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Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Oct 2017To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line...
To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot. ①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (<0.05) , respectively under ATRA exposure. ②The percentages of G(0)/G(1) stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure. ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G(0)/G(1) stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.
Topics: Cell Differentiation; Cell Proliferation; Humans; Mutation; Nuclear Proteins; Nucleophosmin; Tretinoin; U937 Cells
PubMed: 29166739
DOI: 10.3760/cma.j.issn.0253-2727.2017.10.008 -
Molecular Microbiology Feb 1999Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative...
Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.
Topics: Animals; Bacterial Proteins; Cell Survival; Colony Count, Microbial; Endopeptidases; Fimbriae, Bacterial; Guinea Pigs; Hartmannella; Humans; Legionella pneumophila; Lung; Microscopy, Electron; Mutagenesis; Proteins; Spleen; Statistics as Topic; Stem Cells; Temperature; Time Factors; U937 Cells
PubMed: 10048038
DOI: 10.1046/j.1365-2958.1999.01239.x -
Cytotherapy Aug 2016PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of...
BACKGROUND AIMS
PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci.
METHODS
We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells.
RESULTS
PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines.
CONCLUSIONS
This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.
Topics: Adult; Antibody-Dependent Cell Cytotoxicity; Cells, Cultured; Fetal Blood; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; K562 Cells; Leukemia, Myeloid; Lymphocyte Count; Myeloblastin; T-Lymphocytes, Cytotoxic; U937 Cells
PubMed: 27378343
DOI: 10.1016/j.jcyt.2016.05.007 -
AIDS Research and Human Retroviruses Mar 2008Monocytes and macrophages play a prominent role in the establishment of HIV-1 infection, virus dissemination, and development of viral reservoirs. Like T cells,...
Monocytes and macrophages play a prominent role in the establishment of HIV-1 infection, virus dissemination, and development of viral reservoirs. Like T cells, macrophages display immune polarization that can promote or impair adaptive immunity. We hypothesize that dysregulation of monocyte/macrophage activation and differentiation may promote immune dysfunction and contribute to AIDS pathogenesis. Using flow cytometry, we analyzed the frequency of monocyte subsets in human immunodeficiency virus type 1 (HIV-1) infection relative to seronegative controls, focusing on the CD163(+)/CD16(+) monocyte as a likely precursor of the "alternatively activated" macrophage. Individuals with detectable HIV-1 infection showed an increase in the frequency of CD163(+)/CD16(+) monocytes (CD14(+)) when compared to seronegative or HIV-1-infected persons with undetectable viral loads. A positive correlation between increased CD163(+)/CD16(+) monocyte frequency and viral load was revealed that was not seen between viral load and the number of CD4(+) T cells or frequency of CD16(+) monocytes (without CD163 subtyping). We also found a strong inverse correlations between CD16(+) monocytes (r = -0.71, r(2) = 0.5041, p = 0.0097) or CD163(+)/CD16(+) monocytes (r = -0.86, r(2) = 0.7396, p = 0.0003) and number of CD4(+) T cells below 450 cells/microl. An inverse relationship between CD163(+)/CD16(+) and CD163(+)/CD16() monocytes suggests the expanded CD163(+)/CD16(+) population is derived exclusively from within the "alternatively activated" (MPhi-2) subset. These data suggest a potential role for CD163(+)/CD16(+) monocytes in virus production and disease progression. CD163(+)/CD16(+) monocytes may be a useful biomarker for HIV-1 infection and AIDS progression and a possible target for therapeutic intervention.
Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers; CD4 Lymphocyte Count; Disease Progression; Flow Cytometry; HIV Infections; Humans; Lipopolysaccharide Receptors; Macrophages; Monocytes; Receptors, Cell Surface; Receptors, IgG; Viral Load
PubMed: 18373432
DOI: 10.1089/aid.2007.0193 -
The Journal of Experimental Medicine Mar 1975The present communication concerns the effect of azathioprine on the mitotic activity of promonocytes and the production of monocytes. In vitro and in vivo labeling with...
The present communication concerns the effect of azathioprine on the mitotic activity of promonocytes and the production of monocytes. In vitro and in vivo labeling with [3H]thymidine showed that during azathioprine treatment the promonocytes synthesize DNA and that, contrary to expectation, the labeling index increases. Cytospectrophotometric determination of the Feulgen-DNA content of the promonocytes during azathioprine treatment showed an increase in the percentage of tetraploid promonocytes, and determination of the various phases of the cell cycle showed significantly increased DNA synthesis and cell cycle times as compared with the normal steady state. On the basis of these results it can be concluded that azathioprine arrests the cell cycle of the promonocytes late in the DNA synthesis phase or in the postsynthesis (G2) phase and mitosis does not occur. This timing of the effect of azathioprine had not been previously observed. The diminished mitotic activity of the promonocytes during azathioprine treatment depressed monocyte production. During treatment with 3 mg/kg azathioprine the cell cycle time of the promonocytes was on the average 5.5 h longer than in the normal steady state and the rate of monocyte production was reduced by 70%. During an acute inflammatory reaction too, monocyte production is affected by azathioprine. In animals not treated with azathioprine but with an acute inflammation the cell cycle time becomes shorter and the monocyte production increases, but animals treated with (3 mg/kg) azathioprine do not show this effect. The kinetics of the monocyte also changes under the low dosage of azathioprine. As consequence of the diminished production of monocytes, far fewer (about 50%) monocytes enter and leave the circulation than during the normal steady state. During an acute inflammatory reaction the numbers in transit through the circulation are slightly augmented but remain considerably lower than in nonazathioprine-trehat of animals not treated with azathioprine.
