参考文献

Through the regulation of human leukocyte antigen (HLA)-DM (DM) in B cells,HLA-DO (DO) modulates positively or negatively the presentation of specific peptides. Transduction of DO into human blood monocyte-derived dendritic cells (MoDC) has been proposed as a mean of modifying the peptide repertoire of major histocompatibility complex class II molecules. However,maturation of DC induced by inflammatory stimuli or possibly the adenoviral vector itself triggers acidification of vesicles and shuts down transcription of the class II transactivator gene as well as de novo biosynthesis of class II-related molecules and DM activity. In these conditions,it is unclear that transduced DO could alter the peptide repertoire. Our Western blot and reverse transcriptase-polymerase chain reaction analyses revealed that human DC derived from blood monocytes express small amounts of DOalpha. Transduction of DObeta alone resulted in the accumulation of a small pool of DO in DM(+) CD63(+) vesicles and at the plasma membrane of mature DC. The cell-surface increase in class II-associated invariant chain peptide (CLIP)/class II complexes is in line with an inhibitory role of DO on DM. Cotransduction of DOalpha and DObeta only slightly increased CLIP and DO levels at the cell surface. Together with the fact that a large fraction of transduced DO remains in the endoplasmic reticulum,this suggests that DM is limiting in these conditions. DO expression did not affect a mixed lymphocyte reaction but reduced presentation of the exogenous gp100 antigen to a specific T cell clone. These results show that transduced DO modulates antigen presentation in human mature MoDC,evoking the possible use of this chaperone for immunotherapy. View Publication

Patients with sepsis are immune compromised,as evidenced by their failure to clear their primary infection and their propensity to develop secondary infections with pathogens that are often not particularly virulent in normal healthy individuals. A potential mechanism for immunosuppression in sepsis is lymphocyte apoptosis,which may occur by either a death receptor or a mitochondrial-mediated pathway. A prospective study of blood samples from 71 patients with sepsis,55 nonseptic patients,and 6 healthy volunteers was undertaken to quantitate lymphocyte apoptosis and determine cell death pathways and mechanisms of apoptosis. Apoptosis was evaluated by flow cytometry and Western blotting. Lymphocyte apoptosis was increased in CD4 and CD8 T cells,B cells (CD20),and NK cells (CD56) in septic vs nonseptic patients. Samples taken sequentially from 10 patients with sepsis showed that the degree of CD3 T cell apoptosis correlated with the activity of his/her sepsis. In septic patients,apoptotic lymphocytes were positive for active caspases 8 and 9,consistent with death occurring by both mitochondrial-mediated and receptor-mediated pathways. In support of the concept that both death pathways were operative,lymphocyte apoptosis occurred in cells with markedly decreased Bcl-2 (an inhibitor of mitochondrial-mediated apoptosis) as well as cells with normal concentrations of Bcl-2. In conclusion,apoptosis occurs in a broad range of lymphocyte subsets in patients with sepsis and correlates with the activity of the disease. Lymphocyte loss occurs by both death receptor and mitochondrial-mediated apoptosis,suggesting that there may be multiple triggers for lymphocyte apoptosis. View Publication

Anthrax lethal toxin (LT) is a critical virulence factor that cleaves and inactivates MAPK kinases (MAPKKs) in host cells and has been proposed as a therapeutic target in the treatment of human anthrax infections. Despite the potential use of anti-toxin agents in humans,the standard activity assays for anthrax LT are currently based on cytotoxic actions of anthrax LT that are cell-,strain-,and species-specific,which have not been demonstrated to occur in human cells. We now report that T cell proliferation and IL-2 production inversely correlate with anthrax LT levels in human cell assays. The model CD4+ T cell tumor line,Jurkat,is a susceptible target for the specific protease action of anthrax LT. Anthrax LT cleaves and inactivates MAPKKs in Jurkat cells,whereas not affecting proximal or parallel TCR signal transduction pathways. Moreover,anthrax LT specifically inhibits PMA/ionomycin- and anti-CD3-induced IL-2 production in Jurkat cells. An inhibitor of the protease activity of anthrax LT completely restores IL-2 production by anthrax LT-treated Jurkat cells. Anthrax LT acts on primary CD4+ T cells as well,cleaving MAPKKs and leading to a 95% reduction in anti-CD3-induced proliferation and IL-2 production. These findings not only will be useful in the development of new human cell-based bioassays for the activity of anthrax LT,but they also suggest new mechanisms that facilitate immune evasion by Bacillus anthracis. Specifically,anthrax LT inhibits IL-2 production and proliferative responses in CD4+ T cells,thereby blocking functions that are pivotal in the regulation of immune responses. View Publication

