技术资料

B-lymphocyte stimulator (BLyS),a relatively recently recognized member of the tumor necrosis factor ligand family (TNF),is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors,TACI,BCMA,BAFF-R,to regulate B-cell survival,differentiation,and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study,we examined BLYS gene expression,function,and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells,including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL),playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors,NF-kappaB and NFAT,are involved in regulating BLyS expression through at least one NF-kappaB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-kappaB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-kappaB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B. View Publication

PURPOSE This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2alpha and fluprostenol (fluprostenol-isopropyl ester [travoprost]),which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1,a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied. METHODS The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2alpha on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis. RESULTS The ET-1-induced (10(-8) M) contraction in isolated BTM was inhibited by PGF2alpha (10(-6) M) and fluprostenol (10(-6) M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2alpha or fluprostenol. In cultured TM cells,ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2alpha. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM. CONCLUSIONS FP receptor agonists operate by direct interaction with ET-1-induced contractility of TM. This effect is mediated by the FP receptor. Thus,FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1-dependent mechanisms. View Publication

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes,humanization of rodent Abs,or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however,this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g.,affinity and titers) of mAb-producing cell lines,including hybridomas. We reasoned that this process,named morphogenics,could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover,these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing. View Publication

Circulating CD34+ cells are haemopoietic progenitors that may play a role in tissue repair. No data are available on circulating progenitors in chronic obstructive pulmonary disease (COPD). Circulating CD34+ cells were studied in 18 patients with moderate-to-severe COPD (age: mean+/-sd 68+/-8 yrs; forced expiratory volume in one second: 48+/-12% predicted) and 12 controls,at rest and after endurance exercise. Plasma concentrations of haematopoietic growth factors (FMS-like tyrosine kinase 3 (Flt3) ligand,kit ligand),markers of hypoxia (vascular endothelial growth factor (VEGF)) and stimulators of angiogenesis (VEGF,hepatocyte growth factor (HGF)) and markers of systemic inflammation (tumour necrosis factor (TNF)-alpha,interleukin (IL)-6,IL-8) were measured. Compared with the controls,the COPD patients showed a three-fold reduction in CD34+ cell counts (3.3+/-2.5 versus 10.3+/-4.2 cells.microL-1),and a 50% decrease in AC133+ cells. In the COPD patients,progenitor-derived haemopoietic and endothelial cell colonies were reduced by 30-50%. However,four COPD patients showed progenitor counts in the normal range associated with lower TNF-alpha levels. In the entire sample,CD34+ cell counts correlated with exercise capacity and severity of airflow obstruction. After endurance exercise,progenitor counts were unchanged,while plasma Flt3 ligand and VEGF only increased in the COPD patients. Plasma HGF levels were higher in the COPD patients compared with the controls and correlated inversely with the number of progenitor-derived colonies. In conclusion,circulating CD34+ cells and endothelial progenitors were decreased in chronic obstructive pulmonary disease patients and could be correlated with disease severity. View Publication

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine,Vidaza) has recently been approved as an antitumor agent,and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems,which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR),5-aza-2'-deoxycytidine (5-aza-CdR),zebularine,procaine,(-)-epigallocatechin-3-gallate (EGCG),and RG108. Of these,5-aza-CR,5-aza-CdR,zebularine,and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic,as evidenced by the induction of micronuclei. In addition,5-aza-CR,5-aza-CdR,zebularine,and RG108 caused concentration-dependent demethylation of genomic DNA,whereas procaine and EGCG failed to induce significant effects. Finally,the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase,which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs. View Publication

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs,the effects of IL-2 and the TLR-7 agonist,S28690,on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members,production of TNF-alpha and IL-10,and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However,IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C,which inhibited tumor cell proliferation,switched off" IL-10 production� View Publication

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. In the absence of Ikaros,there is a lack of markers defining fate restriction along lympho-myeloid pathways,but it is unclear whether formation of specific progenitors or expression of their markers is affected. Here we use a reporter based on Ikaros regulatory elements to separate early progenitors in wild-type and Ikaros-null mice. We found previously undetected Ikaros-null lympho-myeloid progenitors lacking the receptor tyrosine kinase Flt3 that were capable of myeloid but not lymphoid differentiation. In contrast,lack of Ikaros in the common myeloid progenitor resulted in increased formation of erythro-megakaryocytes at the expense of myeloid progenitors. Using this approach,we identify previously unknown pivotal functions for Ikaros in distinct fate 'decisions' in the early hematopoietic hierarchy. View Publication

Ca(2+) influx through voltage-gated Ca(2+) channels,especially the L-type (Ca(v)1),activates downstream signaling to the nucleus that affects gene expression and,consequently,cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas,during 12 days of fetal bovine serum-induced differentiation,neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients,with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation,cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons,which responded to membrane depolarization with weak Ca(2+) signals. Conversely,Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels,especially the Ca(v)1,and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation. View Publication

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro,bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-),PAX5(+),TdT(+),IgH rearranged)/myeloid (CD11b/Mac1(+),c-fms(+),lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast,young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model." View Publication

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span,granulocyte macrophage colony-stimulating factor,interleukin-4,and calcium ionophore. All B-precursor ALL (N22) and AML (N13),but not T-cell ALL (N3),differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture,donor-derived CTL acquired a memory T phenotype,showing concomitant high CD45RA,CD45RO,CD62L expression. CD8(+) cells,but not CD4(+) cells,were granzyme,perforine,and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%,30:1 E:T ratio),but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%,1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. View Publication

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However,the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g.,the brain) is higher than for treatment of anemia. Notably,a dose-dependent risk of adverse effects has been associated with rhEPO administration,especially in high-risk groups,including polycythemia-hyperviscosity syndrome,hypertension,and vascular thrombosis. Of note,several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity,primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover,we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here,we demonstrate that rhEPO administration in the rat increases systemic blood pressure,reduces regional renal blood flow,and increases platelet counts and procoagulant activities. In contrast,carbamylated rhEPO,a heteromeric receptor-specific ligand that is fully tissue protective,increases renal blood flow,promotes sodium excretion,reduces injury-induced elevation in procoagulant activity,and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands,which appear to elicit fewer adverse effects,may be especially useful in clinical settings for tissue protection. View Publication

Erythropoiesis,the essential process of hematopoietic stem cell development into erythrocytes,is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver,neonatal spleen,and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen,short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype,demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation,including hemoglobinization,cell condensation,and enucleation,and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells. View Publication