Topics: Acute Disease; Animals; Azathioprine; Bone Marrow; Bone Marrow Cells; Cell Count; Cell Division; Cell Nucleus; DNA; Inflammation; Kinetics; Male; Mercaptopurine; Mice; Mitosis; Monocytes; Spectrophotometry; Thymidine; Time Factors; Tritium
PubMed: 1117257
DOI: 10.1084/jem.141.3.531 -
The Journal of Experimental Medicine Jan 1973To elucidate mechanisms underlying the prolonged monocytopenia induced in the peripheral blood of mice by injection of a subcutaneous depot of hydrocortisone acetate,...
To elucidate mechanisms underlying the prolonged monocytopenia induced in the peripheral blood of mice by injection of a subcutaneous depot of hydrocortisone acetate, the effect of this compound on the production of monocytes and their release from the bone marrow was studied. Hydrocortisone was found to cause a rapid reduction of the bone marrow promonocytes to about 65% of their initial number. The number of monocytes in the bone marrow decreased gradually, over a period of 96 h, to 75% of the initial value. The mitotic activity of the promonocytes was not diminished, as judged from the labeling in vitro with [(3)H]thymidine and the DNA-synthesis and cell-cycle times of these cells. The production of monocytes was only moderately diminished, i.e., to about 80% of the normal amount. The release of monocytes from the bone marrow was found to be influenced by hydrocortisone. After in vivo labeling with [(3)H]thymidine the monocyte-labeling indices were initially significantly higher in hydrocortisone-treated than in normal mice. It is concluded that a decreased production of monocytes in the bone marrow cannot account for the prolonged monocytopenia in the peripheral blood after hydrocortisone administration. However, hydrocortisone interferes with the release of newly formed monocytes from the bone marrow, resulting in a prolonged sojourn of these cells in this compartment.
Topics: Animals; Autoradiography; Bone Marrow; Bone Marrow Cells; Cell Count; Cell Division; Cell Movement; Cells, Cultured; DNA; Hydrocortisone; Kinetics; Mice; Mitosis; Monocytes; Tritium
PubMed: 4688316
DOI: 10.1084/jem.137.1.10 -
Journal of Virology Aug 2008Human immunodeficiency virus (HIV)-positive persons are predisposed to pulmonary infections, even after receiving effective highly active antiretroviral therapy. The...
Human immunodeficiency virus (HIV)-positive persons are predisposed to pulmonary infections, even after receiving effective highly active antiretroviral therapy. The reasons for this are unclear but may involve changes in innate immune function. HIV type 1 infection of macrophages impairs effector functions, including cytokine production. We observed decreased constitutive tumor necrosis factor alpha (TNF-alpha) concentrations and increased soluble tumor necrosis factor receptor type II (sTNFRII) in bronchoalveolar lavage fluid samples from HIV-positive subjects compared to healthy controls. Moreover, net proinflammatory TNF-alpha activity, as measured by the TNF-alpha/sTNFRII ratio, decreased as HIV-related disease progressed, as manifested by decreasing CD4 cell count and increasing HIV RNA (viral load). Since TNF-alpha is an important component of the innate immune system and is produced upon activation of Toll-like receptor (TLR) pathways, we hypothesized that the mechanism associated with deficient TNF-alpha production in the lung involved altered TLR expression or a deficit in the TLR signaling cascade. We found decreased Toll-like receptor 1 (TLR1) and TLR4 surface expression in HIV-infected U1 monocytic cells compared to the uninfected parental U937 cell line and decreased TLR message in alveolar macrophages (AMs) from HIV-positive subjects. In addition, stimulation with TLR1/2 ligand (Pam(3)Cys) or TLR4 ligand (lipopolysaccharide) resulted in decreased intracellular phosphorylated extracellular signal-regulated kinase and subsequent decreased transcription and expression of TNF-alpha in U1 cells compared to U937 cells. AMs from HIV-positive subjects also showed decreased TNF-alpha production in response to these TLR2 and TLR4 ligands. We postulate that HIV infection alters expression of TLRs with subsequent changes in mitogen-activated protein kinase signaling and cytokine production that ultimately leads to deficiencies of innate immune responses that predispose HIV-positive subjects to infection.
Topics: Adult; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Cytokines; HIV Infections; Humans; Immunity, Innate; Ligands; Macrophages, Alveolar; Middle Aged; RNA, Viral; Signal Transduction; Toll-Like Receptors; Tumor Necrosis Factor-alpha; U937 Cells
PubMed: 18524817
DOI: 10.1128/JVI.00362-08 -
Cancer Immunology, Immunotherapy : CII Aug 2002Dendritic cells (DC) have been successfully used in clinical pilot studies to induce tumor-specific immunity as well as clinical response in selected patients. However,...
Eliciting cytotoxic T lymphocytes against acute myeloid leukemia-derived antigens: evaluation of dendritic cell-leukemia cell hybrids and other antigen-loading strategies for dendritic cell-based vaccination.
Dendritic cells (DC) have been successfully used in clinical pilot studies to induce tumor-specific immunity as well as clinical response in selected patients. However, DC-based immunotherapy remains a challenge and several parameters need to be examined in order to optimize the induction of anti-tumor immune responses. This study focuses on DC vaccination for leukemia and evaluates the in vitro efficacy of three different strategies for generating antigen-loaded DC-based vaccines for the induction of major histocompatibility complex (MHC) class I-restricted anti-leukemia cytotoxic T lymphocyte (CTL) responses. These included direct fusion of DC with leukemia cells to generate DC-leukemia cell hybrids, and DC pulsed with either apoptotic leukemia cell fragments or whole tumor cell lysates. Using either the U937 cell line or primary human acute myeloid leukemia blasts (AML), DC-leukemia cell hybrids were found to be the most potent in vitro inducers of CTL activity. DC pulsed with apoptotic tumor cell fragments were less efficient, but induced a more potent CTL response compared to tumor lysate-pulsed DC. The CTL responses were both MHC class I-restricted and antigen-specific, as shown by the inability of the CTL to lyse other control targets. The data presented here suggest that the method of antigen loading onto DC may be critical in the design of tumor vaccines.
Topics: Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; Cell Count; Cell Differentiation; Cell Fusion; Culture Media, Conditioned; Dendritic Cells; Histocompatibility Antigens Class I; Humans; Hybrid Cells; Immunophenotyping; K562 Cells; Leukemia, Myeloid, Acute; Lymphocyte Activation; Monocytes; Neoplastic Stem Cells; Phagocytosis; T-Lymphocytes, Cytotoxic; U937 Cells
PubMed: 12111118
DOI: 10.1007/s00262-002-0284-4 -
FEBS Letters Aug 1999Exposure of U937 cells to tert-butylhydroperoxide (tB-OOH) led to cyclosporin A-sensitive mitochondrial membrane permeability transition and necrosis. Pyruvate and...
Exposure of U937 cells to tert-butylhydroperoxide (tB-OOH) led to cyclosporin A-sensitive mitochondrial membrane permeability transition and necrosis. Pyruvate and rotenone, which increase mitochondrial NADH via different mechanisms, prevented these responses and the cells which received these treatments proliferated with kinetics similar to those observed in untreated cells. In contrast with these results, cells rescued by cyclosporin A were unable to proliferate. Thus, mitochondrial NADH plays a pivotal role in preventing upstream events which result in the onset of mitochondrial membrane permeability transition and death in cells exposed to tB-OOH. These events appear to be critical for recovery of the ability of the cells to proliferate.
Topics: Apoptosis; Cell Count; Cell Division; Cell Survival; Cyclosporine; DNA Fragmentation; Enzyme Inhibitors; Humans; Kinetics; Necrosis; Pyruvic Acid; Rotenone; Time Factors; U937 Cells; Uncoupling Agents; tert-Butylhydroperoxide
PubMed: 10486581
DOI: 10.1016/s0014-5793(99)01027-3 -
Immunology Jul 1990In mice given an intravenous injection of Mycobacterium bovis (BCG), the bacilli proliferated in the spleen, liver and lungs but the peritoneal cavity remained sterile....
In mice given an intravenous injection of Mycobacterium bovis (BCG), the bacilli proliferated in the spleen, liver and lungs but the peritoneal cavity remained sterile. The numbers of blood monocytes and alveolar macrophages were increased during the first 2 weeks of the infection, whereas the number of peritoneal macrophages remained constant. To study whether factor-increasing monocytopoiesis (FIM) plays a role in the regulation of the monocytosis during the BCG infection, the activity of this factor in the serum of mice at various intervals during the infection was determined. Previous studies have shown that FIM stimulates monocyte production by its effect on the mitotic activity of monoblasts and promonocytes in the bone marrow. The FIM activity of the serum reached a maximum on Day 4 and remained elevated during the first 21 days of the BCG infection. Since FIM is synthesized and secreted by macrophages that have phagocytosed opsonized particles, it is highly probable that FIM occurring in serum originates from macrophages that have ingested BCG. The results of the present study led to the conclusion that FIM plays a role in the monocytosis developing during infection with BCG.
Topics: Animals; Cell Count; Leukocytosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Monocytes; Mycobacterium Infections; Mycobacterium bovis; Peptides
PubMed: 2199369
DOI: No ID Found