HHOX11L2) , NK2 homd NKX2-5 ,27, and  cluster ; and proter  pathway, most commonly altered by PTEN deletion/mutations, PTPN2 deletion, PIK3R1 or v-akt murine thymoma viral oncogene homolog 1 (AKT1) mutations; (ii) the IL-7R/JAK/STAT pathway, via activating mutations in the interleukin 7 receptor α-chain gene (IL-7Rα), Janus kinase 1 (JAK1), JAK3 or signal transducer and activator of transcription 5B (STAT5B); (iii) the RAS/mitogen-activated protein kinase (RAS/MAPK) signaling through Kirsten rat sarcoma viral oncogene homolog (KRAS) and neurofibromin 1 (NF1) mutations; and (iv) via chimeric ABL1 fusion genes such as nucleoporin 214kDa-Abelson murine leukemia viral oncogene homolog 1 (NUP214)-ABL1 and ETS Variant 6-Abelson murine leukemia viral oncogene homolog 1 (ETV6)-ABL1; (2) altered epigenetic regulation through mutations affecting (i) PHF6; (ii) PRC2 components EZH2, SUZ12 and EED; (iii) KMT2A  reviewed in  [−/−Tcf7 lymphomas showed upregulated expression of myocyte enhancer factor 2c (Mef2c) [Wnt signaling plays an important role in normal hematopoiesis ,230, ando et al. , a constg levels , suggestg levels ,237. In of NOTCH . In factin T-ALL , and mor (Mef2c) , which h (Mef2c) . In conc (Mef2c) . In thesPTEN deletion, rendered T-ALL cells resistant to gamma secretase inhibition by upregulating glycolysis [Metabolic homeostasis is a fundamental trait of cells which becomes altered in cancer in order to satisfy the heightened demand for metabolites necessary for growth and proliferation. Oncogenic mutations can directly alter cellular metabolism in a cell-intrinsic way, favoring malignant transformation. Moreover, microenvironmental cues such as hypoxia, nutrient availability, oxidative stress and cross-talk from neighboring cells all affect cancer cell metabolism and contribute to determine metabolic heterogeneity within the tumor . It is tycolysis .Recent evidence suggests that AMPK confers cancer cells with the ability to cope with metabolic stresses by regulating glucose metabolism. AMPK is a heterotrimeric serine/threonine kinase composed of an α-catalytic subunit and two regulatory subunits β and γ which regulate its activation and substrate specificity . AMPK isT-ALL is an aggressive hematological disease for which few therapeutic options are available in the case of primary resistant or relapsed disease, highlighting the need for better risk stratification and the necessity to identify more effective targeted therapies . Althoug