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Biomedical subjects

I D Bernstein

Publications and source records attributed to I D Bernstein.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of flt3 ligand on acute myeloid and lymphocytic leukemic blast cells from children.

A ligand for the flt3 tyrosine kinase receptor (flt3R) has recently been cloned. Forty-three cases of childhood acute myeloid leukemia (AML) and 27 cases of childhood acute lymphocytic leukemia (ALL) were examined by flow cytometric analysis for cell-surface flt3R and proliferative response in vitro to flt3 ligand (flt3L). Flt3R was commonly expressed on the cell surface of leukemic cells from all AML subclasses and B-ALL, but we did not detect cell-surface flt3R on T-ALL. Flt3L alone induced the proliferation of the monocytic AML-M5 cells and some erythroleukemic AML-M6 cells. Some isolated instances of weak proliferative responses were also noted in other AML subclasses. Interleukin-4 (IL-4) alone inhibited the proliferation of a group of AML-M5 cells and, when combined with flt3L, suppressed the proliferative effect of flt3L. In general, B-ALL and T-ALL cells failed to respond to flt3L alone or in the presence of combinations of IL-2, IL-3, or IL-7.

Acute Disease↗
PubMed1995

Primitive human hematopoietic precursors express Bcl-x but not Bcl-2.

Bcl-2 and its homologue, bcl-xL, encode membrane-associated proteins that suppress programmed cell death of hematopoietic cell lines after growth factor withdrawal, and are expressed in hematopoietic precursor cells. To better understand the maintenance of long-term survival in the hematopoietic stem cell population, we evaluated the expression patterns of Bcl-2 and Bcl-x in primitive hematopoietic precursor populations. Hematopoietic precursor cells expressing CD34 (CD34+) and lacking maturation-linked surface antigens (lin-) were isolated from adult human bone marrow using two-color immunofluorescence cell sorting and fractionated on the basis of forward light scatter characteristics into blast-sized and small to medium lymphocyte-sized cell populations. Bcl-2 expression was shown in 78% to 90% of CD34+ lin- blast-sized cells versus less than 10% of small to medium lymphocyte-sized CD34+ lin- cells by immunohistochemical analysis. Small to medium lymphocyte-sized CD34+ lin- cells were further enriched for primitive precursors by selecting cells that lacked expression of CD38 (CD34+ lin- CD38-). In parallel experiments, only 1% to 4% of CD34+ lin- CD38- cells expressed Bcl-2, whereas 45% to 56% of these cells generated colony-forming cells. In contrast, > or = 94% of cells in all bone marrow subpopulations studied expressed Bcl-x protein. Both alternatively spliced bcl-x transcripts, bcl-xL and bcl-xs, were present. Our data show that the most primitive hematopoietic precursors express Bcl-x but not Bcl-2. Thus, the functional bcl-2 homologue, bcl-xL, may be essential for the long-term survival of the hematopoietic stem cell population.

ADP-ribosyl Cyclase↗
PubMed1995

Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors.

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.

3T3 Cells↗
PubMed1995

Cellular biology of acute myelogenous leukemia.

The acute myelogenous leukemias (AMLs) are a heterogeneous group of disorders with diverse morphologic, histochemical, and antigenic characteristics. In this article, we discuss the heterogeneous nature of AML with regard to its cellular level of origin, cell surface antigen expression, and hematopoietic growth factor responsiveness.

Antigens, Neoplasm↗
PubMed1995

Cell surface expression of the multidrug resistance P-glycoprotein (P-170) as detected by monoclonal antibody MRK-16 in pediatric acute myeloid leukemia fails to define a poor prognostic group: a report from the Childrens Cancer Group.

Expression of the multidrug resistance (MDR-1) gene product, P-glycoprotein (P-170), and the stem cell antigen, CD34, at diagnosis were determined using monoclonal antibodies (MoAbs) MRK-16 and 12.8 respectively, in 130 pediatric acute myeloid leukemia (AML) patients entered onto Childrens Cancer Group (CCG) study CCG-2891. Fluorescein isothiocyanate (FITC) as a second step reagent was employed for the measurement of P-170 expression since it is commonly used in clinical laboratories. Nine of 30 (30%) infant ( < 1 year of age) de novo specimens expressed P-170 at levels > or = 20% of control cells. In contrast, eight of 100 (8%) AML samples from older children ( > or = 1 year of age) expressed the multidrug resistance surface protein at diagnosis. With the exception of one infant, all de novo samples that expressed P-170 also expressed CD34. Pediatric patients of any age with positive P-170 expression using MoAb MRK-16 with a FITC-conjugated second step reagent fared no worse than remaining patients treated on the same treatment with regard to induction failure, incidence of relapse, event-free survival, or overall survival. Further investigation is necessary to determine whether P-170 assay systems with greater sensitivity will distinguish pediatric AML patients with poor prognosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Cell surface expression of c-kit receptors by childhood acute myeloid leukemia blasts is not of prognostic value: a report from the Childrens Cancer Group.