We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed,mRNAs for two,CXCL10 and CXCL11,ligands for the chemokine receptor CXCR3,were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity,but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells,and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex,including venules,as detected by in situ hybridization,whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3,these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes,macrophages,and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells,contributing to the immunopathology of AIDS. View Publication

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

The limited number of progenitor stem cells in umbilical cord blood (UCB) enforces the optimization and strict control of all the procedures involved in its therapeutic use--ie,collection,processing,cryopreservation,thawing,and transportation--to ensure graft potency at transplantation. For this reason,international UCB standards recommend storage of a cell sample attached to the UCB unit as a quantitative and functional control of the unit selected for transplantation. To validate the use of the sample attached to the UCB unit as a quality-control tool for the final product,UCB units (n = 20) stored in liquid nitrogen with the Bioarchive system were analyzed. The UCB units and their attached segments were thawed,and the number and viability of total nucleated cells,mononucleated cells,CD45 + cells,and CD34+ cells were determined,as were colony-forming cell counts. There was no significant difference between UCB units and segments for any of the parameters assessed. Additionally,the linear correlation coefficient (R2) in these paired samples was 0.85 and 0.78 for CD34+ cells and colony-forming cells,respectively. In conclusion,the cell sample in the tube segment physically linked to the transplant UCB bag predicts the total cell content and functionality of the unit and may serve as a source for final quality control of the UCB unit before transplantation. View Publication

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts,an effect achieved either alone or in combination with differentiating agents,such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore,valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4,a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary,we here show that valproic acid,known to induce differentiation or apoptosis in leukemic blasts,stimulates the proliferation of normal HSC,an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. View Publication

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however,other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets,purified ALDH(br) CD34+,ALDH(neg) CD34+,and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice,and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison,ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development. View Publication

PURPOSE: The PRL-3 mRNA is consistently elevated in metastatic samples derived from colorectal cancers. We sought to generate a specific PRL-3 monoclonal antibody (mAb) that might serve as a potential diagnostic marker for colorectal cancer metastasis. EXPERIMENTAL DESIGN: PRL-3 is one of three members (PRL-1,PRL-2,and PRL-3) in a unique protein-tyrosine phosphatase family. Because the three PRLs are 76% to 87% identical in their amino acid sequences,it poses a great challenge to obtain mAbs that are specific for respective phosphatase of regenerating liver (PRL) but not for the other two in the family. We screened over 1,400 hybridoma clones to generate mAbs specific to each PRL member. RESULTS: We obtained two hybridoma clones specifically against PRL-3 and another two clones specifically against PRL-1. These antibodies had been evaluated by several critical tests to show their own specificities and applications. Most importantly,the PRL-3 mAbs were assessed on 282 human colorectal tissue samples (121 normal,17 adenomas,and 144 adenocarcinomas). PRL-3 protein was detected in 11% of adenocarcinoma samples. The PRL-3- and PRL-1-specific mAbs were further examined on 204 human multiple cancer tissues. The differential expressions of PRL-3 and PRL-1 confirmed the mAbs' specificity. CONCLUSIONS: Using several approaches,we show that PRL-3- or PRL-1-specific mAbs react only to their respective antigen. The expression of PRL-3 in textgreater10% of primary colorectal cancer samples indicates that PRL-3 may prime the metastatic process. These mAbs will be useful as markers in clinical diagnosis for assessing tumor aggressiveness. View Publication

The application of genome-wide expression analysis to a large-scale,multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition,variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject,among different healthy subjects,among different leukocyte subpopulations,and the effect of traumatic injury,were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast,genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly,traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects,and can be studied with confidence in human disease. View Publication