Novel 2 Marker Sandwich Immunoassay for the Detection of Precursor- and Mature Granulocyte-Derived Exosomes in Peripheral Blood: A Preliminary Case Study

Introduction: In order to assess hematopoietic precursor cells, bone marrow biopsies are routinely conducted, which is painful for the patient and is not applicable for frequent testing. However, precursor, maturating, and matured cells are known to produce exosomes, which are released into peripheral blood. By analyzing these exosomes in plasma, bone marrow condition may be assessed noninvasively. Myeloid precursor cells express different surface markers than when they have matured. It is reasonable to speculate that these markers would be incorporated into exosomes. In this study, we developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) by using a combination of monoclonal antibodies against 2 different leukocyte antigens, to account for the number of exosomes in plasma. Methods In 6 patients undergoing chemotherapy, venous blood was drawn at regular intervals before, during, and after respective chemotherapies was given. Various clones of monoclonal antibodies against CD11b, CD33, CD34, CD35, CD81, and CD117 were obtained, and antibodies were biotinylated by EZ link Sulfo-NHS-LC-Biotin. Anti-brain antibodies were switched with anti-leukocyte antibodies, and exosome activity was trended. Results CD11b+ and CD 117+ plates showed small changes during chemotherapy (within +/- 50% of the initial value), while plasma WBC count showed a dramatic decrease during chemotherapy. In the recovery phase of WBC after chemotherapy, CD11b+ and CD16+showed a greater than 2-fold increase, with the up-trend taking place earlier than its plasma WBC counterparts. CD 117+ CD33+ also showed an up-trend, but was slower than their CD11b+ and CD16+ counterpart. Finally, each exosome value was divided by other markers to calculate the ratio, and compared between pre and post chemotherapy blood. In half of the cases, there was not a difference, versus an almost double premature/mature ratio in post-chemotherapy blood in the other half of patients. Conclusion From data collected from pre-chemotherapy studies, the lack of correlation between ELISA data compared to plasma WBC data, indicates that plasma exosome analysis might not be suitable for the monitoring of acute bone marrow suppression. Additionally, in the recovery phase, surface markers correlation with WBC was present in only some of the markers, indicating that the assay sensitivity is dependent on the marker combinations. Overall, both myeloid precursor- and mature granulocyte-derived exosomes were increased during the recovery from chemotherapy in 3 out of 5 patients, and exosome profiles were different after chemotherapy in 2 out of 5 patients. Disclosures Mitsuhash: NanoSomiX:Current Employment.

Genetically distinct leukemic stem cells in human CD34− acute myeloid leukemia are arrested at a hemopoietic precursor-like stage

Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34−, there are multiple, nonhierarchically arranged CD34+ and CD34− LSC populations. Within CD34− and CD34+ LSC–containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34− LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34− mature granulocyte–macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.

Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog

The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.

Large Field Virtual Microscopy as a Tool for Quality Control and Blast Definitions in MDS and Leukemia. From the International Working Group on MDS.