The prognostic significance of c-kit receptor expression on leukemic blast cells was determined in 122 children with acute myeloid leukemia (AML) entered onto Childrens Cancer Group protocol 213. Clinical and laboratory characteristics as well as outcome were analyzed according to the percentage of blast cells expressing c-kit receptors and the relative number of c-kit receptors per cell as determined by indirect immunofluorescence. c-kit receptor expression was strongly associated with the expression of the CD34 antigen. However, contrary to findings in adult patients with AML, c-kit receptor expression by childhood AML blast cells was not predictive of a poor response to therapy.

Acute Disease↗
PubMed1994

A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors.

Members of the Notch gene family have been shown to mediate cell-fate decisions by multipotent precursors in a number of different systems. To determine whether members of this family might play a similar role in hematopoiesis, we asked if homologues of the Notch gene are expressed in human hematopoietic precursors. Using degenerate oligonucleotides corresponding to conserved amino acid sequences in known Notch homologues as primers for the polymerase chain reaction (PCR), we demonstrated that at least one Notch homologue is expressed in human bone marrow CD34+ cells, a population enriched for hematopoietic precursors. Cloning and sequencing of the PCR products identified this Notch homologue as TAN-1, a member of the Notch family previously cloned from a T-cell leukemia with a translocation involving this gene. Subsequent evaluation of bone marrow hematopoietic cells for TAN-1 expression using a reverse transcription-PCR assay confirmed the expression of TAN-1 in CD34+ hematopoietic precursors, including the immature subset that lacks expression of lineage-associated antigens (CD34+lin-). These findings, together with the known role of Notch homologues in other systems, suggest that members of the Notch family, including TAN-1, may be involved in mediating cell-fate decisions during hematopoiesis.

Animals↗
PubMed1994

GATA-1 is expressed in acute erythroblastic leukaemia.

Previous studies have demonstrated the expression of GATA-1 (a DNA-binding nuclear protein) in erythrocytes, megakaryocytes, eosinophils, basophils, mast cells, early marrow progenitor cells and in mouse and human erythroid leukaemia cell lines. We studied 31 bone marrow specimens from patients with acute myeloid leukaemia (AML) for GATA-1 expression by reverse transcriptase/polymerase chain reaction (RT/PCR) analysis. GATA-1 expression was detected in all of the patients with erythroleukaemia, and in one of nine patients with megakaryoblastic leukaemia, but absent from 17 patients with French-American-British (FAB) M1-5 leukaemia. In AML, GATA-1 expression is indicative of differentiation to the erythroid and possibly megakaryocytic lineages, analogous to its expression in normal haemopoiesis.

Bone Marrow↗
PubMed1994

Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes.

Cytogenetic studies have previously identified abnormalities of chromosome band 11q23 in many cases of infant acute leukemia. Recent studies by ourselves and others have demonstrated breakpoint clustering in acute leukemias bearing translocations involving 11q23, and a Drosophila trithorax gene homologue (called MLL, HRX, or ALL-1) has been shown to span the 11q23 breakpoints of these translocations. To determine if this gene is affected in infant acute myeloid leukemia (AML), we have analyzed 26 infant AML cases for molecular alterations of this 11q23 gene. 15 out of 26 cases studied (58%) showed rearrangement of the MLL gene at the molecular level, and these rearrangements were clustered within an approximately 11-kb region containing nine exons of this gene. Moreover, 14 of the 15 cases with 11q23 rearrangements (93%) had myelomonocytic or monocytic phenotypes (M4 or M5 FAB subtypes, respectively), both of which are associated with a poor prognosis in childhood AML. In contrast, only 1 of 11 nonrearranged cases had an M4 or M5 phenotype (P = 0.00002). Rearrangement also correlated significantly with hyperleukocytosis (P = 0.02), another clinical parameter associated with poor outcome in this disease. Our results demonstrate that molecular rearrangements of MLL are common in M4 or M5 infant AML, and suggest that alteration of this gene may result in abnormal control of proliferation and differentiation in monocytic progenitor cells.

Acute Disease↗
PubMed1994

Chemotherapy for induction of remission of childhood acute myeloid leukemia followed by marrow transplantation or multiagent chemotherapy: a report from the Childrens Cancer Group.

