The A20 protein is a known inhibitor of apoptosis that here is shown to be a novel cancer stem cell-promoting factor associated with poor glioma patient survival. A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFα-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFα-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-κB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted  Glioblastomas are the most common and aggressive primary brain tumors in adults, with a median survival of only 12–15 months. Glioblastomas display a cellular hierarchy with a subset of cells having stem cell–like properties, including the capacity to self-renew and propagate tumors. Specific ablation of cancer stem cells is widely thought to be critical for effective and long-lasting treatment of cancers. We report the identification of the antiapoptotic protein A20  as a novel regulator of glioma stem cell survival. Glioma stem cells overexpress A20 relative to non-stem glioma cells, and this protects them from cell death, whereas depletion of A20 attenuates glioma stem cell survival and tumor growth. Interrogation of a molecular glioma database reveals that A20 levels correlate with decreased survival in patients. These data indicate that A20 is a tumor enhancer in the context of glioma, which importantly contrasts with its known function as a tumor suppressor in the context of lymphoma. Therefore, A20 may be a context-specific regulator of cancer stem cell survival and growth. Tumors are aberrant organ systems that display a complex interplay between neoplastic cells and recruited vascular, inflammatory, and stromal elements To identify molecular targets in cancer, the majority of analyses completed to date compare bulk tumor to normal tissues and may therefore underestimate the importance of genes and proteins expressed within the cancer stem cell subpopulation. For example, comparison of GSCs to non-stem glioma cells or bulk tumor has resulted in greater understanding of the importance of HIF2α A20 expression in some cancers: A20 is elevated in undifferentiated nasopharyngeal carcinoma A20 null mice also suggested a link to stem cell biology: the epidermal and dermal layers of −/−A20 mice are significantly thicker than that of wild-type controls, demonstrating A20 regulates skin cell fate A20 mRNA expression or copy number is associated with poor glioma patient survival.We speculated that the cell survival and NF-κB regulator A20, or Tumor Necrosis Factor α inducible protein 3 (TNFAIP3), was one molecular target with a greater role in glioma than currently understood A20 mRNA levels in comparison to matched non-stem glioma cells  when cells were isolated from a patient specimen passaged short term in immunocompromised mice  or direcA20 expression using lentiviral transduced short hairpin RNAs (shRNAs) (Sigma Mission shRNA). To control for potential off-target shRNA effects, two different sequences of shRNA directed against A20 and a nontargeting shRNA were used when cell numbers permitted. Transduction with A20 shRNA reduced A20 protein levels in GSCs in comparison to the nontargeting control, but did not alter Olig2 expression . Thus, knockdown of A20 inhibits cell growth due in part to decreased proliferation associated with increased cell death and cell-cycle arrest.Although our findings demonstrated A20 was consistently up-regulated in GSCs, no studies to date have suggested a functional role for A20 in cancer stem cells. As A20 was linked to cell survival in some reports pression . A20 tarver time . In contma cells . The difl growth –S3F. To argeting , supportargeting  and a coA20-directed shRNA increased the percentage of Annexin V–positive cells when compared to nontargeting control shRNA. Caspases, including caspase 3 and caspase 7, are cysteine-aspartic acid proteases that are activated during apoptosis A20 targeting compared to control. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining detects DNA fragments that occur in the last phase of apoptosis A20  in accordance with Institutional Animal Care and Use Committee–approved protocols. For all cell numbers transplanted, the median survival of mice injected with GSCs derived from either T3359  tissue  and The Cancer Genome Atlas (TCGA), which respectively contain information from multiple brain tumor types or glioblastoma only. Using REMBRANDT, we found that up-regulation of survival . When thsurvival . However) tissue . As elevl n = 4; . It is aA20 is induced or the pathways through which A20-expressing cells regulate survival. We therefore utilized the TCGA glioblastoma database to evaluate the presence of common glioblastoma mutations  may be beneficial for glioma therapy. However, the increased survival of mice upon A20 restoration to A20-deficient lymphoma cells demonstrates that targeting A20 may be harmful for other tumor types. As we do not fully understand the mechanisms that cause A20 to have differential effects on tumor growth and cancer cell behaviors, further elucidation of A20 molecular and biological signals is warranted.Our data implicate A20 as an important mediator of cancer stem cell biology by demonstrating that A20 is involved in glioma maintenance through the regulation of GSC growth and survival. The increased survival of mice bearing intracranial tumors upon Primary human brain tumor patient specimens were obtained from patients providing informed consent under protocols approved by the Duke University or Cleveland Clinic Institutional Review Boards. All animal experiments were performed in accordance with a Duke University or Cleveland Clinic Institutional Animal Care and Use Committee–approved protocol.http://www.worthington-biochem.com/PDS/default.html). Cells were then cultured in Neurobasal medium supplemented with B27 without vitamin A, l-glutamine, sodium pyruvate (Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF), and 10 ng/ml epidermal growth factor (EGF) (R&D Systems) for at least 6 h to recover surface antigens. Cells were then labeled with an allophycocyanin (APC)-conjugated CD133 antibody (Miltenyi Biotec), and sorted by fluorescence-activated cell sorting (FACS). Alternatively, cells were separated microbead-conjugated CD133 antibodies and magnetic columns (Miltenyi Biotec). CD133-positive cells were designated as GSCs whereas CD133-negative cells were designated as non-stem glioma cells. Consistent with previously defined methods for GSC and non-GSC cell culture As previously described A20 and Olig2, individual gene primers were ordered from Integrated DNA technologies and Master Mixes were purchased from SuperArray Bioscience Corporation. mRNA levels were measured using an ABI-7900 system (Applied Biosystems). Sequences for primer sets were as follows:Total RNA was prepared using the RNeasy kit (Qiagen), and reverse transcribed into cDNA using a SuperScript III First-Strand Synthesis Kit (Invitrogen). To investigate expression of A20: Forward 5′-AGT GTT CCC AGG TGG CCT TAG AAA-3′; Reverse 5′-TCT CAG CCA AGA CGA TGA AGC AGT-3′. Olig2: Forward 5′-GGT AAG TGC GCA ATG GTA AGC TGT-3′; Reverse 5′-TAC AAA GCC CAG TTT GCA ACG CAG-3′.Cells, neurospheres, or tumor sections were fixed with 4% paraformaldehyde, washed with Tris-buffered saline, and incubated with polyclonal mouse anti-A20 (Santa Cruz Biotechnology) and goat anti-Sox2 (Santa Cruz Biotechnology) where indicated. Primary antibodies were incubated for 16 h at 4°C followed by detection with Alexa 488 donkey anti-mouse (Invitrogen) and Alexa 568 donkey anti-rabbit (Invitrogen) secondary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen), and slides were mounted using Fluoromount . Images were taken with a Leica SP-5 confocal microscope.Equal amounts of cell lysate were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and detected using an enhanced chemiluminescence system (Pierce Biotechnology) with antibodies against A20 (Abcam or Santa Cruz Biotechnology), Olig2 (R&D Systems), and Tubulin (Sigma).Tumors were dissociated as described earlier and bulk or isolated GSCs, and non-stem glioma cells were fixed in 4% paraformaldehyde and subjected to FACS analysis. FACS analysis was performed on a FACS Aria with 100-µm nozzle and low sheath pressure. Human-specific anti-CD133 (293C3) conjugated to allophycocyanin5 (APC) (Miltenyi) was used with anti–A20-PE generated using the Lightning-Link PE kit (Innova Biosciences) in combination with an A20 antibody (Abcam).A20 and a scrambled nontargeting control (SHC002) were purchased from Sigma. These vectors were cotransfected with the packaging vectors psPAX2 and pCI-VSVG (Addgene) or the ViraPower Lentiviral Expression System packaging mix (Invitrogen) into 293FT cells by Lipofectamine 2000 (Invitrogen) to produce the virus. Efficiency of different lentiviral shRNA clones in cells was determined by Western blot analysis and real-time PCR. The sequence of the shRNAs utilized for shRNA1 (NM_006290.2-635s1c1), shRNA2 (NM_006290.2-2104s1c1), and shNRA3 (NM_006290.2-957s21c1) was as follows: 5′-CCGGCACTGGAAGAAATACACATATCTCGAGATATGTGTATTTCTTCCAGTGTTTTTG-3′ and 5′-CCGGGAAGCTCAGAATCAGAGATTTCTCGAGAAATCTCTGATTCTGAGCTTCTTTTTG-3′; and 5′-GTACCGGGATGAAGGAGAAGCTCTTAAACTCGAGTTTAAGAGCTTCTCCTTCATCTTTTTTG-3′.Lentiviral shRNA clones (Sigma Mission RNAi) targeting GSCs infected with lentivirus expressing the indicated shRNAs for 24 h were plated in 96-well plates at 1,000 cells per well. Cell titers were determined after the indicated number of days after plating using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).GSCs plated in six-well plates at 100,000 cells per well were infected with lentivirus expressing the indicated shRNAs for 48 hours. To determine the percentage of cells in each phase of the cell cycle, cells were fixed with ethanol and stained with propidium iodide followed by cell-cycle analysis. To detect apoptotic cells, Annexin V-FITC staining was performed with the Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen) according to the manufacturer's instructions. For experiments in which GSCs and matched non-stem glioma cells were treated with TNFα, cells were plated at a density of 100,000 cells per well in a six-well plate and treated for 72 h with 5 ng/ml TNFα.GSCs infected with lentivirus expressing the indicated shRNAs for 24 h were plated at 1,000 cells per well and caspase 3/7 activity measured with a commercially available kit (Promega) after an additional 24 h. Relative caspase activity was then determined by correcting for cell titers determined as indicated above. When TNFα treatment was combined with shRNA treatments, cells were infected with lentivirus expressing the indicated shRNAs for 24 h followed by TNFα treatment for 24 h.GSCs infected with lentivirus expressing the indicated shRNAs for 36 h were stained for TUNEL using an Apo-BrdU-Red In Situ DNA Fragmentation Assay Kit (Biovision) according to the manufacturer's instructions.GSCs infected with lentivirus expressing the indicated shRNAs for 24 h were plated in 24-well plates at 10 cells per well and the percentage of wells containing neurospheres quantified at indicated times. For secondary sphere formation, neurosphere forming cells from the first plating were trypsinized and plated at 10 cells per well in 24-well plates. Neurospheres were imaged with an Olympus CK40 digital camera mounted to a light microscope and neurosphere size was calculated using ImageJ software. When TNFα treatment was combined with shRNA treatments, cells were infected with lentivirus expressing the indicated shRNAs for 24 h followed by treatment with 5 ng/ml TNFα treatment.Intracranial transplantation of GSCs into nude mice was performed as described t-test or ANOVA using GraphPad InStat 3.0 software. For repeated measures ANOVA and in vivo studies where Kaplan-Meier curves and log-rank analysis were performed, MedCalc software was used.Significance was tested by Figure S1A20 is elevated in SSEA-1+ fractions of human glioma cells where SSEA-1, but not CD133, is informative for tumorigenic potential. Equal amounts of lysates from the human glioma cell lines 1228 and 905 sorted for the expression of SSEA-1 were probed for the expression of A20 by Western blot. α-Tubulin was utilized as a loading control.