技术资料

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells,thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently,novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study,we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis,and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion,TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival. View Publication

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule,the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals,testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed,there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover,the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor,AG490. 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

The infantile neuronal ceroid lipofuscinosis (INCL),a rare (one in 100 000 births) but one of the most lethal inherited neurodegenerative storage disorders of childhood,is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in s-acylated (palmitoylated) proteins and facilitates their degradation and/or recycling. Thus,PPT1-deficiency leads to an abnormal intracellular accumulation of s-acylated proteins causing INCL pathogenesis. Although neuronal apoptosis is the suggested cause of neurodegeneration in this disease,the molecular mechanism(s) remains poorly understood. We recently reported that one of the major pathways of neuronal apoptosis in PPT1-knockout (PPT1-KO) mice that mimic INCL,is mediated by endoplasmic reticulum (ER) stress-induced caspase-12 activation. ER stress also increases the production of reactive oxygen species (ROS),disrupts Ca(2+) homeostasis and increases the potential for destabilizing mitochondrial membrane. Mitochondrial membrane destabilization activates caspase-9 present in this organelle,and can mediate apoptosis. We report here that the levels of superoxide dismutase (SOD),most likely induced by ROS,in human INCL as well as PPT1-KO mouse brain tissues are markedly elevated. Moreover,we demonstrate that activated caspase-3 and cleaved-PARP,indicative of apoptosis,are also increased in these tissues. Using cultured neurospheres from PPT1-KO and wild-type mouse fetuses,we further demonstrate that the levels of ROS,SOD-2,cleaved-caspase-9,activated caspase-3 and cleaved-PARP are elevated. We propose that: (i) ER stress due to PPT1-deficiency increases ROS and disrupts calcium homeostasis activating caspase-9 and (ii) caspase-9 activation mediates caspase-3 activation and apoptosis contributing to rapid neurodegeneration in INCL. 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

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

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV,purified HPCs were exposed to KSHV,and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells,and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection,persistence,and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo. View Publication

Novel dihydropyrrolopyrazole-substituted benzimidazoles were synthesized and evaluated in vitro as inhibitors of transforming growth factor-beta type I receptor (TGF-beta RI),TGF-beta RII,and mixed lineage kinase-7 (MLK-7). These compounds were found to be potent TGF-beta RI inhibitors and selective versus TGF-beta RII and MLK-7 kinases. Benzimidazole derivative 8b was active in an in vivo target (TGF-beta RI) inhibition assay. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines,complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(ptextless0.0001). In t(12;21) samples,efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels,suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H,blocking the CD55 and CD59 complement inhibitors,and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL,alone or in combination with cytotoxic drugs. 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

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