技术资料

IL-3,IL-5,and GM-CSF are related hematopoietic cytoines that are important for allergic inflammation. The receptors for human IL-5,IL-3,and GM-CSF are members of the hematopoietin receptor superfamily and are comprised of a cytokine-specific alpha chain and the common beta chain that is shared among these cytokines for signaling. Each of these cytokines contributes to the differentiation and function of leukocyte subpopulations and have clinical importance in protective immunity and in the pathophysiology of a spectrum of immunologic diseases that are as diverse as allergy and asthma,pulmonary alveolar proteinosis,neurodegenerative diseases,and malignancies. Delineating the biology of these cytokines is enabling the development of new strategies for diagnosing and treating these diseases and modulating immune responses. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However,comparing the two assays,cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells,p = 0.0001). Overall,60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells� View Publication

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo,selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study,we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment,at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore,99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR,compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice,demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity,and CFUs were stained intensely for AGT protein,indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge,these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment. View Publication

The tumor necrosis factor (TNF)-like ligand BAFF/BLyS (B-cell activating factor of the TNF family/B-lymphocyte stimulator) is a potent B-cell survival factor,yet its functional relationship with other B-cell surface molecules such as CD19 and CD40 is poorly understood. We found that follicular dendritic cells (FDCs) in human lymph nodes expressed BAFF abundantly. BAFF up-regulated a B cell-specific transcription factor Pax5/BSAP (Pax5/B cell-specific activator protein) activity and its target CD19,a major component of the B-cell coreceptor complex,and synergistically enhanced CD19 phosphorylation by B-cell antigen receptor (BCR). BAFF further enhanced B-cell proliferation,immunoglobulin G (IgG) production,and reactivity to CD154 by BCR/CD19 coligation and interleukin-15 (IL-15). Our results suggest that BAFF may play an important role in FDC-B-cell interactions through the B-cell coreceptor complex and a possibly sequential link between the T cell-independent and -dependent B-cell responses in the germinal centers. View Publication

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer,and in particular,unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties,we have begun to target EphA2 on tumor cells using agonistic antibodies,which mimic the consequences of ligand binding. In our present study,we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells,which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand,as well as this subset of EphA2 antibodies. Finally,we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together,these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells. View Publication

Singlet oxygen ((1)O(2)) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of (1)O(2) is possible in cells in vitro and tissues in vivo. Here,we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10,25,or 50 mWcm(-2) and,(1)O(2) luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells,the photobleaching,and the pO(2) in the cell suspensions were also monitored. There were large variations in cell survival and (1)O(2) generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However,in all of the cases,cell kill correlated strongly with the cumulative (1)O(2) luminescence and allowed direct estimation of the (1)O(2) per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of (1)O(2) luminescence measurement as a dosimetric tool for PDT,as well as confirming the likely role of (1)O(2) in porphyrin-based PDT. View Publication

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART),and replication capacities correlated with pre-ART and post-STI viral set points. Of note,no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage,genetic subtype,and sensitivity to neutralizing antibodies. In contrast,viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates,suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary,our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection,and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection. View Publication

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is an essential transcription factor for granulocytic differentiation. C/EBPalpha mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region,resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein,which may be responsible for the differentiation block observed in AML. To test this hypothesis,we introduced a cDNA encoding an N-terminal mutated C/EBPalpha (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34+ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly,mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBPalpha protein sequences,expression levels,or inefficient targeting of relevant cells. Taken together,our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs. View Publication

CD4+ T cells are required for immunity against many viral infections,including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1,whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors,although it is not known whether these factors are produced during primary antigen-specific responses. Here,we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of naïve CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22,CCL22),and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3),macrophage inflammatory protein-1 beta (CCL4),and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus. View Publication