(0.06 MB TIF)Click here for additional data file.Figure S2Flow cytometry demonstrates A20 colocalizes with a glioma stem cell marker. (A and B) Flow cytometry of fixed bulk tumor cells isolated directly from T3832 (A) or T3946 (B) patient specimens demonstrates significant co-staining of the glioma stem cell marker CD133 and A20. (C and D) Flow cytometry analysis of glioma stem cell-enriched (C) and -depleted (D) cultures from a T4105 patient specimen passaged short term in immunocompromised mice demonstrates passage of cells in vitro maintains coexpression of the glioma stem cell marker CD133 and A20 in glioma stem cells with reduced CD133 and A20 expression in non-stem glioma cells.(1.83 MB TIF)Click here for additional data file.Figure S3A20 preferentially decreases the growth of glioma stem cells. Cell growth as measured with Trypan Blue staining demonstrated that A20 shRNA decreases the growth of non-stem glioma cells isolated from T4121 (A) or T08-836 cells (B). An asterisk (*) indicates p<0.01 with ANOVA comparison to non-targeting shRNA. Matched glioma stem cells with A20 targeting are shown in A20 shRNA1 (C and E) or A20 shRNA3 (D and F) demonstrates significantly greater reductions in cell number in the glioma stem cell fractions. An asterisk (*) indicates p<0.001 with t-test comparison to non-stem glioma cells.(0.47 MB TIF)Click here for additional data file.Figure S4A20 protects GSCs from TNFα-induced apoptosis. GSC enriched cultures isolated from a T4597 glioma xenograft were infected with non-targeting shRNA or shRNA directed against A20 and treated with 5 ng/ml TNFα. (A) Relative caspase activity increased with A20 knockdown and was further increased by TNFα treatment. (B) Neurosphere formation decreased with A20 knockdown and was further decreased by TNFα treatment. An asterisk (*) indicates p<0.05 with ANOVA comparison to similarly treated nontargeting control cells. A number sign (#) indicates p<0.05 with ANOVA comparison of TNF to untreated cells infected with the same shRNA.(0.13 MB TIF)Click here for additional data file.Figure S5Tumors resulting from implantation of A20 knockdown GSCs express A20. Immunofluorescence of paraffin-embedded sections of tumors resulting from implantation of GSCs infected with nontargeting shRNA or A20 shRNA1 demonstrates A20 is expressed in both tumor types.(0.41 MB TIF)Click here for additional data file.Figure S6Glioblastoma genetic subsets and expression of A20. (A) Table demonstrating the number of samples in the TCGA database analyzed as having low, intermediate, or high A20 expression. The number of samples in each group indicated as having mutations in p53, EGFR, PTEN, or NF1 is also shown. (B) Analysis of the percentage of patients with mutations in p53, EGFR, PTEN, or NF1 for the groups have differential A20 expression is shown. The percentage of patients with p53 or NF1 mutations is increased in the set of patients with high A20 expression. (C) Some TNFα signaling mediator mRNAs are elevated in the set of patients with high A20 expression. Analysis of the expression of multiple TNF receptor and NF-κB family members demonstrated elevated expression was often observed in patient samples with elevated A20.(0.51 MB TIF)Click here for additional data file.


CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive.Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis.To test the tumorigenic potential of cyclin D1b via previously unrevealed actions.Our study indicates that cyclin D1b participates into MM pathogenesis  CCND1 gene generates two mRNA species by alternative splicing . The first set of animals (five mice per clone) was sacrificed at eight weeks  was inoculated in Matrigel into the lower flank of nude mice. The day after, 10 μM of either scrambled siRNA (5'-aat tct ccg aac gtg cac gt-3') or siRNA targeting VEGF (5'-aag gag acc ctg atg aga tc-3') were mixed with AteloGene™  according to manufacturer's instructions. The mixture (150 μl) was s.c. injected wrapping up the cells at the injection site. Chemical tyrosine kinase (TK) inhibitors targeting VEGFR2/3 ) and all FGFR ), a gift of F. Bono, were dissolved in 5% glucose in physiological serum. SAR and SSR were i.v. injected biweekly at 40 mg/kg each, starting at day 1 following inoculation of cells. Each group contained 5 mice. At day 11, volume of tumors was measured as before and the growth of tumors monitored thereafter. The tumor evolution was calculated as the ratio between the volume of tumors at each time point and the volume of the tumors of non treated mice at day 11. Statistical analysis for tumor evolution in each group was done with the Student's t test. During the experiments, mice had free access to food and water and all the experiments were performed at the Common Service of Animal Experimentation , in accordance to the declaration of Helsinki on animal welfare and with the approval of the ethics committee of the University of Paris 11/CNRS (responsible person V. Dommergue-Dupont).Female, six week-old Finefix-fixed paraffin embedded 4 μm-sections were deparaffinized in toluene twice for 5 min and rehydrated by using graded EtOH concentrations. After antigen retrieval in citrate buffer pH 6.2 , immunohistochemical labeling with anti-CD138 or anti-CD34 antibodies (Abs) was performed with the Vector Vectastain Elite kit  and 3',3' Diaminobenzidine (DAB) as chromogen. Sections were counterstained with hemalun.http://www.microarray.fr:8080/merge/index) were diluted to a final concentration of 50 mM in 50% dimethyl sulfoxide, 100 mM potassium phosphate (pH 8.0) and printed onto hydrogel-coated slides  using a microGrid II arrayer . Total RNAs (200 ng) were amplified by linear PCR and labelled with Cy3 using Bioprime Array CGH Genomic Labelling System Kit (Invitrogen). Total RNA from one culture of LP-1cl1 cells was similarly amplified, labelled with Cy5 and used as a reference probe for hybridization. Each Cy3-labelled probe was co-hybridized with the Cy5 reference probe on microarrays in a G2545A oven  at 60°C for 18 h. Microarrays were washed  and scanned with a G2565B scanner (Agilent). Raw data were extracted from scanned microarray images (.tif) using Feature Extraction Software v9.5 (Agilent) and normalized using the Quantile method adapted to bicolour microarrays. All the protocols used can be obtained by contacting the microarray and sequencing platform of the IGBMC (web site: http://www-microarrays.u-strasbg.fr/). In order to select genes that are differentially expressed among the three biological groups , we performed an analysis of variance using Cy5/Cy3 log2 ratios. To limit the error due to multiple tests, we used permutation of samples for controlling the false discovery rate [p-value less than 0.01 were considered to be significant. Moreover, we filtered out genes with a fold change (FC). The FC between LP-1K and LP-1cl1 was calculated as the median value of the 4 replicates ratios in the LP-1K samples over the median value of the 4 replicates ratios in the LP-1cl1 samples. Three FC were calculated: LP-1K vs. LP-1cl1, LP-1D1b vs. LP-1cl1 and LP-1K vs. LP-1D1b and a threshold equal to 2 was used for selecting three lists of significant genes. To design Venn diagram, we used the VENNY software http://bioinfogp.cnb.csic.es/tools/venny/ and individual gene expression profiles were generated with the TigrMev 4_03 software http://www.tm4.org/mev.html. To determine functional relationships between genes, we used DAVID Bioinformatics Resources http://david.niaid.nih.gov.For each cell line , total RNA was extracted from four independent cultures with Trizol reagent  according to the manufacturer' instructions and used for expression analysis on a 25K human oligonucleotide microarray covering most of the known human transcripts. The 50 mers 5'-amino modified oligonucleotides from the RNG/MRC oligonucleotide collection  . Their sequences are reported in the Additional File ® system (Roche Diagnostics) using the LightCycler® FastStart DNA master SYBR Green I kit (Roche Diagnostics) according to the manufacturer's instructions. Cycles were as follows: a 10 min initial cycle at 95°C, followed by 45 cycles of 10 sec of denaturation at 95°C, 5 sec of annealing at 58°C, and 10 sec of extension at 72°C. The specificity of the fluorescence was verified by the melting curve analysis after each reaction. The relative abundance of each target was normalized to 36B4 expression and the quantification of each mRNA compared to 36B4 was done using the comparative threshold method (Ct).To validate the microarray data, we used RNAs previously used for microarray hybridization. Primers for 7 LP-1K or LP-1D1b cells in 30 μl Matrigel (BD Biosciences) were deposited after gentle laceration of the surface. Digital pictures were taken under a stereomicroscope (Nikon SMZ1500) at day 2, 4, 6 of tumor development. Twenty eggs were used for each condition.Fertilized chicken eggs  were handled as described previously . On embrs.c. into a first set of five nude mice. Eight weeks after injection, tumors were present at the site of inoculation in 4/5 mice for LP-1K, 5/5 mice for LP-1 Myc and 3/5 mice for LP-1D1b  and binding protein 2 . CDKN2B mRNA was decreased both in LP-1K and LP-1D1b cells. However, at the protein level, no major differences were seen between LP-1cl1 and LP-1D1b whereas p15INK4B disappeared totally in LP-1K cells , tumor necrosis factor receptor (TNFR), hepatocyte growth factor receptor (HGFR), interleukin-21 receptor (IL-21R) etc.) or signalization molecules belonging to the phosphoinositol-3 kinase (PI3K)/AKT, Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB could be altered in LP-1 derived cells Table . The STAThe large number of genes and pathways altered by cyclin D1b and/or cyclin K expression precludes a