PURPOSE: In an effort to evaluate the usefulness of bone marrow transplantation, the Childrens Cancer Group (CCG) initiated a multiinstitutional study comparing bone marrow transplantation versus chemotherapy after successful induction of remission for previously untreated children and young adults with acute myeloid leukemia. PATIENTS AND METHODS: From 1979 to 1983, 508 patients were entered onto this study and 490 were treated. After induction, patients with an HLA mixed leukocyte culture (MLC)-compatible sibling underwent bone marrow transplantation. Patients not eligible for bone marrow transplantation were eligible for randomization to two chemotherapy maintenance regimens. All patients undergoing bone marrow transplantation were conditioned with cyclophosphamide and total-body irradiation (TBI). Methotrexate was used to prevent or modify graft-versus-host disease (GVHD). RESULTS: Three hundred eighty-one patients achieved bone marrow remission (78%). Eighty-nine patients had an HLA/MLC-compatible sibling donor and were eligible for bone marrow transplantation, and 252 had no match. Comparison of survival estimates for patients eligible for transplantation versus not eligible at 3 years (52% v 41%), 5 years (50% v 36%), and 8 years (47% v 34%) showed a significant difference in favor of bone marrow transplantation (P < .05). Disease-free survival (DFS) demonstrated similar results. Application of a cure model to the results showed a better outcome for those eligible for transplantation (P = .04). Patients randomized between the two chemotherapy regimens did not show any significant difference between those treated with a continuous maintenance versus a cyclic regimen (P = .16). CONCLUSION: Children and young adults who successfully achieved a remission with multiple-agent chemotherapy who had an HLA/MLC-compatible donor and were thus eligible for an allogeneic bone marrow transplant had better survival than those not eligible for transplantation.

Acute Disease↗
PubMed1994

Isolation of human hematopoietic stem cells.

Human hematopoietic stem cells are contained within a population of marrow cells that expresses the CD34 antigen but not other antigens associated with commitment to specific lineages. Evidence that stem cells capable of maintaining long-term hematopoiesis are within this CD34+ lineage-negative (Lin-) population is reviewed, including in vivo studies in humans and nonhuman primates. In vitro studies of the CD34+ Lin- population have indicated that the blast-sized cells, which are presumably in cycle, proliferate and give rise to colony-forming cells in the presence of combinations of growth factors, including c-kit ligand and interleukin-3 (IL-3). Recent studies have examined the factors required for the growth of the quiescent subset of the CD34+ Lin- cells, identified as small to medium lymphocyte-sized cells that resist treatment with 4-hydroperoxycyclophosphamide, a known characteristic of the marrow-repopulating cell. These studies have shown that an interaction with marrow stromal cells is required, in addition to c-kit ligand and IL-3, to induce these cells to proliferate and form multiple colony-forming cells. These studies have further indicated that this effect of stroma is mediated by a soluble factor(s). This activity may represent a novel factor(s) and/or a novel combination of growth factors.

Antigens, CD↗
PubMed1993

Ontogeny of hematopoietic stem cell development: reciprocal expression of CD33 and a novel molecule by maturing myeloid and erythroid progenitors.

We have identified a molecule expressed by human marrow granulocyte/monocyte colony-forming cells (CFU-GM), erythroid colony-forming cells (CFU-E), and erythroid burst-forming units (BFU-E), but not their precursors detectable in long-term bone marrow culture. This antigen, detected by flow microfluorimetry using monoclonal antibody 7B9, is coexpressed with CD33 on many CD34+ CFCs, although only the 7B9 antigen was detected on a portion of BFU-E and CFU-E, whereas only CD33 was found on a portion of CFU-GM. Antibody 7B9 appears to be useful for isolating subsets of progenitors based on their common or selective expression of 7B9 antigen and CD33.

Antibodies, Monoclonal↗
PubMed1993

Hematopoietic precursors resistant to treatment with 4-hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity.

We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC-treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC-resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.

ADP-ribosyl Cyclase↗
PubMed1993

Strain-dependent leakiness of mice with severe combined immune deficiency.

Mice with immunodeficiency provide an excellent in vivo model for cell transfer experiments. In this study, we compare the extent of immune deficiency of the original CB17 severe combined immune-deficient (SCID) mice with that of two other strains of immune-deficient mice, the recently developed C3H SCID mice and the beige/nude/X-linked immune-deficient (BNX) mice. Detectable levels of serum lg (higher than 0.4 microgram/ml) were found in 79% of CB17 SCID mice studied (n = 24) and in all BNX mice (n = 12); some leaky CB17 SCID mice had normal levels of Ig. In contrast, only 15% of C3H SCID mice (n = 61) had detectable serum lg; the highest Ig level in this strain was 9.6 micrograms/ml. Age had no effect on serum Ig concentrations of C3H SCID mice; in contrast, all old (> 1-year-old) CB17 SCID mice studied had detectable levels of serum Ig. Transfer of syngeneic, normal, neonatal thymocytes increased serum Ig of SCID mouse origin to near-normal levels in all CB17 SCID mice but had no effect on serum lg concentrations in C3H SCID mice. Treatment with anti-asialo-GM-1 antiserum to abrogate NK cell activity increased serum Ig levels in 37% of CB17 SCID mice but had no effect on Ig production in C3H SCID mice. Flow cytometric analysis failed to identify mature T or B cells in C3H SCID mice; in contrast, some leaky CB17 SCID mice had detectable numbers of T and B cells in the peritoneal cavity. After immunization with bacteriophage phi X 174, neither C3H nor CB17 SCID mice, including leaky mice, produced specific antibody to phage. In contrast, BNX mice produced small but significant amounts of anti-phage antibody. These results indicate that, of the three strains of immune-deficient mice, C3H SCID mice have the most severe immune defect. We predict that C3H SCID mice will be best suited for cell transfer experiments.

Animals↗