Distinct ongoing Ig heavy chain rearrangement processes in childhood B- precursor acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is thought to arise from the clonal expansion of a single transformed precursor cell. However, an oligoclonal Ig heavy chain (IgH) rearrangement pattern has been observed in 30% of ALL patients and was shown to be the result of ongoing rearrangement events. The extent and nature of these ongoing rearrangement processes in individual patients has so far remained obscure. We performed a detailed analysis of leukemic VHDJH rearrangements in three children with B-precursor ALL at diagnosis and one B-lymphoid blast crisis of a child with Ph+ chronic myeloid leukemia at diagnosis and relapse. The children were selected because they presented with multiple IgH rearrangements on Southern blot analysis. Polymerase chain reaction analysis of leukemic cells from two B-precursor ALL patients showed exclusively two groups of related sequences resulting from VH gene replacement events. Most VH gene replacements involved 3' located acceptor VH genes. Analysis of cells from the other B-precursor ALL patient showed exclusively related sequences as a result of VH gene joinings to a pre-existing DJH rearrangement. In the B-lymphoid blast crisis, a single germline precursor cell had generated multiple unrelated rearrangements and additional groups of related rearrangements resulting from VH to DJH joinings. Direct proof for the VH to DJH joining mechanism was obtained by amplification of the expected preexisting DJH rearrangements. Our findings suggest that the pattern of ongoing rearrangements in an individual patient reflects the IgH rearrangement status of the precursor cell at the time of malignant transformation. Sequence analysis of VHDJH rearrangements at diagnosis may therefore allow a prediction of the reliability of complementarity determining region 3 probes for the detection of minimal residual disease.     

Divalent cation-dependent and independent surface expression of thrombospondin on thrombin-stimulated human platelets

Thrombospondin (TSP), a platelet alpha-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concentrations (1 X 10(8)/mL), a divalent ion-dependent, low-capacity mechanism predominates. At higher cell concentrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for greater than 90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endogenous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet- surface expression of the endogenous TSP pool.     

Platelet membrane glycoprotein IIb heavy chain forms a complex with glycoprotein IIIa that binds Arg-Gly-Asp peptides

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-IIIa heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioiodinated platelets were treated with 1% 2-mercaptoethanol for 18 hours at 4 degrees C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipeptide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-IIIa complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-IIIa complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-IIIa complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-IIIa complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.     

Purging leukemic cells from simulated human remission marrow with alkyl- lysophospholipid

Alkyl-lysophospholipids (ALP) are analogues of 2- lysophosphatidylcholine that have been reported to have selective antitumor activity. These compounds could potentially be useful in purging bone marrow of leukemic cells in autologous marrow transplantation in acute leukemia. To determine the efficacy of pharmacological purging by ALP, we have designed a human assay system to mimic the conditions expected in the clinical setting of autotransplantation using remission marrow. A simulated remission marrow (SRM) was prepared by mixing normal marrow cells and HL60 cells in a ratio of 1,000:1. The effect of cryopreservation on ALP-treated normal, HL60, and SRM cells was examined. In separate experiments, ALP significantly reduced the number of clonogenic HL60 cells with no effect on normal marrow progenitors. The effect of ALP was more apparent after cryopreservation. Incubation of HL60 cells with 50 micrograms/mL ALP for four hours followed by cryopreservation resulted approximately in a 3 log reduction of clonogenic HL60 cells. ALP also selectively purged the small number of leukemic cells from SRM. In SRM, the data suggested that ALP had indirect cytotoxic activity on leukemic cells by enhancing the cytotoxic activity of monocytes in addition to its direct effect. We found no evidence that clonogenic HL60 cells decreased because of induction of differentiation by ALP. These data indicated that treatment of marrow cells with ALP offers an efficient means to eliminate leukemic cells from the graft.     

Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell- free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spontaneous regression of a monoclonal proliferation of large granular lymphocytes associated with reversal of anemia and neutropenia

A 43-year-old male with a phenotypically homogeneous, expanded subset of T cells presented in 1981 with anemia and neutropenia. The surface antigen phenotype of 99% of the peripheral blood lymphocytes was T3+, T8+, T4-, and they were morphologically large granular lymphocytes (LGL). The same cells comprised 37% of the marrow nucleated cells. Eight months after he presented, the peripheral blood T8+, LGL diminished spontaneously, and the anemia and neutropenia completely resolved. The patient remains hematologically normal as of October 1984. To determine if the T8+, LGL represented a clonal expansion, DNA from peripheral blood lymphocytes collected and cryopreserved when the patient was neutropenic and anemic, and when he was hematologically normal, was analyzed for clonal T-cell antigen receptor gene rearrangements. Using Southern blot analysis, a clonal DNA rearrangement was demonstrated, and this clone diminished but was still demonstrable in peripheral blood lymphocytes collected in 1984. The above observations implicate the expanded T8+, LGL in the pathogenesis of the neutropenia and anemia, yet the exact mechanism remains to be elucidated.     

Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.     

In vitro studies of lactoferrin and murine granulopoiesis

Human lactoferrin (LF) has been reported to inhibit in vitro granulopoiesis by means of decreasing colony-stimulating activity production by monocytes. We performed a series of experiments to determine if the reported experimental results could be replicated using highly purified murine LF and murine target cells. Three different types of experiments were performed. (1) Medium was conditioned by lung, femoral shaft, and adherent peritoneal cells in the presence and absence of LF, and the granulopoietic stimulating activity in the conditioned media was assayed by means of a 7-day agar colony assay and a 3-day liquid slide chamber assay, which quantitates 3H-TdR incorporation into DNA. (2) In cultures stimulated by an underlayer of adherent peritoneal cells, marrow cell colony formation in agar was determined after 7 days of culture in the presence or absence of LF. (3) LF was added to 3-day liquid marrow cell cultures that had been stimulated by lung or femoral shaft conditioned media. In all experimental situations, highly purified, iron-saturated LF in concentrations up to 10(-7) M had no effect on in vitro granulopoiesis. These results do not support LF's reputed regulatory role in granulopoiesis.