thorough analysis in this manuscript. We focused on two discrete functions of cyclins D-type identified by the microarray analysis and well-known as support for tumorigenic process: cell migration and angiogenesis.ITGB7 coding for integrin β7, recognized as a major promoter of MM cell proliferation trough interactions with stroma cells [Kip1 and its phosphorylation of a Ser10 residue [Kip1 in LP-1-derived cell clones  at the vicinity of the injection site or biweekly, chemical FGFR or VEGFR inhibitors, SSR and SAR respectively. As shown Figure LP-1 cells such as myeloma cell lines synthesize angiogenic factors such as vascular endothelial growth factor (VEGF) (data not shown). Cyclin D1b and/or cyclin K expression in LP-1 cells impacted on proangiogenic and antiangiogenic gene expression Table . Comparein vivo studies failed to reveal a strong oncogenic potential of the conventional cyclin D1, referred to cyclin D1a [in vitro [in vivo [CCND1 gene disrupting the phosphorylation at Thr286 and thereby leading to nuclear accumulation of cyclin D1 have been described in endometrial and esophageal carcinomas further reinforcing this notion [Kip1 and does not control cell migration [Cyclin D1 is overexpressed in a broad range of solid malignancies, expressed in lymphoid tumors such as MM and MCL and not in their normal counterparts. However, clin D1a ,12. By cin vitro -6 and to[in vivo ,8. Theses notion ,26. Howes notion ,18, retaigration . Here weIn vivo, cyclin D1 inhibits oxidative glycolysis, lipogenesis, and mitochondrial gene activity in the mammary epithelium [LDHA , GAPDH  and ALDOA  is decreased. These enzymes catalyze important energy-yielding steps in carbohydrate metabolism. The expression of genes coding for key enzymes involved in oxidative glycolysis such as pyruvate kinase , phosphoglycerate kinase 1 , enolase 1  in LP-1D1b cells; enolase 2  in LP-1K cells are down-regulated. This suggests a reduction of glycolysis in tumor cells and, therefore, such as in mammary tumor cells, a paradoxical role of cyclin D1 [The tumorigenic properties of cyclins D1b and K are not conferred by an exacerbated proliferation. LP-1D1b and LP-1K cells display the same proliferation properties and cyclin D1b or cyclin K expressions have no major impact on cell cycle regulation. Conversely, genes involved in metabolism, signal transduction, transport, transcriptional and translational regulations are profoundly altered by cyclin D1b and/or cyclin K. ithelium ,28. In byclin D1 . Indeed,yclin D1 . HoweverCEBPG (FC: +3.08), coding for a close related transcription factor C/EBPγ whose function in myeloma cells remains to be established. But it is tempting to speculate some functional redundancy between the two factors. Among the transcription factors altered by cyclin K and/or cyclin D1b expressions, besides MYC, MAF, ETS family members, well-known as major oncogenic actors in plasma cells [The roles of cyclin D1 in regulating signal transduction, transcription and translation and their relevance in the cellular transformation process are documented . Among tma cells , severalEIF4EBP1, FC: +3.35; EIF3EIP, FC: +2.79; EIF4A2, FC: +2.50; EIF3F, FC: +2.05; EIF1, FC: +2.02). By contrast, in LP-1K, EIF3A and EIF5 are downregulated . A more active translation likely explains the faster growth of LP-1D1b-derived tumors compared to LP-1K tumors.Eukaryotic initiation factors (eIFs) control translation at the limiting step of initiation and several of them have been recognized as major actors in transformation processes . In LP-1-/- mouse embryonic fibroblasts, cyclins D1a and b have unique properties with regard to cell migration [Kip1 and inhibits RhoA-induced ROCK kinase activity promoting cell migration while cyclin D1b fails to stabilize p27Kip1 and has no effect on cell migration. Our results confirm that cyclin D1b does not affect LP-1 cells migration. Although cyclin K resembles cyclin D1a in agreement with its known biological functions: binding to CDK4/6, phosphorylation of pRb; one prominent feature of its structure is the impairment of p27Kip1 binding [Kip1, the lack of migration capacity and an enhanced clonogenic potential in vitro. Experiments assessing the metastatic potential of LP-1-derived cells in vivo are ongoing.Clinical observations indicate that cyclin D1 overexpression in human cancers correlate with metastasis. In cyclin D1igration . Cyclin  binding . Accordinude mice after s.c. injection of LP-1-derived cells show the same different vascularization. Indeed, LP-1 MM cells overexpressing cyclin D1b markedly promote tumor angiogenesis. Cyclin D1 regulates vascular endothelial growth factor (VEGF) production and thereby, growth of vascular endothelial cells and tumor [FGFR3 in LP-1D1b cells. Activation of the fibroblast growth factor 3 (FGFR3) expressed by myeloma cells and its ligand FGF present in the mouse could sustain in vivo angiogenesis such as in the bone marrow milieu [IL6, FGF9 and FGFR3. It is tempting to speculate that the expression of FGFR3 triggers premalignant cells to enter a malignant state as observed in our model.Cyclin K/D1b-expressing cells, grafted onto the CAM of chicken embryo, generate within a few days tumors whose vascularization is significantly different. Tumors obtained in nd tumor . The inhw milieu . The expw milieu . This stCCND1, several genes have been recognized as major actors of MM tumorigenesis: CCND2, MAF, FGFR3, ITGB7 and CXCR3 [BCL2, BNIP3, FGFR3, MCL1, RAN and XBP1. BCL2 protein is the archetype of apoptosis regulatory molecules; it is an integral outer mitochondrial membrane protein that blocks the apoptotic death. BCL2 is often overexpressed in transformed cells of the B lymphoid lineage, in malignant compared to normal plasma cells [BNIP3 gene is repressed in MM cells through the methylation of its promoter [MCL1 encodes two proteins belonging to the BCL2 family with either pro- or antiapoptotic functions; its overexpression has been detected in blood sample from a myeloma patient but not in his twin [FGFR3 gene and, in turn, the constitutive activation of several signaling pathways in 80% of t+ MM patients. Five genes  are altered by cyclin D1b alone and six  by both cyclins D1b and K. Those findings question the relevance of cyclin D1b expression in MM pathogenesis.Besides nd CXCR3 . All of nd CXCR3 -42. Sevema cells . By conthis twin . The rolCCND1 alternative splicing was regulated by a G/A polymorphism at the exon 4/intron 4 boundary [We have previously shown that both isoforms of cyclin D1a and b mRNAs are present in MM cells and their relative levels similar. However, cyclin D1a isoform is predominant both in MM cell lines and primary cells . It has boundary ,44. Thisboundary ,46. TheyThe authors declare that they have no competing interests.in vivo experiments; VM performed Q-PCR and IHC assays, GT performed western blots, confocal microscopy and flow cytometry; J-ML and JW-B performed CAM assays; DD and BJ performed microarray analyses and validated statistically the results; GA made computational analyses; VM, J-MR, JW-B, DD and BJ critically revised the manuscript; BS designed experiments, analyzed and interpreted the data, drafted the manuscript. All authors approved the final version of the manuscript and its submission for publication.VM and J-MR performed Table S1. Sequences of primers used for validation of microarray data by Q-PCR.Click here for fileFigure S1. Hierarchical clustering of cyclin K-altered genes. Clustering was visualized with TigrMev 4_03 software http://www.tm4.org/mev.html. Sequences with FC>3 were selected. Expression levels are shown for either upregulated genes (red) or downregulated genes (green). The name of probes is indicated in the treeview.Click here for fileFigure S2. Hierarchical clustering of cyclin D1b-altered genes. See legend of Additional File Click here for fileFigure S3. Hierarchical clustering of cyclin K- and D1b-altered genes. See legend of Additional File Click here for file


Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic. H5N1 influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. H5N1 influenza vaccines are now under development but none are yet available for human use http://www.who.int/csr/disease/swineflu) based on the rapid worldwide spread of the novel swine-origin pandemic influenza A (H1N1) 2009 virus (H1N1/09). As vaccine manufacturers scramble to produce new H1N1 vaccines for the upcoming influenza season, the limited global supply of the vaccine will require both prioritizing target groups for vaccination and exploring other interventions that can help reduce H1N1/09 virus transmission and disease severity, including the administration of antiviral agents (http://h1n1.nejm.org/). Passive immunization (the transfer of specific immunoglobulins/antibodies (Abs) to a previously non-immune recipient host) could offer an alternative strategy to prevent and treat influenza virus infection. Even after targeted vaccines become available, passive immunization could still have prophylactic effects and provides an additional countermeasure against influenza Highly pathogenic avian influenza A virus (HPAIV) of the H5N1 subtype that has emerged since 2004, resulted in more than 430 cases of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% (Pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (CF) A number of attempts have been made to develop human monoclonal Abs (mAbs) against H5N1 influenza haemagglutinin (HA) using Epstein-Barr virus (EBV) immortalization of B cells isolated from patients infected with H5N1 www.fao.org/docs/eims/upload/214194/rushton-comp.pdf). Therefore, many countries including China, Indonesia, Thailand, and Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines that controls the H5N1 epidemic to some extent The recent epidemic of HPAIV H5N1 virus has resulted in serious economic losses to the poultry industry, mostly in Southeast Asia (d) mice were purchased at 6 to 8 weeks of age from Charles River Co.  or the Jackson Laboratory . All mice were maintained in specific pathogen–free barrier facilities. All experiments and animal procedures conformed to protocols approved by the Institutional Animal Care and Use Committees of Seoul National University, Yonsei University, Konkuk University, Seoul, Korea, and the United States Centers for Diseases Control and Prevention (US CDC), Atlanta, GA., USA. Hy-Line Leghorn hens purchased from Kyunggi Poultry Farm were housed in an animal facility at Konkuk University. All the hens were kept in rooms lightened for 16 h per day with constant temperature of 25°C.Female wild-type (WT) BALB/cAnNCrl (H-2Madin-Darby canine kidney (MDCK) cells  were maintained in standard complete Dulbecco's modified Eagle's medium (D-MEM)  containing 5% fetal bovine serum (FBS) and antibiotics.50 of the H5N2 virus was determined in mice for challenge experiment. Avian H5N1 viruses were propagated in the allantoic cavity of 10-day-old embryonated hen's eggs at 37°C for 24 h to 30 h. The H5N1 human influenza isolate was incubated for an additional 10 h to 18 h. Allantoic fluid was pooled from multiple eggs, clarified by centrifugation, and frozen at −70°C until use. All experiments with HPAI (VN1203) virus were conducted under Biosafety Level 3 containment, including enhancements (BSL3+) required by the U.S. Department of Agriculture and the Select Agent Influenza virus strains, A/PR/8/34 (H1N1) (PR8) and A/Philippines/2/82/X-79 (H3N2) (A/Philippines) were prepared as previously reported Eggs laid by hens raised in the poultry unit of Konkuk University, Seoul, Korea and purchased from randomly selected supermarkets in Hanoi, Vietnam and Seoul, Korea and farms in Vietnam were used in experiments.Twenty-five-week-old domestic Leghorn hens were immunized intramuscularly with heat-inactivated A/PR/8/34 (H1N1) mixed with Freund's adjuvant (FA) . 