All current classifications for myelodysplastic syndromes (MDS) require the identification of the blast population. From the FAB (1976) to the WHO (1999) new criteria such as the results of cytogenetic analysis have been introduced but the percentage of blasts remains a major factor in diagnosis, subclassification and prognosis. The definition of a blast cell is still based on those proposed in 1976 by the FAB group, but it is unclear that it has been applied in the same manner worldwide. Since the WHO has changed the definition for AML (minimum criterion is 20% blasts) and since RAEB has been divided into two groups (5 to 9 and 10 to 19% blasts respectively) it has become clear that the definition of blasts of granulocyte lineage should be more precise. Definition: Experts in morphology from the IWG on MDS have proposed that all cells from undifferentiated blast (without granules) to but not including the promyelocyte would qualify as “blasts”. A promyelocyte starts with the appearance of a “clearly visible Golgi zone” independently of the number of granules. Mature granulocyte starts with disappearance of cytoplasmic basophilia and more mature nucleus. Methodology: To verify agreement between experts on this new definition, a unique digital picture was produced from the bone marrow smear of an AML patient with FAB M2 by the association of 150 consecutive native pictures (600x800 pixels). The mosaic picture is 8340x2386 pixels (about 30 M in jpeg format or 16 M in pdf). All the 265 nucleated cells have been numbered and a drop down menu for choices was included. The picture was placed on the server of the MDS Foundation (http://www.mds-foundation.org/goasguenfollowup) and after evaluation the results were automatically sent to the MDS foundation center and registered. The five experts evaluated exactly the same cells (numbered from 1 to 265) and results were submitted for statistical analysis. Results: for 176 cells (66.6%) there was complete concordance (5/5) and for 60 others (22.7%) concordance was 4/5. If we consider that concordance of at least 4/5 is acceptable, we obtained 89.4% good concordance. Moreover, 23 cells (8.7%) had a concordance of 3/5 while only 5 (1.89%) had a concordance of 2/5. We conclude that these definitions of blasts and promyelocytes are reproducible and may help to standardize the classification of AML and MDS. All experts would have produced the same diagnosis since standard deviations of the percentages of various cell types were very low. Conclusion: the production of large field digital pictures may be very useful for education and quality control in morphology. In addition, use of such images may help to provide a new approach to difficult cells and may be useful in proposing new criteria for classification. Table: Minimum, maximum and mean percentage of cells for 5 experts % of cells Minimum Maximum Mean Standard deviation Blasts 58.1 61.7 59.9 1.54 Promyelocytes 12.2 22.3 17.7 4.82 Matures cells 14.4 21.6 18.8 3.12

Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

Synthesis of granulocyte colony-stimulating factor and its requirement for terminal divisions in chronic myelogenous leukemia.

In this paper we demonstrate that maturing neoplastic cells from patients with chronic myelogenous leukemia (CML) constitutively produce G-CSF and are also receptive for this molecule. G-CSF functions as an autocrine growth factor in stable phase CML, and thus is responsible for divisions of maturing leukemic cells leading to an expansion of the compartment of mature cells. This observation is well in line with in vivo features of CML in stable phase, i.e., the hyperplasia of the mature granulocyte compartment. In acute blastic phase of CML expression of the G-CSF gene seems to be less common and not related to autonomous blast growth.

Recombinant human granulocyte colony-stimulating factor as an activator of human granulocytes: potentiation of responses triggered by receptor- mediated agonists and stimulation of C3bi receptor expression and adherence

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N- formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl- methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.

Recombinant human granulocyte colony-stimulating factor as an activator of human granulocytes: potentiation of responses triggered by receptor- mediated agonists and stimulation of C3bi receptor expression and adherence

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N- formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl- methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.

Subpopulation heterogeneity in human acute myeloid leukemia determined by monoclonal antibodies

The leukemic population in 63 patients with acute myeloid leukemia (AML) was studied with 15 monoclonal antibodies that detect lineage- related and stage-related antigens on normal hemopoietic cells. Indirect immunofluorescence and fluorescence-activated cell sorting showed that subpopulations of leukemic cells reacted with some or all antibodies, but the percentage of cells reacting with a single antibody varied widely among patients. The composite antigenic phenotype of the various cases, as determined by immunofluorescence assay, did not correlate with the French-American-British morphological classification. Furthermore, some cells in each case failed to express any antigen normally expressed on myelomonocytic precursors from the level of the early CFU-GM to the mature granulocyte or monocyte. In double-fluorescence experiments, the individual cells expressed none, one, or both antigens. These results demonstrate that there is considerable subpopulation heterogeneity in AML. This heterogeneity may considerably limit or complicate the use of monoclonal antibodies for diagnosis, prognosis, and treatment of acute nonlymphocytic leukemia (ANLL).