Impact of FLT3 Receptor (CD135) Detection by Flow Cytometry on Clinical Outcome of Adult Acute Myeloid Leukemia Patients.

BACKGROUND: The significance of FMS-like tyrosine kinase 3 (FLT3)-ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3-ITD mutation, and to identify its role in minimal residual disease. PATIENTS AND METHODS: CD135 was measured by flow cytometry on leukemic blasts of 257 adults with de novo AML. High expression of CD135 ≥ 20% was correlated with clinical, laboratory, and other prognostic factors that influenced treatment outcome. FLT3-ITD mutation was tested by PCR. RESULTS: The frequency of CD135 expression was 138 (53.7%) of 257. FLT3-ITD was detected in (21.4%). Positive CD135 expression was associated with high total leukocyte count (P = .006), platelet count (P = .003), monocytic leukemia (P < .001), and CD34 (P = .008) and CD117 (P = .006) expression. CD135 expression ≥ 25% was a predictor of FLT3-ITD mutation (P = .03). CD135 overexpression was a negative predictor of complete remission and of postinduction minimal residual disease at days 14 and 28 (P < .001). CD135 had an adverse impact on overall and disease-free survival (68.5% vs. 15%, P = .002). Multivariate analysis indicated CD135 was the sole independent prognostic factor for overall survival (hazard ratio = 2.49; 95% confidence interval, 1.855-3.345; P < .001). CONCLUSION: CD135 is emerging as a prognostic factor, a new marker for minimal residual disease, and a potential novel therapeutic target of AML.

Gene expression and proliferation biomarkers for antidepressant treatment resistance.

The neurotrophic hypothesis of depression suggests an association between effects on neuroplasticity and clinical response to antidepressant drug therapy. We studied individual variability in antidepressant drug effects on cell proliferation in lymphoblastoid cell lines (LCLs) from n=25 therapy-resistant patients versus n=25 first-line therapy responders from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. Furthermore, the variability in gene expression of genes associated with cell proliferation was analyzed for tentative candidate genes for prediction of individual LCL donor's treatment response. Cell proliferation was quantified by EdU (5-ethynyl-2'-deoxyuridine) assays after 21-day incubation of LCLs with fluoxetine (0.5 ng µl-1) and citalopram (0.3 ng µl-1) as developed and described earlier. Gene expression of a panel of candidate genes derived from genome-wide expression analyses of antidepressant effects on cell proliferation of LCLs from the Munich Antidepressant Response Signature (MARS) study was analyzed by real-time PCR. Significant differences in in vitro cell proliferation effects were detected between the group of LCLs from first-line therapy responders and LCLs from treatment-resistant patients. Gene expression analysis of the candidate gene panel revealed and confirmed influence of the candidate genes ABCB1, FZD7 and WNT2B on antidepressant drug resistance. The potential of these genes as tentative biomarkers for antidepressant drug resistance was confirmed. In vitro cell proliferation testing may serve as functional biomarker for individual neuroplasticity effects of antidepressants.

Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.

Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are critical for initiating and controlling the immune response. However, study of DC, particularly pDC, function is hampered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation preferentially favor the production of cDCs over pDCs. Here, we demonstrated that pDCs could be efficiently generated in vitro from common lymphoid progenitors (CLPs) using Flt3 ligand (FL) in three different culture systems, namely feeder-free, BM-feeder and AC-6-feeder. This was in stark contrast to common DC progenitors (CDPs), in which cDCs were prominently generated under the same conditions. Moreover, the efficiency and function of pDCs generated from these three systems varied. While AC-6 system showed the greatest ability to support pDC development from CLPs, BM-feeder system was able to develop pDCs with better functionality. pDCs could also be expanded in vivo using hydrodynamic gene transfer of FL, which was further enhanced by the combined treatment of FL and IFN-α. Interestingly, IFN-α selectively promoted the proliferation of CLPs and not CDPs, which might contribute to enhanced pDC development. Together, we have defined conditions for in vitro and in vivo generation of pDCs, which may be useful for investigating the biology of pDCs.

Lineage-related and particle size-dependent cytotoxicity of chitosan nanoparticles on mouse bone marrow-derived hematopoietic stem and progenitor cells.

Chitosan nanoparticles (CSNPs) have potential applications in stem cell research. In this study, ex vivo cytotoxicity of CSNPs on mouse bone marrow-derived (MBMCs) hematopoietic stem and progenitor cells (HSPCs) was determined. MBMCs were exposed to CSNPs of different particle sizes at various concentrations for up to 72 h. Cytotoxicity effect of CSNPs on MBMCs was determined using MTT, Live/Dead Viability/Cytotoxicity assays and flow cytometry analysis of surface antigens on HSCs (Sca-1(+)), myeloid-committed progenitors (CD11b(+), Gr-1(+)), and lymphoid-committed progenitors (CD45(+), CD3e(+)). At 24 h incubation, MBMCs' viability was not affected by CSNPs. At 48 and 72 h, significant reduction was detected at higher CSNPs concentrations. Small CSNPs (200 nm) significantly reduced MBMCs' viability while medium-sized particle (∼400 nm) selectively promoted MBMCs growth. Surface antigen assessment demonstrated lineage-dependent effect. Significant decrease in Sca-1(+) cells percentage was observed for medium-sized particle at the lowest CSNPs concentration. Meanwhile, reduction of CD11b(+) and Gr-1(+) cells percentage was detected at high and intermediate concentrations of medium-sized and large CSNPs. Percentage of CD45(+) and CD3e(+) cells along with ROS levels were not significantly affected by CSNPs. In conclusion, medium-sized and large CSNPs were relatively non-toxic at lower concentrations. However, further investigations are necessary for therapeutic applications.

Arsenite Interacts with Dibenzo[def,p]chrysene (DBC) at Low Levels to Suppress Bone Marrow Lymphoid Progenitors in Mice.

Arsenite (As(+3)) and dibenzo[def,p]chrysene (DBC), a polycyclic aromatic hyrdrocarbon (PAH), are found in nature as environmental contaminants. Both are known to individually suppress the immune system of humans and mice. In order to determine their potential interactive and combined immunosuppressive effects, we examined murine bone marrow (BM) immune progenitor cells' responses following combined oral exposures at very low levels of exposure to As(+3) and DBC. Oral 5-day exposure to DBC at 1 mg/kg (cumulative dose) was found to suppress mouse BM lymphoid progenitor cells, but not the myeloid progenitors. Previously established no-effect doses of As(+3) in drinking water (19 and 75 ppb for 30 days) produced more lymphoid suppression in the bone marrow when mice were concomitantly fed a low dose of DBC during the last 5 days. The lower dose (19 ppb) As(+3) had a stronger suppressive effect with DBC than the higher dose (75 ppb). Thus, the interactive toxicity of As(+3) and DBC in vivo could be As(+3) dose dependent. In vitro, the suppressive interaction of As(+3) and DBC was also evident at low concentrations (0.5 nM), but not at higher concentrations (5 nM) of As(+3). These studies show potentially important interactions between As(+3) and DBC on mouse BM at extremely low levels of exposure in vivo and in vitro.

Chemokine treatment rescues profound T-lineage progenitor homing defect after bone marrow transplant conditioning in mice.

Development of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.

Synergistic anticancer effects of Pam3CSK4 and Ara-C on B-cell lymphoma cells.

PURPOSE: The low immunogenicity of many cancer cells and the immunosuppression by various cancers and anticancer therapies have been an obstacle in the development of efficacious immunotherapies. Our goal was to test whether Toll-like receptor (TLR) agonists and anticancer chemotherapeutic agents synergize in rendering tumor cells more immunogenic. EXPERIMENTAL DESIGN: We treated B-cell lymphoma cells with the TLR1/2 agonist Pam3CSK4 and the genotoxic anticancer agent 1-ß-D-arabinofuranosylcytosine (Ara-C). The effects on the immunogenicity of tumor cells were measured in transfer experiments and in vitro studies. RESULTS: The treatment of B-cell lymphoma cells with the TLR1/2 agonist Pam3CSK4 enhanced the anticancer effects of the genotoxic agent Ara-C. Mice injected with cotreated tumor cells survived longer than mice challenged with Pam3CSK4 or Ara-C-treated cells. Administration of Pam3CSK4 or Ara-C reduced the tumor load of mice injected with tumor cells. Cotreatment had no effect on the rate of apoptosis or proliferation of Ara-C-treated cells, but upregulated the expression of several immunomodulatory molecules. Consistent with an increased immunogenicity of Pam3CSK4 and Ara-C-treated B-cell lymphoma cells, rejection of cotreated tumor cells required natural killer cells and T cells. We demonstrate that the upregulation of immunomodulatory molecules in response to Pam3CSK4 and Ara-C depended in part on NF-κB. CONCLUSION: TLR agonists can increase the efficacy of conventional cancer therapies by altering the immunogenicity of B-cell lymphoma cells.

Arsenite selectively inhibits mouse bone marrow lymphoid progenitor cell development in vivo and in vitro and suppresses humoral immunity in vivo.

It is known that exposure to As(+3) via drinking water causes a disruption of the immune system and significantly compromises the immune response to infection. The purpose of these studies was to assess the effects of As(+3) on bone marrow progenitor cell colony formation and the humoral immune response to a T-dependent antigen response (TDAR) in vivo. In a 30 day drinking water study, mice were exposed to 19, 75, or 300 ppb As(+3). There was a decrease in bone marrow cell recovery, but not spleen cell recovery at 300 ppb As(+3). In the bone marrow, As(+3) altered neither the expression of CD34+ and CD38+ cells, markers of early hematopoietic stem cells, nor CD45-/CD105+, markers of mesenchymal stem cells. Spleen cell surface marker CD45 expression on B cells (CD19+), T cells (CD3+), T helper cells (CD4+) and cytotoxic T cells (CD8+), natural killer (NK+), and macrophages (Mac 1+) were not altered by the 30 day in vivo As(+3) exposure. Functional assays of CFU-B colony formation showed significant selective suppression (p<0.05) by 300 ppb As(+3) exposure, whereas CFU-GM formation was not altered. The TDAR of the spleen cells was significantly suppressed at 75 and 300 ppb As(+3). In vitro studies of the bone marrow revealed a selective suppression of CFU-B by 50 nM As(+3) in the absence of apparent cytotoxicity. Monomethylarsonous acid (MMA(+3)) demonstrated a dose-dependent and selective suppression of CFU-B beginning at 5 nM (p<0.05). MMA(+3) suppressed CFU-GM formation at 500 nM, a concentration that proved to be nonspecifically cytotoxic. As(+5) did not suppress CFU-B and/or CFU-GM in vitro at concentrations up to 500 nM. Collectively, these results demonstrate that As(+3) and likely its metabolite (MMA(+3)) target lymphoid progenitor cells in mouse bone marrow and mature B and T cell activity in the spleen.

Brief report: deficient thymic output in rheumatoid arthritis despite abundance of prethymic progenitors.

OBJECTIVE: To determine the frequencies of common lymphoid progenitors (CLPs) and recent thymic emigrants (RTEs) in patients with rheumatoid arthritis (RA) and healthy control subjects. METHODS: Flow cytometry was performed to determine the frequencies of CLPs and RTEs in the peripheral blood of 101 control subjects and 51 patients with RA. Thirteen of these patients were also analyzed longitudinally for 6 months after initiation of treatment with a tumor necrosis factor (TNF) inhibitor. RESULTS: A significant correlation between the frequencies of CLPs and RTEs was observed in healthy control subjects. The frequencies of both CLPs and RTEs decreased with age and correlated inversely with absolute lymphocyte numbers in peripheral blood. In patients with RA, the frequencies of RTEs were significantly decreased compared with the frequencies in control subjects. Importantly, the frequencies of CLPs were significantly higher in patients with RA compared with control subjects. Therapeutic TNF blockade further increased the frequency of CLPs, thereby normalizing thymic output, as indicated by an increase in the number of RTEs. CONCLUSION: Thymic insufficiency in RA is not attributable to an inadequate supply of progenitor cells to the thymus. Thus, insufficient numbers of RTEs could result from inadequate thymic T cell neogenesis, or alternatively, could be a consequence of high CD4+ T cell turnover, homeostatic proliferation, and subsequent dilution of the RTE population.

Bcl11a controls Flt3 expression in early hematopoietic progenitors and is required for pDC development in vivo.

Bcl11a is a transcription factor known to regulate lymphoid and erythroid development. Recent bioinformatic analysis of global gene expression patterns has suggested a role for Bcl11a in the development of dendritic cell (DC) lineages. We tested this hypothesis by analyzing the development of DC and other lineages in Bcl11a (-/-) mice. We found that Bcl11a was required for expression of IL-7 receptor (IL-7R) and Flt3 in early hematopoietic progenitor cells. In addition, we found severely decreased numbers of plasmacytoid dendritic cells (pDCs) in Bcl11a (-/-) fetal livers and in the bone marrow of Bcl11a (-/-) fetal liver chimeras. Moreover, Bcl11a (-/-) cells showed severely impaired in vitro development of Flt3L-derived pDCs and classical DCs (cDCs). In contrast, we found normal in vitro development of DCs from Bcl11a (-/-) fetal liver cells treated with GM-CSF. These results suggest that the persistent cDC development observed in Bcl11a (-/-) fetal liver chimeras reflects derivation from a Bcl11a- and Flt3-independent pathway in vivo.

Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells' sensitivity to DNA interstrand crosslinking drugs.

Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients.

Inhibitory activities of microalgal extracts against Epstein-Barr virus DNA release from lymphoblastoid cells.

This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitt's lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC(50)<0.01 µg/ml) with a high therapeutic index (>28000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC(50)=2.9 µg/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1'a was most active in reducing the cell-free EBV DNA (EC(50)=1.38 µg/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV.

[Distribution of individuals by spontaneous frequencies of lymphocytes with micronuclei. Particularity and consequences].

By micronucleus (MN) assay with cytokinetic cytochalasin B block, the mean frequency of blood lymphocytes with MN has been determined in 76 Moscow inhabitants, 35 people from Obninsk and 122 from Chelyabinsk region. In contrast to the distribution of individuals on spontaneous frequency of cells with aberrations, which was shown to be binomial (Kusnetzov et al., 1980), the distribution of individuals on the spontaneous frequency of cells with MN in all three massif can be acknowledged as log-normal (chi2 test). Distribution of individuals in the joined massifs (Moscow and Obninsk inhabitants) and in the unique massif of all inspected with great reliability must be acknowledged as log-normal (0.70 and 0.86 correspondingly), but it cannot be regarded as Poisson, binomial or normal. Taking into account that log-normal distribution of children by spontaneous frequency of lymphocytes with MN has been observed by the inspection of 473 children from different kindergartens in Moscow we can make the conclusion that log-normal is regularity inherent in this type of damage of lymphocytes genome. On the contrary the distribution of individuals on induced by irradiation in vitro lymphocytes with MN frequency in most cases must be acknowledged as normal. This distribution character points out that damage appearance in the individual (genomic instability) in a single lymphocytes increases the probability of the damage appearance in another lymphocytes. We can propose that damaged stem cells lymphocyte progenitor's exchange by information with undamaged cells--the type of the bystander effect process. It can also be supposed that transmission of damage to daughter cells occurs in the time of stem cells division.

Development of small-molecule PUMA inhibitors for mitigating radiation-induced cell death.

PUMA (p53 upregulated modulator of apoptosis) is a Bcl-2 homology 3 (BH3)-only Bcl-2 family member and a key mediator of apoptosis induced by a wide variety of stimuli. PUMA is particularly important in initiating radiation-induced apoptosis and damage in the gastrointestinal and hematopoietic systems. Unlike most BH3-only proteins, PUMA neutralizes all five known antiapoptotic Bcl-2 members though high affinity interactions with its BH3 domain to initiate mitochondria-dependent cell death. Using structural data on the conserved interactions of PUMA with Bcl-2-like proteins, we developed a pharmacophore model that mimics these interactions. In silico screening of the ZINC 8.0 database with this pharmacophore model yielded 142 compounds that could potentially disrupt these interactions. Thirteen structurally diverse compounds with favorable in silico ADME/Toxicity profiles have been retrieved from this set. Extensive testing of these compounds using cell-based and cell-free systems identified lead compounds that confer considerable protection against PUMA-dependent and radiation-induced apoptosis, and inhibit the interaction between PUMA and Bcl-xL.

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors.

The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.

Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice.

In this study we used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6h) of lymphoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in DMBA-treated mice. The duration of these changes were consistent with the blood levels of DMBA and its metabolites that were achieved by either IP or oral DMBA administration. CFU-GM and CFU-preB activities returned to control levels by 2 and 7 days after oral DMBA exposure, respectively, but remained suppressed through 7 days after IP DMBA administration. The continued presence of low levels of DMBA in the bloodstream following IP administration was associated with sustained suppression of CFU-preB, total bone marrow lymphoid cells and peripheral blood lymphocytes. The changes noted above were not observed in Cyp1b1 null mice, demonstrating the need for local DMBA metabolism in the bone marrow by Cyp1b1 to impair bone marrow CFU-preB and CFU-GM. Furthermore, these data provide evidence that myeloid-lineage cells are restored more quickly than lymphoid-lineage cells after DMBA exposure.