5 µg of antigen was suspended in 250 µl of phosphate-buffered saline (PBS) and emulsified with an equal volume of complete FA. Incomplete FA was used for boosting immunizations. The hens were immunized three times with two weeks between the immunizations. The hen sera were collected eight weeks after the initial immunization, and eggs laid after last immunization were collected continuously. In some cases, immunized hens were boosted within a 3–4 months' interval to keep in hyperimmunized condition for a longer time period.g for 15 min at 4°C). Solid ammonium sulfate was added to reach 60% saturation (390 g/l) and the mixture was stirred in the cold for 15 min. Precipitate was collected by centrifugation and washed once with 60% saturated ammonium sulfate (SAS). The protein precipitate was dissolved in PBS and dialyzed three times against at least 10 volumes of PBS. Dialyzed IgY was adjusted to the original egg yolk volume  pasteurized at 60°C for 30 minutes and stored at 4°C. The purity of the IgY preparations was determined by sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining and is consistent with that obtained by others A rapid and simple water dilution method for extraction of IgY from egg yolk was adapted from the work by Akita and Nakai 50) titers were determined by inoculating groups of eight mice intranasally with serial 10-fold dilutions of virus as previously described 50) of A/PR/8/34 (H1N1) virus, 1,000 pfu (5× LD50) of A/Philippines (H3N2), 10× LD50 of VN/1203 (H5N1) or 5× LD50 A/Aquatic bird/Korea/W81/2005 (H5N2) resuspended in 50 µl PBS per animal. Ketamine-anesthetized mice were treated intranasally with 50 µl of IgY before or after infection. Death was defined when animals presented with more than a 25% weight loss, which required euthanasia and used as the endpoint in these studies.Fifty percent lethal dose (LD50) was determined by serial titration of virus stock in eggs, and EID50/ml values were calculated according to the method of Reed and Muench 50) was performed on MDCK cells by detection of viral protein according to Rowe et al. 5/ml) were incubated in Dulbecco's modified Eagle's medium containing 5% FBS and 1× of antibiotic-antimycotic solution (GIBCO) (complete DMEM) in 96-well Nunclon™ Surface plates  for 3 h at 37°C with 5% CO2. Cells were washed with serum free medium before adding serial dilutions of virus-containing samples in 100 µl of DMEM containing 2.5 µg/ml of trypsin. Plates were incubated for 18 h at 37°C with 5% CO2 before fixation of the cells with cold 80% acetone for 10 min. Plates were then washed with PBS/Tween 20 before addition of anti-NP IgG antibody  diluted 1/4,000 in PBS containing 1% bovine serum albumin and incubated at room temperature for 1 h. Goat anti-mouse IgG horseradish peroxidase-conjugated antibody  was added for 1 h at room temperature. Reaction was developed by adding 100 µl of freshly prepared TMB  substrate  to each well, and the plates were incubated at room temperature for approximately 5 min. The reaction was stopped with 50 µl of 1 M sulfuric acid. The absorbance was measured at 450 nm (A450) with SPECTRAmax photometer . Wells having an absorbance reading greater than 3 standard deviations above the mean absorbance of wells containing only MDCK cells were scored positive for virus growth. The TCID50 of each stock virus was calculated by the method of Reed and Muench The 50% egg infectious dose  assays performed on MDCK cells following the procedure as previously described 50 of viruses in U-bottom 96-well microtiter plates at 37°C for 1 h. At the end of incubation, freshly prepared 1% chicken red blood cells (CRBC) were added, and plates were mixed by agitation, covered, and allowed to set for 1 h at room temperature. The HI titers were determined by the reciprocal of the last dilution which contained non-agglutinated CRBC. Positive and negative control samples were included on each plate.Samples were treated with receptor destroying enzyme II  at a final dilution of 1∶3 before being tested in HI assay. Two-fold serially diluted samples were incubated with equal volume containing 100 TCIDTM Nunc-Immuno plates  were coated overnight with purified IgY  at a concentration of 10 µg/ml. Dilutions of serum were incubated 2 h on coated and blocked ELISA plates. Bound immunoglobulins were detected with goat anti-mouse Ig (H+L) horseradish peroxidase-conjugated antibody  At the end of the incubation (2 h at 37°C), TMB substrate was added and the reaction was stopped with an equal volume of 1 M sulfuric acid. The color developed was measured in a SPECTRAmax photometer at 450 nm. The reproducibility of the assay was ascertained by applying a control hyperimmune mouse serum on each plate. Assay results were expressed as end-point titration values which are determined by the last dilutions that are above cutoff for assay (OD 450 nm reaches plateau).The standard ELISA was performed for detection of anti-IgY in the sera of IgY-immunized mice. 96-well MaxiSorpt-test or an unpaired Mann Whitney U test available in Microsoft Excel software .The data are expressed as the mean ± one standard error of the mean (SEM) and compared using a two-tailed student's We first tested the possibility that yolks from commercially available eggs in Vietnam, where mass vaccination of poultry against avian influenza H5N1 is mandatory, contain H5N1-speficific IgY. We determined H5-specific HI titers in the sera and yolks of the eggs obtained from a farm in Vietnam that was participating in a national mass vaccination program. IgY preparation was restored in PBS to the original volume of yolk. Indeed, H5-specific HI titers determined in yolks were comparable to those seen in sera of vaccinated hens . Next, wWe first used a mouse-adapted, low pathogenic avian influenza A virus (LPAIV) strain A/Aquatic bird/Korea/W81/2005 (H5N2), which shares 94.4% nucleotide sequence homology with HA (H5) but has different NA (N2) from the one used for mass immunization in Vietnam  for challenge experiments in BALB/c mice. As shown in Based on these results, we further examined whether protection against infection with HPAIV H5N1 strain, A/Vietnam/1203/2004, which was isolated from a fatal case, could be achieved. Animals treated intranasally with H5N1-specific IgY before infection displayed mild weight loss and recovered completely by the end of the first week after inoculation . Of noteWe further examined the protective effect of IgY prepared from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza A H1N1 PR8 virus. PR8 virus is a common laboratory mouse-adapted influenza strain that serves as a model virus for current pandemic H1N1/09 virus and can be handled safely under Biosafety Level 2 (BSL2) conditions. We found substantial levels of haemagglutination inhibition (HI) and virus neutralization (VN) Abs in the sera and yolks derived from immunized hens . When naAlthough raw eggs are consumed widely in many countries and uncooked egg components are used in the preparation of many foods, reports on the presence of anti-IgY Abs in humans have been limited to only two studies. One study demonstrated the presence of anti-IgY Abs in sera obtained from normal individuals We next asked whether pre-existing anti-IgY Abs prevent virus-specific IgY-mediated protection. We immunized mice with normal IgY or immune IgY specific for particular subtypes. Three weeks later, serum Abs specific for IgY were determined by ELISA. One hundred percent of the immunized mice generated anti-IgY Abs at the level comparable to that of IgY-treated mice . Such IgEscherichia coli IgY reduced mortality in newborn piglets Streptococcus mutans decreased caries when used as a mouth rinse Helicobacter pylori IgY reduced Helicobacter infections Pseudomonas aeuruginosaPseudomonas aeuruginosa in cystic fibrosis (CF) patients http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm094047.htm) that contains egg components could be a proof for safe intranasal delivery of IgY.Several animal studies and a number of human studies of IgY against different pathogens have demonstrated preventive and prophylactic effects of IgY. Rotavirus-specific IgY provided protection against infection with bovine rotavirus both in calves and in mice, whereas anti-The efficacy of IgY seems to be specific for infection with viruses of the same HA type, since H5N1-specific IgY provided protection against infections with HPAIV H5N1 and H5N2 strains but failed to protect against infection with H1N1 PR8 virus that shares the same type of NA but not HA.P. aeuruginosa infection in the lungs of CF patients requires long-term repeated applications of anti-bacterial IgY Although IgY treatment induced significant Ab response towards IgY, the anti-IgY, however did not interfere with the protective effectiveness of virus-specific IgY. The findings suggest that IgY treatment could be applied to persons who have developed anti-IgY Abs and that such a treatment strategy could be repeated if multiple treatment is required and necessary to protect infections against other pathogens later on. Indeed, the current protocol for treatment of The approach of using specific IgY for prevention and therapy of influenza virus infection offers an alternative to current immunotherapy, which uses HPAIV H5N1 convalescent plasma IgY are relatively stable. We found no change in protective activity after at least 13 months' storage at 4°C, and lyophilization does not affect activity, making production of IgY practical. The use of IgY immunotherapy has many advantages since IgY does not activate the human complement system or human Fc-receptors, which all are well-known cell activators and mediators of inflammation Thus, we show that consumable eggs available in the markets of countries that impose mandatory H5N1 mass vaccination of poultry, offer an enormous source of valuable, affordable, and safe virus-specific IgY, which can be used for prevention and protection against potential H5N1 pandemic influenza. Our study provides a proof-of-concept for use of influenza virus-specific IgY in passive immunization against influenza outbreaks, including current H1N1 pandemic.


Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 . Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes  is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119. Alphavirus genus in the family Togaviridae and is maintained in a natural enzootic cycle between mosquitoes and rodent hosts, although equines and humans may also be infected in an epizootic cycle Venezuelan equine encephalitis virus (VEEV) is a member of the VEEV has potential as a bioweapon, principally because of its low human infective dose, easy production, and capability for effective transmission by aerosolization Alphaviruses have a positive-sense, single-stranded RNA genome of approximately 11.45 kb enclosed within an icosahedral nucleocapsid surrounded by a lipid bilayer derived from the infected cell's plasma membrane. Two integral membrane glycoproteins, E1 and E2, are embedded in the lipid envelope and are assembled as heterodimers into 80 trimeric spikes on the virus surface c,d,e,f,g,h) involved in VEEV neutralization c and E2h are the most conserved on the E2 glycoproteins of heterologous VEEVs We have previously analyzed the antigenic structure of both the VEEV E1 and E2 glycoproteins using murine (m) monoclonal antibodies (MAbs) and defined six E2 epitopes  exhibited potent neutralizing activity, were converted to fully human IgG1 molecules. Two F5 hMAb neutralization-escape VEEV variants were isolated. Sequencing of the E1 and E2 protein genes of these variant viruses identified a unique human VEEV neutralization domain as the likely binding site of the F5 hMAb. Alphavirus cryoEM maps, with associated markers, support proposing a probable surface-accessible location on VEEV E2 for the F5 native (n) IgG binding site.Fully human (h) MAbs would be the best choice for clinical treatment of human infections; however, little is known about the immunologic specificities of the human antibody repertoire to VEEV, and no protective hMAbs have yet been isolated, characterized, or implemented. In this report we have presented a map of the human VEEV antibody response and determined the human immunodominant epitopes on the VEEV E1 and E2 proteins. We used phage-display technology to isolate VEEV-specific hFabs from bone marrow donors known to have circulating antibodies for VEEV The VEE complex viruses used in this study were Trinidad donkey , vaccine strain TC-83 (1-AB), P676 (1-C), 3880 (1-D), Mena II (1-E), Everglades (EVE)(2), Mucambo (MUC) (3-A), Pixuna (PIX) (4), Cabassou (CAB) (5), and AG80-663 (Rio Negro) (6), which were obtained from the Division of Vector-Borne Infectious Diseases (DVBID), Centers for Disease Control (CDC), Fort Collins, CO. Viruses grown in Vero cells were purified by equilibrium density-gradient centrifugation Total RNA was obtained from bone marrow and blood samples supplied by two military donors  using Tri-reagent BD  according to the manufacturer's instructions. Blood utilized in this project was obtained under protocol 01-124 approved by Scripps Clinic, IRB#00001283, Assurance Number FWA00000467. Bone marrow samples were obtained from a commercial source. Donor sera ELISA titers to VEEV TC-83 were 1∶500–3000.Messenger RNA was isolated using Oligotex spin columns  following the manufacturer's protocol. To amplify mRNA, first strand cDNA was synthesized using SuperScript II reverse transcriptase  according to the manufacturer's protocol. Second strand cDNA was synthesized according to the method fully described in the patent WO2005/060641A2 E. coli cells, strain ER 2738, and amplified by adding helper phage, strain VCSM13, to the infected bacteria for each round of panning. Individual colonies were produced by plating infected bacteria.Fab-bearing phage from all libraries were panned through one to four rounds of enrichment against inactivated VEEV TC-83 coated in 96-well plates overnight at 4°C. Wells were washed with water and blocked 1 h at 37°C with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The phage library was added to the wells and incubated at 37°C for 2 h. Wells were washed 10 times with PBS containing 0.05% Tween-20 (PBS-T). The bound Fab-phage were eluted with 2M glycine, pH 2.2, neutralized with 2M Tris base, used to infect log phase Screening by ELISA on inactivated VEEV TC-83 virus was performed in high throughput mode using a Tecan robot  for large numbers of colonies (>1000) picked using a Q-pix instrument . Individual colonies were grown overnight in deep-well microtiter dishes in a Hi-Gro high-speed incubator-shaker . Aliquots were removed and stored as stocks containing 15% glycerol or 10% DMSO. After centrifugation of the deep-well dishes, Fab-containing supernatants were collected for ELISA screening. Alkaline phosphatase (AP)-labeled goat anti-human Fab  was used to detect expressed Fab bound to antigen. Miniprep DNA (Qiagen) from positive colonies was sequenced across the light and heavy chains using standard primers for the phagemid expression vectors. Sequences were analyzed using DNAstar software  to identify and classify unique candidates. For soluble Fab expression and purification, the gene III fusion region of the phagemid was removed from positive, unique candidates by subcloning. At this stage it was possible to insert an oligonucleotide that encoded a combination epitope tag consisting of an influenza virus hemagglutinin (HA tag) and 6 histidine residues (His tag) for detection and purification with anti-HA and/or Ni-NTA Panning and screening of additional libraries were done on inactivated VEEV TC-83, either with or without epitope masking by non-neutralizing hMAbs F2 engineered (e) IgG and H6 eIgG to increase the chances of isolating Fabs with neutralizing capability. For panning with epitope masking, wells coated with VEEV TC-83 were incubated with F2 eIgG (50 µg/ml) and H6 eIgG (10 µg/ml) in 1% BSA-PBS at 37°C for 30 min followed by addition of library phage and incubation at 37°C for 1.5 h.E. coli cells with induction by isopropyl-β-D-thiogalactopyranoside and purified on a goat anti-human IgG F(ab′)2 affinity column. Purified F5 Fab for use as a positive control in ELISA was prepared from F5 eIgG by papain digestion using an ImmunoPure Fab Preparation Kit (Pierce) according to the manufacturer's instructions.Fabs selected for characterization were cloned into a Fab expression vector, PAEV1, using Eco RI/Spe I restriction sites available in the vector . Two FabFour hFabs  were converted to full-length IgG1s in a two-step cloning process . The reg5 cells/ml) with 64 µg of MAb expression vector DNA using Effectene (Qiagen) according to the manufacturer's protocol. The culture supernatant was harvested 5–7 days after transfection and each MAb was purified on a protein A column using FPLC. Stable cell lines producing F5 nIgG were established by selecting transfected 293-EBNA cells with puromycin (10µg/ml). The transfected cells were initially grown in 24-well plates and medium was screened by ELISA using a goat anti-human IgG F(ab′)2 antibody-AP conjugate  for detection. The 2 wells with the highest antibody expression were chosen for single cell selection in four 96-well plates. Wells containing single-cell colonies were tested by ELISA and the best producers were chosen for expansion. One of these clones, IE9, was grown in 1.5 L and 6 L cultures that produced 25 mg and 50 mg MAb, respectively, after protein A column purification. The purified antibody showed specific binding to inactivated VEEV TC-83 that was comparable to that of F5 nIgG prepared from transient transfection of 293-EBNA cells.HMAbs were produced by transfecting 120 ml of 293-EBNA cells . An absorbance ratio (A405 test sample/A405 negative control) >2 was considered to be positive.Indirect ELISAs were used to verify VEEV binding by purified hFabs and to determine the cross-reactivity of selected hFabs and hMAbs to six VEEV subtypes and four varieties of subtype 1. ELISAs were performed essentially as previously described 2-specific] as a detector and Sigma 104 phosphatase substrate. Reactions were read at 405 nm. Dilutions of MAb conjugates that resulted in an A405 of approximately 1.0 were also determined for use in CBAs. For competitions using MAb-HRP conjugates, wells were coated with 1 µg VEEV TC-83 overnight and then blocked with 3% goat serum in PBS for 1 h at 37°C. MAb or Fab competitors were added at predetermined dilutions, serially diluted 1∶2 in PBS-T, and incubated at room temperature for 30 min. A previously determined constant dilution of MAb-HRP in blocking buffer was added to each serially-diluted competitor and incubated for 1 h at 37°C. One hundred µl premixed tetramethyl benzidine substrate  was added to each well, the reaction was stopped in 15–30 min with 50 µl 1N H2SO4, and plates were read at 450 nm.Competitive binding assays (CBAs) were used to determine the ability of F5 nIgG to block the binding of anti-VEEV neutralizing mMAb-horseradish peroxidase (HRP) conjugates, and, conversely, the ability of purified mMAbs or hFabs to block the binding of F5 nIgG-HRP to virus. A dilution of each unconjugated mMAb, hMAb, or hFab that resulted in approximately 70% binding to VEEV TC-83 was determined by titration in an indirect ELISA, using either AP-conjugated goat anti-mouse IgG (Fc-specific) or goat anti-human IgG , [3c epitope defined by F5 nIgG does not spatially overlap any of the other epitopes defined by the panel of hFabs  on the surface of the spike above the more cryptic locations of the E2 N-terminus (VE2pep01) and aa 241–265 (VE2pep13). Such an arrangement would be in agreement with the current knowledge of the structure of the E2 glycoprotein, the location of specific markers, and functional attributes of specified epitopes. We are now in collaboration to obtain structural data on Fab-virion complexes to determine actual binding sites of F5 nIgG and other human and murine MAbs.Although the crystal structure of the alphavirus E2 glycoprotein has not been solved, cryoEM reconstructions of E2 have been reported  epitope . Previouin vivo.The VEEV neutralizing ability of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. When administered prophylactically, as little as 100 ng of Hy4 was able to protect 90% of mice challenged intraperitoneally with virulent VEEV Figure S1Cloning of Fabs into the expression vector PAEV1.(A) Light and heavy chains of selected Fabs were inserted into vector PAEV1 at Eco RI/Spe I sites. (B) Fabs with light chains containing Eco RI sites were inserted into vector PAEV1 (containing the light and heavy chains of Fab L1A7) at Xba I/Age I sites.(0.06 MB PPT)Click here for additional data file.Figure S2Schematic presentation of conversion of Fabs to engineered IgG (eIgG). A fragment containing mammalian control elements was inserted into each Fab at Not I and Xho I sites in the PAX243 Fab vector. The Sfi I to Age I fragment containing the light chain, mammalian control elements, and the heavy chain was transferred to an IgG expression vector containing mammalian elements upstream of the light chain as well as IgG CH1, hinge, CH2, and CH3 regions downstream from the heavy chain. Final construct of eIgG retained the engineered Xba I, Sac I, and Xho I sites.(0.02 MB PPT)Click here for additional data file.Figure S3Conversion of engineered (e) F5 eIG to native (n) F5 nIgG. This conversion was done in a 3 step process: (1) Hind III in the mammalian control elements for the heavy chain was changed to Asc I by site-specific mutagenesis and overlap PCR. (2) Xba I and Sac I sites from the light chain and the Xho I site from the heavy chain were converted to native sequences by site-specific mutagenesis and overlap PCR. (3) The engineered Xho I site was changed to native sequence by site-specific mutation and overlap PCR.(0.07 MB PPT)Click here for additional data file.Figure S4MAbs binding to native Venezuelan equine encephalitis virus TC-83. Human MAbs: F5 nIgG ♦, H6 eIgG •, and G1eIgG ▴; murine MAb 3B4C-4 ▪.(0.07 MB PPT)Click here for additional data file.Figure S5Binding of purified Fabs to inactivated Venezuelan equine encephalitis virus TC-83. Solid lines are Fab binding to VEEV TC-83: KR2B12, black inverted triangles; KR2A3, pink diamonds; LR3H11, blue triangles; K1B11, magenta circles; KR2C2, black squares; K2E2, green circles; K1H3, orange diamonds; L1A7, cyan squares; and F5, red squares. The black circle/dashed line is a representative Fab (K1B11) binding to the ovalbumin negative antigen control.(0.07 MB PPT)Click here for additional data file.Table S1Primers used in reverse transcription PCR and sequencing reactions.(0.03 MB DOC)Click here for additional data file.Table S2ELISA binding of purified human and murine Venezuelan equine encephalitis virus (VEEV) MAbs to either native or 0.3% β-propiolactone (BPL)-treated VEEV TC-83.(0.03 MB DOC)Click here for additional data file.


RASSF10 is a predicted gene yet to be experimentally verified. Here we cloned, characterised and demonstrated expression of RASSF10 in normal human bone marrow. We also determined the methylation status of CpG islands associated with RASSF1–10 in a series of childhood acute lymphocytic leukaemias (ALL) and normal blood and bone marrow samples.The Ras-assocation family (RASSF) of tumour suppressor genes (TSGs) contains 10 members that encode proteins containing Ras-assocation (RA) domains. Several members of the RASSF family are frequently epigenetically inactivated in cancer, however, their role in leukaemia has remained largely uninvestigated. Also, RASSF6 and RASSF10 were the only RASSF members with a high frequency of leukaemia-specific methylation. RASSF6 was methylated in 94% (48/51) B-ALL and 41% (12/29) T-ALL, whilst RASSF10 was methylated in 16% (8/51) B-ALL and 88% (23/26) T-ALL. RASSF6 and RASSF10 expression inversely correlated with methylation which was restored by treatment with 5-aza-2'deoxycytidine (5azaDC).COBRA and bisulphite sequencing revealed RASSF6 or RASSF10 in cancer. These data show epigenetic inactivation of the candidate TSGs RASSF6 and RASSF10 is an extremely frequent event in the pathogenesis of childhood leukaemia. This study also warrants further investigation of the newly identified RASSF member RASSF10 and its potential role in leukaemia.This study shows the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and represents the first report of inactivation of  RASSF1–6, that contain C-terminal RA  and Sav/RASSF/Hippo (SARAH) protein interaction domains. Previously we and others had demonstrated that several RASSF members, namely RASSF1A, RASSF2, RASSF4 and RASSF5A are frequently epigenetically inactivated by promoter region CpG island hypermethylation in a broad range of solid tumour types , a RASSF9 homologue [GenBank:CG13875] and a RASSF10 homologue [GenBank:CG32150] in Drosophila melanogaster distinct from the RASSF1–6 Drosophila homologue [GenBank:CG4656-PA] [Mutation of the Ras superfamily of proteins, or of upstream and downstream signalling components, plays a significant role in the pathogenesis of most forms of cancer. Ras proteins interact with a wide variety of downstream effectors to regulate several signalling pathways important for normal cellular growth . Howeverur types . These pur types . Very reRASSF6 and RASSF10 in B-ALL and T-ALL respectively. These findings support the candidacy of RASSF6 and RASSF10 as TSGs involved in the pathogenesis of childhood leukaemias.To date the role of the RASSF family in childhood leukaemia has remained uninvestigated and inactivation of the 'N-terminal' RASSF members has not been investigated in any cancer. In this report we describe investigation of the RASSF family for inactivation by promoter CpG island hypermethylation in a large series of childhood B- and T-ALL and find very frequent promoter hypermethylation and loss of expression of RASSF1–6, in a large series of childhood ALL to ascertain whether epigenetic inactivation of these genes occurs in childhood leukaemia. Surprisingly, we found that RASSF1–5 were infrequently methylated whilst RASSF6 was methylated in B-ALL , T-ALL  and 5/5 unclassified childhood leukaemias but not in normal blood or bone marrow control samples  reactivated RASSF6 expression . Analysis of the sequence flanking this alternative ATG (GCCATGG) support its candidacy as a genuine translation initiation site since it matches the consensus 'Kozak' sequence of A/GCCATGG in which the important -3 and +4 A/G and G residues respectively (underlined) were conserved [ also predicted the ATG at Chr11:12,987,700 is the translation initiation site within the RASSF10 gene. This suggested the RASSF10 gene contains a shorter open reading frame encoding a 507 amino acid protein. However, since the IMAGE clone may be incomplete we confirmed the transcription start site of the RASSF10 gene using 5'RACE. We first demonstrated expression of the RASSF10 transcript in DNaseI-treated normal human bone marrow RNA using RT-PCR  T-ALL and 8/51 (16%) B-ALL but was unmethylated in normal bone marrow and 21 normal blood samples  and loss of RASSF10 expression in SUP-T1 (completely methylated) and in both cases expression was upregulated following 5azaDC and TSA treatment . RASSF8 methylation was found to be infrequent, occurring in 2/6 leukaemia cell lines, 2/19 (10%) childhood T-ALLs, and 4/46 (9%) B-ALL with no methylation detectable in normal bone marrow or normal blood (0/7). Methylation of the RASSF8 CpG island correlated with loss of RASSF8 expression which was restored following treatment with 5azaDC  provide further evidence of expression in brain, skeletal muscle, pancreas, liver and trachea. Interestingly, we demonstrate that current genome browser predictions of the RASSF10 gene and protein may require revision and provide evidence of alternative transcription and translation inititation sites that produce a much shorter protein of 507 amino acids. We also found RASSF10 was very frequently inactivated in T-ALL (88%). RASSF10 CpG island methylation correlated with loss of RASSF10 expression which was restored following 5azaDC and TSA treatment. It is particularly interesting that RASSF6 methylation occurs very frequently in B-ALL whilst RASSF10 methylation is more restricted to T-ALL. It is unclear at present whether this represents independent routes to inactivation of the same pathway or whether it reflects inactivation of separate pathways important in the development of these different subtypes of leukaemia. Nevertheless, inactivation of RASSF6 or RASSF10 by promoter DNA hypermethylation appears to be an event associated with the majority of childhood leukaemias.ignaling . OrtholoRASSF6 is also inactivated in cancer, but specifically in leukaemias. Given the emerging role of RASSF6 as a Ras effector and possible TSG involved in the regulation of apoptosis it is likely that loss of expression may be involved in the ability of leukaemic cells to evade apoptosis. Our data also suggests that of the newly identified 'N-terminal' RASSF genes, RASSF10 is an interesting candidate for further analysis in other types of cancer and for functional investigation to ascertain its role in leukaemias.In summary, we show that the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and show for the first time that Twelve leukaemia cell lines , U937, Jurkat (JKT), TALL-1, NALM1, NALM6, NALM16, NALM17, THP-1, SUP-T1 and MOLT-4) and 89 primary childhood ALL comprising 51 B-cell ALL (B-ALL), 32 T-cell ALL (T-ALL), 1 pre-B-ALL and 5 unclassified leukaemias were analysed. In addition a total of 21 normal blood samples and 1 normal bone marrow  sample were used as controls. All DNA samples were obtained with informed consent from patients and family members.RASSF1A and RASSF2 were determined using MSP as described previously [RASSF3, RASSF4 and RASSF5A were determined by combined bisulphite restriction analysis (COBRA) as described in Hesson et al., [et al., [et al., [RASSF6, primers were designed to amplify the entire CpG island encompassing the first exons of RASSF6A (NM_177532) and RASSF6B (NM_201431) from bisulphite modified DNA using semi-nested bisulphite PCR  for 2 h at 65°C or 2 U BstUI restriction enzyme (CGCG) for 2 h at 60°C before visualisation on a 2% agarose gel. Samples selected for bisulphite sequencing were cloned (RASSF6) into the CloneJET vector (Fermentas) according to manufacturers instructions or sequenced directly (RASSF8 and RASSF10). Up to 12 individual colonies were chosen for colony PCR using the RASSF6 COBRA primers. Products were then sequenced to ascertain the methylation status of individual alleles and to determine the methylation index (MI). The MI was calculated as a percentage using the equation; number of CpG dinucleotides methylated/total number of CpG dinucleotides sequenced × 100.Bisulphite modification was performed as described previously . Promoteeviously . The met et al., , Hesson [et al.,  and Eckf[et al., . For RAS2. Cells were treated with 5 μM of the DNA demethylating agent 5azaDC (Sigma) freshly prepared in ddH2O and filter-sterilised. The medium (including 5 μM 5azaDC) was changed every day for 5 days. Cells were also treated on day 4 with 0.1 μM TSA for 24 hrs. RNA was prepared using RNA bee (AMS biotechnology) according to manufacturers' instructions. cDNA was generated from 1 μg total RNA using SuperScript III (Invitrogen) and polyN primers. We analysed expression of RASSF6 using primers specific for exons 2 and 4 that amplify both the A and B isoforms (F = 5'-ATGATGGCTCACCAGTACCC-3' and R = 5'-GGTCGTTTTACTCCCCAGAA-3'). We analysed expression of RASSF10 using the primers 5'-CCATGACCCAGGAGAAACAG-3' (F) and 5'-TGCTGGCGAATTGTGTGGTC-3' (R). Since RASSF10 is a single exon gene we first DNaseI (Fermentas) treated all RNA samples prior to cDNA synthesis and included control experiments in which reverse transcriptase was omitted (No RT controls) in all samples analysed. We analysed expression of RASSF8 using the primers F = 5'-TCCATTGAGAAACAGCTGGA-3' and R = 5'-TGGCACAAATCAAAAAGGAA-3'. In all cases a GAPDH control was included using conditions described previously [RASSF6, RASSF8 and RASSF10 were amplified from 50 ng cDNA using 0.8 μM of each primer, 3 mM MgCl2, 0.25 mM dNTPs and 1 U Fast start Taq (Roche).Leukaemia cell lines were maintained in RPMI1640 (Sigma) supplemented with 10% FCS, 2 mM Glutamine, 20 mM HEPES, 1 mM Sodium Pyruvate and 12.6 mM Glucose Monohydrate at 37°C, 5% COLeukaemia lines THP-1 and CEM were treated with 5azaDC as above. Protein extracts were then prepared by lysis in 50 mM TrisHCl pH7.5, 1 mM EDTA, 1 mM EGTA, 50 mM Sodium Fluoride, 5 mM Sodium Pyrophosphate, 1 mM Sodium Orthovanadate, 0.27 M Sucrose, 1% Triton and protease inhibitor cocktail (Roche). Twenty micrograms of protein extracts were resolved by SDS-PAGE, transferred to PVDF membrane and probed for the presence of RASSF6 using 1 μg/mL anti-RASSF6 rabbit-raised polyclonal antibody (ProteinTech Group).DNaseI (Fermentas) treated according to manufacturers instructions. We synthesised cDNA using a 5'/3' RACE kit  and the RASSF10 antisense primer 5'-TGCTGGCGAATTGTGTGGTC-3'. The 5' UTR of the RASSF10 transcript was obtained using nested PCR with the nested antisense RASSF10-specific primers 5'-GCTGTTTCTCCTGGGTCATG-3' and 5'-TATCTTCTTTTCCGAAGGATCCATG-3' used in combination with a polyT-anchor primer. Control experiments in which reverse transcriptase was omitted were also performed to eliminate DNA contamination. PCR products were gel extracted, sequenced and the sequences obtained aligned against the human genome browser.One microgram of total RNA from bone marrow (AMS Biotechnology) was PCR primers were used to amplify exons 2 and 3 of the K- and N-Ras genes from genomic DNA. The primers were as follows: K-Ras exon 2 F 5'-TTTGTATTAAAAGGTACTGGTGGAG-3'; K-Ras exon 2 R 5'-CCTTTATCTGTATCAAAGAATGGTC-3'; K-Ras exon 3 F 5'-CTGTGTTTCTCCCTTCTCAGG-3'; K-Ras exon 3 R 5'-AGAAAGCCCTCCCCAGTCCT-3'; N-Ras exon 2 F 5'-GATGTGGCTCGCCAATTAAC-3'; N-Ras exon 2 R 5'-GAATATGGGTAAAGATGATCCGAC-3'; N-Ras exon 3 F 5'-GTTAGATGCTTATTTAACCTTGGC-3'; N-Ras exon 3 R 5'-TGTGGTAACCTCATTTCCCC-3'. PCR products were directly sequenced using ABI BigDye cycle sequencing kit (Perkin-Elmer).RASSF: Ras-association family; TSG: tumour suppressor gene; RA: Ras-association domain; ALL: Acute Lymphocytic Leukaemia; 5azaDC: 5-aza-2'deoxycytidine; TSA: Trichostatin A; SARAH: Sav/RASSF/Hpo domain; MSP: Methylation-specific PCR; COBRA: Combined Bisulphite Restriction Analysis; RACE: Rapid Amplification of cDNA Ends.The authors declare that they have no competing interests.LBH acquired the majority of data , wrote the manuscript and contributed to the design and concept of the study. TLD performed bisulphite modification and contributed to COBRA analysis and mutation screening. WNC performed COBRA, bisulphite sequencing and expression analysis pertaining to RASSF8. DC, MG, RC and ATB provided leukaemia DNA samples and cell lines. ADC and ERM contributed to the concept of the study. FL conceived the studies, oversaw the experimental work and helped draft the manuscript and established all the collaborations. All authors have read and approved the final manuscript.RASSF methylation profile. The data provided represent analysis of RASSF members and additional data relating to RASSF6 and RASSF10 in childhood leukaemia. Figure S1: RASSF methylation profile of leukaemia cell lines. The methylation status of RASSF1A and RASSF2 was determined using MSP whereas the methylation status of RASSF3-6 were determined using COBRA. RASSF1A, RASSF5A and RASSF6 were hypermethylated in leukaemia cell lines whereas RASSF2 and RASSF3 showed no evidence of methylation. RASSF4 was methylated in both leukaemia cell lines and normal blood. For MSP assays M = methylated specific PCR; U = unmethylated specific PCR. For COBRA assays U = undigested PCR product; T = TaqI digested PCR product. Figure S2: RASSF methylation profile of primary leukaemias. RASSF genes showing evidence of hypermethylation in leukaemia cell lines were investigated for methylation in B-ALL and T-ALL leukaemias. RASSF1A and RASSF5A were infrequently methylated in T-ALL (2/12 and 2/24 respectively). RASSF4 was methylated in 25/25 T-ALL but in only 7/50 B-ALL leukaemias, however given the low level methylation of RASSF4 observed in normal blood this gene was not investigated further. RASSF6 was methylated in 48/51 (94%) B-ALL and 12/29 (41%) T-ALL but not in normal bone marrow (BM) or blood control samples. For MSP assays M = methylated specific PCR; U = unmethylated specific PCR. For COBRA assays U = undigested PCR product; T = TaqI digested PCR product. RASSF6 CpG islandFigure S3: Bisulphite sequencing of the . As in figure RASSF6 expression in normal tissues and leukaemia cell linesFigure S4: . A, RASSF6 mRNA was detected in all tissues analysed. B, Restoration of RASSF6 mRNA expression in methylated leukaemia cell lines following treatment with 5azaDC and TSA showing the additional cell lines JKT and REH. GAPDH was used as a control for equal loading. RASSF8 CpG island infrequently inactivates RASSF8 in leukaemiasFigure S5: Methylation of the . A, Direct bisulphite sequencing of RASSF8 CpG island PCR products. Filled black circle represents a completely methylated CpG dinucleotide, open circle represents a completely unmethylated CpG dinucleotide and filled gray circle represents a partially methylated CpG dinucleotide. B, Methylation of the RASSF8 CpG island correlates with loss or downregulation of RASSF8 expression which is restored following 5azaDC treatment.Click here for fileRASSF6 methylation indexes and PCR primers used within this study. The data provided summarise RASSF6 methylation index data in cell lines, primary tumour and control samples (Table S1). PCR primers used in this study are also listed (Table S2). Table S1: A summary of the methylation index of the RASSF6 CpG island in leukaemia cell lines, B-ALL, T-ALL and normal blood and bone marrow control samples. Table S2: MSP and COBRA primer sequences used within this study.Click here for file


Aims: To compare two progressive approaches [once-daily insulin glargine plus ≤3 mealtime lispro (G+L) vs. insulin lispro mix 50/50 (LM50/50) progression once up to thrice daily ] of beginning and advancing insulin in patients with type 2 diabetes (T2D) and inadequate glycaemic control on oral therapy, with the aim of showing non-inferiority of PP to G+L.Methods: Patients were randomized to PP (n = 242) or G+L (n = 242) in a 36-week, multinational, open-label trial. Dinnertime insulin LM 50/50 could be replaced with insulin lispro mix 75/25 if needed for fasting glycaemic control.Results: Baseline haemoglobin A1c (HbA1c) were 9.5% (PP) and 9.3% (G+L); p = 0.095. Change in A1C (baseline to endpoint) was −1.76% (PP) and −1.93% (G+L) (p = 0.097) [between-group difference of 0.17 ]. Non-inferiority of PP to G+L was not shown based on the prespecified non-inferiority margin of 0.3%. A1C was lower with G+L at weeks 12 , 24 , but not at week 36 . There were no significant differences in percentages of patients achieving A1C ≤7%, overall hypoglycaemia incidence and rate or weight change. Total daily insulin dosages at endpoint were higher with PP vs. G+L , likely due to more injections .Conclusions: Both treatments progressively improved glycaemic control in patients with T2D on oral therapy, although non-inferiority of PP to G+L was not shown. Higher insulin doses were observed with PP with no between-treatment differences in overall hypoglycaemia or weight gain. Intensive glycaemic control can delay the onset and slow the progression of diabetes-related complications in patients with type 2 diabetes (T2D) ,2. The rRecent studies support the clinical utility of initiating insulin therapy early in patients with T2D by adding a single injection of basal insulin to an existing oral regimen in order to achieve and maintain target hemoglobin A1C (HbA1c) levels ≤7% –8. As onPremixed formulations that contain both basal and prandial insulin could also be used in starting or progressing insulin therapy ,11 but nIn this study, two progressive approaches to starting and intensifying insulin therapy are compared in patients with T2D and inadequate glycaemic control on oral therapy: premix insulin progression (PP) with once- then twice- or thrice-daily LM50/50 insulin administration vs. basal insulin glargine initiated once daily then supplemented with one to three prandial insulin (lispro) injections [insulin glargine plus insulin lispro (G+L)] as needed to meet glycaemic targets.2, were taking a thiazolidinedione dose greater than what was indicated in combination with insulin, or were taking glucose-lowering agents other than metformin, sulphonylurea or thiazolidinedione, had functional capacity class III/IV cardiac disease, impaired renal function, active liver disease, or serum alanine transaminase levels >2 times the upper limit of normal.This 36-week, randomized, open-label, active-controlled trial was conducted in nine countries  in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines. All patients provided written informed consent. Eligible patients were men and women, 30–80 years, with T2D, A1C 7.5–12.0% using ≥2 oral antihyperglycaemic medications (OAMs) for ≥90 days, insulin naïve, capable and willing to use insulin injection devices and self-monitoring of blood glucose (SMBG) levels. Patients were excluded if they had ≥1 episode of severe hypoglycaemia within the prior 6 months, body mass index (BMI) >40 kg/mPatients were randomized to treatment by country and stratified within country by sulphonylurea use and A1C (≤8.5 and >8.5%) through an interactive telephone system. Most patients started on either one 10 U injection of LM50/50 administered with the locally available insulin pen, mostly HumaPen® Luxura™ pen  within 15 min prior to the evening meal or one 10 U injection of insulin glargine (Lantus®) administered with the locally available insulin pen, mostly OptiPen® Pro 1 or Lantus® OptiSet®  at approximately the same time each morning. In either group, 12 U was the starting dose if fasting blood glucose (FBG) was ≥10 mmol (180 mg/dl). Insulin dose adjustments were made utilizing regimen-specific insulin dose titration algorithms  and 2 toHypoglycaemia events were assessed as to incidence (proportion of patients who experience hypoglycaemia), rate (per person per 30 days) and severity. Hypoglycaemia was defined as any time a patient experienced an associated sign or symptom, or had a BG level of <3.9 mmol/l (<70 mg/dl), even if it was not associated with signs, symptoms or treatment. Nocturnal hypoglycaemia was defined as any hypoglycaemic event that occurred between bedtime and waking. Severe hypoglycaemia was defined as an episode with symptoms consistent with hypoglycaemia in which the patient required the assistance of another person, and was associated with either a BG level of <2.8 mmol/l (50 mg/dl) or prompt recovery after oral carbohydrate, glucagon or intravenous glucose.The primary efficacy measure was change in A1C from baseline to endpoint. Secondary efficacy measures were A1C, percentages of patients achieving A1C ≤6.5, <7 and ≤7.0% and 7-point SMBG profiles over time; insulin dose ; number of injections per day; and safety, including hypoglycaemia (described earlier), weight change and treatment-emergent adverse events (TEAEs). Blood, urine and serum samples were collected at screening (week 2). A1C was analysed by a central laboratory .The sample size for the primary analysis was calculated on the basis of a two-sided test for non-inferiority with a 5% significance level. Assuming a standard deviation of 1.1% for A1C, 213 patients completing the study per treatment group would provide 80% power to meet the prespecified non-inferiority limit of 0.3%. Patients who completed a 36-week A1C measurement constituted the per-protocol analysis population .ancova) model with treatment, country, baseline A1C, baseline A1C stratum and sulphonylurea use as covariates. If the upper limit was below 0.3% (PP−G+L), then PP was non-inferior to G+L. Secondary outcomes  were analysed by ancova model, on the intent-to-treat (ITT) dataset, with treatment, country, baseline A1C stratum, sulphonylurea use and baseline as covariates. The percentages of patients achieving A1C goals  were analysed by logistic regression analysis with terms for treatment, country, sulphonylurea use and baseline A1C.The primary outcome was analysed by an analysis of covariance (anova) with treatment, country, baseline A1C stratum and sulphonylurea use as covariates. Categorical safety variables were compared between groups with Fisher's exact test.Safety assessments were based on the entire randomly assigned population. The proportion of patients reporting at least one hypoglycaemic event or a severe hypoglycaemic event was compared using Fisher's exact test. Hypoglycaemic rate and severe hypoglycaemic rate were analysed using ranked analysis of variance  were similar between groups at all time points except fasting, which was significantly lower in the G+L vs. PP group  . At endpAmong patients in the PP group, at endpoint (n = 234): 75 (32.1%) patients were on LM50/50 once daily (A1C: 7.52), 65 (27.8%) patients were on LM50/50 twice daily , 17 (7.3%) patients were on LM50/50 twice daily , 66 (28.2%) patients were on LM50/50 thrice daily (A1C: 7.61) and 11 (4.7%) patients were on LM50/50 (morning) + LM50/50 (mid-day) + LM75/25 (75% insulin lispro protamine suspension/25% insulin lispro) (evening) (A1C: 7.27). Among patients in the G+L group at endpoint (n = 235): 108 (46.0%) patients were on insulin glargine once daily (A1C: 7.25), 62 (26.4%) patients were on G+L once daily (A1C: 7.24), 48 (20.4%) patients were on G+L twice daily (A1C: 7.69) and 17 (7.2%) patients were on G+L thrice daily (A1C: 6.85). Thus, only patients treated with G+L thrice daily achieved a mean A1C <7.0%; only a small number of patients were advanced to this regimen.Weight-adjusted total daily insulin dosages (LSM ± s.e.) at endpoint were significantly greater for the PP vs. G+L group . Most patients ended up taking one or two injections . Mean number (LSM ± s.e.) of insulin injections at endpoint was significantly greater in the PP vs. G+L group . Body weight (LSM ± s.e.) change from baseline at endpoint was similar in both groups .The incidence of overall (over the treatment period) all hypoglycaemia , nocturnal hypoglycaemia  and severe hypoglycaemia  was similar in both groups. The incidence of all hypoglycaemia at endpoint [last observation carried forward (LOCF)] broken down by number of injections is provided in There was no statistically significant difference between the two groups for the overall all or overall nocturnal hypoglycaemic rate. Higher rates (mean ± s.d.) of hypoglycaemic episodes were observed with patients in the G+L group at LOCF endpoint , corresponding to hypoglycaemia occurring between the 24th and 36th week of treatment.Overall, 88 (36.4%) patients in the PP group and 92 (38.0%) patients in the G+L group experienced at least one TEAE during the study (p = 0.778). A small percentage of patients in each group experienced a serious adverse event during the study: 11 (4.5%) patients in the PP group and 10 (4.1%) patients in the G+L group (p = 1.00). There were three deaths reported in the study ; none were considered to be related to study drug or device .More evidence for the use of specific insulin regimens in T2D is needed beyond the introduction of the starting insulin regimen. This study evaluated two progressive regimens for advancing insulin therapy in insulin-naïve patients with T2D continuing prestudy OAMs. Both regimens started with once-daily insulin injections and provided increasing mealtime insulin coverage with additional injections, and both resulted in clinically relevant decreases in A1C over the course of the study. While there was no statistically significant difference between groups in A1C reduction at endpoint and non-inferiority was not shown, important observations in each approach may be noteworthy to consider during clinical management.During the first 24 weeks, while patients in the G+L group were injecting once-daily insulin glargine and adding another injection, patients' mean A1C was significantly lowered in the G+L vs. PP group. From the 24th to 36th week, when additional mealtime insulin injections were not introduced but dose titrations were allowed, A1C increased slightly in the G+L group and, by endpoint, there were no significant differences between the groups. Most studies evaluating basal insulin added to OAMs in T2D note a decrease in A1C from baseline up to ∼12 weeks, after which they stabilize ; whereasIn the PP group, A1C decreased from baseline to 24 weeks almost in parallel with the G+L group, although levels were slightly higher. While PP patients had higher A1C at the 12th and 24th week, patients in this group were able to maintain their A1C levels between the 24th and 36th week; by the end of week 36, there was no difference in A1C between groups. The PP regimen more effectively lowered evening postprandial values compared to G+L therapy. A greater number of patients in the PP vs. G+L group were receiving more than one injection by the end of week 24, which may explain why A1C levels were maintained in this group.The higher rate of hypoglycaemia in the G+L vs. PP group occurred after week 24, a point after which no additional mealtime lispro injections could be introduced. Because A1C increased slightly in the G+L group between weeks 24 and 36, the increased rate of hypoglycaemia is unlikely to be the result of lower glucose levels but may have resulted from increasing the insulin dose without adding injections. In contrast, the greater number of injections in the PP group may have allowed administration of higher doses overall without increasing hypoglycaemia. This finding is consistent with the rationale behind multiple daily insulin (MDI) injections , where each injection provides a unique opportunity for more flexible dose adjustment  to improOur findings are consistent with those of Rosenstock et al.  in whichAn important difference between the two studies is the mean A1C levels at study endpoint. With the intensive insulin therapies in the Rosenstock et al. study, mean A1C at endpoint was <7.0% with both treatments; whereas in the present study, mean A1C at endpoint was 7.6% while patients were on a median of two insulin injections. The difference in achieved mean A1Cs at endpoint again highlights the potential for improvement in glycaemic control by advancing prandial insulin to cover all meals.Relatively few other studies have evaluated progressive insulin advancement in the context of a clinical trial. The Orals Plus Apidra and LANTUS (OPAL) study  evaluateA limitation of the present study is the open-label design, which was unavoidable given the cloudy vs. clear appearance of the insulin preparations that prevented blinding. This may have contributed to inequality in the numbers of injections given that most practitioners use premix insulin twice daily and insulin glargine once daily. While the protocol allowed titration up to three to four injections, most patients only went up to two injections in spite of endpoint A1Cs not being <7%. It could be argued that tighter control should have been implemented considering that lower A1Cs were achievable with three to four insulin injections, as shown in Rosenstock et al. . The proThe slight worsening of glycaemic control in the G+L group after 24 weeks emphasizes the importance of being vigilant about getting patients to achieve glycaemic control. The wide variability in the change in A1C  reflects the broad differences in patients' responses to these insulin regimens. Other factors may need to be studied to better understand this. The apparent resistance to increasing numbers of injections is a reminder that there are barriers to intensifying insulin therapy that also may need to be considered and addressed to more effectively aim for better glycaemic control earlier in the natural history of T2D. One of these barriers is clinical inertia or failure of health-care providers to initiate or advance therapy in a patient who is not at the recommended therapeutic goal ,20.In summary, this study shows that both the PP and G+L regimens are efficient in lowering A1C even if non-inferiority of PP to G+L could not be shown. These findings support the belief that most patients with T2D will likely need more than one type of insulin ,10, and


The results of the Rietveld refinement, based on high resolution synchrotron X-ray powder diffraction data, show that the title compound crystallizes in the same structure as other halogenoapatites with general formula A               5(YO4)3               X  in the space group P63/m. The structure consists of isolated tetrahedral AsO4               3− anions (the As atom and two O atoms have m symmetry), separated by two crystallographically independent Sr2+ cations that are located on mirror planes and threefold rotation axes, respectively. One Sr atom is coordinated by nine O atoms and the other by six. The chloride anions ] chloride, Sr DOI: 10.1107/S1600536809005054/br2096Isup2.rtv            Rietveld powder data: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: