Biomedical subjects

A L Petzer

Publications and source records attributed to A L Petzer.

30 records · Page 2Linked to original sources

PubMed1997

Cytokine manipulation of primitive human hematopoietic cell self-renewal.

Previous studies have shown that primitive human hematopoietic cells detectable as long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs) can be amplified when CD34(+) CD38(-) marrow cells are cultured for 10 days in serum-free medium containing flt3 ligand (FL), Steel factor (SF), interleukin (IL)-3, IL-6, and granulocyte colony-stimulating factor. We now show that the generation of these two cell types in such cultures is differentially affected at the single cell level by changes in the concentrations of these cytokines. Thus, maximal expansion of LTC-ICs (60-fold) was obtained in the presence of 30 times more FL, SF, IL-3, IL-6, and granulocyte colony-stimulating factor than could concomitantly stimulate the near-maximal (280-fold) amplification of CFCs. Furthermore, the reduced ability of suboptimal cytokine concentrations to support the production of LTC-ICs could be ascribed to a differential response of the stimulated cells since this was not accompanied by a change in the number of input CD34(+) CD38(-) cells that proliferated. Reduced LTC-IC amplification in the absence of a significant effect on CFC generation also occurred when the concentrations of FL and SF were decreased but the concentration of IL-3 was high (as compared with cultures containing high levels of all three cytokines). To our knowledge, these findings provide the first evidence suggesting that extrinsically acting cytokines can alter the self-renewal behavior of primary human hematopoietic stem cells independent of effects on their viability or proliferation.

ADP-ribosyl Cyclase↗

PubMed1996

Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia.

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC-IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph-, respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.

Adult↗

PubMed1996

BCR-ABL expression in different subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic myeloid leukemia.

In patients with chronic myeloid leukemia (CML), the leukemic (BCR-ABL+/Ph+) clone typically includes cells belonging to all of the myeloid lineages and frequently some B cells. From such observations it has been inferred that the initial BCR-ABL gene rearrangement event occurs in a pluripotent hematopoietic stem cell and that the clone subsequently generated is maintained by a subpopulation of neoplastic, BCR-ABL-expressing cells that retain at least some of the defining properties of normal hematopoietic stem cells. To test this hypothesis directly, we isolated various subpopulations of CD34+ cells from fresh or cryopreserved samples of peripheral blood from 5 CML patients with high white blood cell counts, 4 of which were selected because of their exclusive content of Ph+ progenitors (both colony-forming cells and long-term culture-initiating cells [LTC-IC]). Cells in each of the CD34+ subpopulations isolated were examined for the presence of BCR-ABL mRNA using a reverse transcriptase-polymerase chain reaction technique that reproducibly gave a positive signal from single K562 cells. BCR-ABL mRNA was detected in 117 of 147 samples (80%) in which actin mRNA was demonstrable. This included 60% to 90% of a large number of individually analyzed CD34+ cells including 46 single CD34+CD71-CD38- cells and 27 single CD34+CD71+CD38+ cells from 3 patients. In 2 of these cases, the same populations also contained a very high frequency of Ph+ LTC-IC. Our findings demonstrate BCR-ABL gene expression in neoplastic cells with functional as well as surface marker characteristics of very primitive normal hematopoietic cells. This implicates the BCR-ABL gene product directly in the acquisition by these cells of properties that alter their interactions with the microenvironment and deregulate their proliferation control.

ADP-ribosyl Cyclase↗

PubMed1996

Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of > 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However, this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.

ADP-ribosyl Cyclase↗

PubMed1996

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

Animals↗

PubMed1995

Growth-inhibitory effect of 2-CdA on myeloid CFU-GM progenitors in normal human long-term bone marrow cultures and its compensation by G-CSF or IL-3.

As neutropenia is a common side effect of treatment with 2-chlorodeoxyadenosine (2-CdA), we investigated the myelosuppressive action of 2-CdA in Dexter-type human long-term bone marrow cultures (LTBMCs). LTBMCs were incubated with varying doses of 2-CdA (5 to 20 nM/L) during the first week. At 20 and 10 nM/L 2-CdA, we found a marked reduction in colony-forming unit-granulocyte/macrophage (CFU-GM) production throughout the culture period of 7 weeks (maximum reduction to 3.5% of untreated control cultures with 20 nM/L and to 27.2% with 10 nM/L, respectively). Even the lowest 2-CdA dose tested (5 nM/L) strongly reduced the number of CFU-GM progenitors during the first 3 weeks (maximum reduction to 52.4% of untreated controls), but this effect was transient, and values had recovered to normal within in 5 weeks. 2-CdA was also shown to cause a dose-dependent decrease in long-term culture-initiating cell (LTCIC) detections after 5 weeks in culture (49.6% of control cultures with 10 nM/L 2-CdA and 14% with 20 nM/L 2-CdA, respectively). When 2-CdA was added to LTBMCs initiated on preformed irradiated stromal feeder layers, similar results on CFU-GM production were obtained, indicating that the effects observed were not secondary to effects on the formation of a supportive layer. In addition, IL-6-concentrations in the supernatant of LTBMCs measured at various intervals after the addition of fresh medium with or without 2-CdA showed no significant decrease in cultures treated with 2-CdA. As neutropenia has been shown to be associated with a small but significant risk of fatal infection, we subsequently investigated the reversal potential of the 2-CdA effect by addition of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or rh interleukin-3 (rhIL-3). The weekly addition of 100 ng/mL rhG-CSF counteracted the 2-CdA-mediated decrease in CFU-GM numbers during the entire period of 7 weeks, reaching statistical significance from weeks 3 to 7 (p < 0.05). Addition of rhIL-3 (100 ng/mL) showed an enhancement of CFU-GM output in 2-CdA-treated cultures that resulted in their numbers exceeding those in control cultures (without 2-CdA) from weeks 1 to 5 (p < 0.05) with a maximum increase of 5.1-fold over the parallel control value at week 3.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Chloroadenosine↗

PubMed1993

Determination of soluble CD21 as a parameter of B cell activation.

In this study we established a novel solid-phase immunoassay for CD21 using the time-resolved fluorescence of lanthanide chelates. The capture assay was able to detect concentrations of as low as 100 pg of CD21 antigen per millilitre of sample and was used for quantitative determination of CD21 in lysates of different cell lines as well as in patient serum specimens. CD21 was measured in lysates of tonsils and cell lines of B, T cell and myelomonocyte lineage, and appeared to consist of monomeric antigen under the detergent conditions used. Elevated levels of soluble CD21 were observed in serum of patients with Epstein-Barr virus (EBV) infection, a disease known to be associated with polyclonal B cell activation, and in infection with the lymphotropic rubella virus. Significantly increased levels were also found in malignancies which are associated with EBV. In patients with nasopharyngeal carcinoma (NPC), a correlation with the titre of EBV-specific IgA was observed, thus supporting a possible role of soluble CD21 as a marker for disease activity in certain malignancies. Our data suggest that measurement of soluble CD21 could serve as a marker for activation of the immune system and diseases involving the B cell lymphoid system. Possible mechanisms and functions of soluble CD21 are discussed.

B-Lymphocytes↗

PubMed1993

Effect of deoxycytidine on 2-chloro-deoxyadenosine-mediated growth inhibition of normal human erythroid and myeloid progenitor cells.

The cytotoxic effect of chlorodeoxyadenosine (CdA) on lymphocytes and monocytes requires phosphorylation by the enzyme deoxycytidine kinase and can be antagonized by coadministration of deoxycytidine (dCyt), a competitive substrate of deoxycytidine kinase. It has also been shown for lymphocytes that coadministration of 3-aminobenzamide (3-ABA), an inhibitor of the enzyme poly-(ADP ribose) synthetase, is activated by CdA-mediated DNA strand breaks, consumes intracellular nicotinamide-dinucleotide (NAD) and can antagonize the lethal effect of CdA. Recent in vitro studies have shown that not only growth of lymphocytes and monocytes, but also colony formation by erythroid and myeloid progenitors derived from normal human bone marrow, is inhibited by CdA in a dose-dependent manner. In this study we examined the effect of various doses of dCyt (10(-6) to 10(-3) M) on CdA-mediated growth inhibition of erythroid and myeloid progenitor cells in vitro. Our results show that colony formation by human bone marrow-derived progenitor cells--CFU-E (colony-forming unit erythroid), BFU-E (burst-forming unit erythroid) and CFU-GM (colony-forming unit granulocyte/macrophage)-in semisolid medium is protected by a high, but clinically achievable and non-toxic, concentration of dCyt (> 10(-4) M) against the inhibitory effects of coadministered high concentrations of CdA. The protective effect of dCyt was markedly different on the various subclasses of progenitor cells, however. Thus, with coadministration of 10(-4) M dCyt, the CFU-E colony formation could be restored to almost 100% despite the presence of high concentrations of CdA (160 nM) compared to control cultures, whereas the colony formation of BFU-E and CFU-GM was restored to only 50%. At a concentration of 10(-3) M dCyt, colony formation of BFU-E and CFU-GM was raised to 80% of control cultures even in the presence of high concentrations of CdA (160 nM). Further experiments in which 3-ABA was coadministered to CdA-treated cultures showed that in all concentrations tested (0.3 to 5 mM) 3-ABA was not able to prevent CdA-mediated cytotoxicity on bone marrow progenitors. Based on these studies, we suggest that the CdA toxicity on CFU-E is mainly mediated by phosphorylation by deoxycytidine kinase, whereas additional mechanisms may be operative in BFU-E and CFU-GM. Considerable biochemical differences seem to exist between hematopoietic stem cells on the one hand and lymphocytes and monocytes from peripheral blood on the other.

Benzamides↗

PubMed1991

Inhibitory effect of 2-chlorodeoxyadenosine on granulocytic, erythroid, and T-lymphocytic colony growth.

Previous studies have shown that 2-chloro-2'-deoxyadenosine (CdA) is markedly toxic to normal and malignant human lymphocytes in vitro and in vivo. Recent clinical trials have shown that CdA is a very promising drug for the treatment of lymphoid malignancies. The present investigations were designed to test the effect of CdA on the in vitro clonal growth of both myeloid progenitors and T-lymphocyte colony-forming cells (CFU-TL) obtained from normal human bone marrow and peripheral blood. Cells were exposed to CdA in doses up to 1280 nmol/L. To reduce indirect effects of CdA mediated by accessory cells, monocyte- and T-lymphocyte-depleted bone marrow cells were used for our investigations. The results show a marked inhibition of myeloid progenitor and lymphocyte colony-forming cells in a dose-dependent manner, correlating with maturation stage in that the immature progenitor cells are more sensitive to this drug. Furthermore, our studies suggest that a sequence of metabolic events previously described for lymphocytes may be operative in myeloid progenitor cells because a minimal exposure time of 48 hours is required to obtain a marked inhibition. CdA toxicity was proposed to be linked with phosphorylation by deoxycytidine-kinase (E.C. 2.7.1.74), the levels of which have been found to be high in lymphocytes, but low in granulocytes. However, the marked inhibition of myeloid progenitor cells shown in these studies suggests that other factors such as modulation of the effect of CdA by the ambient levels of other deoxynucleosides might influence the apparent sensitivity of myeloid cells.

2-Chloroadenosine↗

PubMed1989

C3bi-binding protein on Candida albicans: temperature-dependent expression and relationship to human complement receptor type 3.

We investigated in detail the previously described capacity of pseudohyphae of Candida albicans to bind C3-coated particles. We show that the expression of the C3bi receptor of C. albicans was dependent upon the growth temperature of the fungi. C. albicans grown at 30 degrees C bound strongly to EAC1423bi, whereas those cells grown at 38.5 degrees C were completely devoid of this capacity. The molecule responsible for the attachment of EAC1423bi was heat labile and trypsin sensitive. Several, but not all, monoclonal antibodies to the alpha-chain of human complement receptor type 3 (CR3) stained C. albicans, and this reactivity was expressed in parallel with the capacity of C. albicans to bind EAC1423bi, i.e., both were dependent on the growth temperature of the fungi and were trypsin sensitive. In contrast to CR3, the binding of EAC1423bi to C. albicans did not require the presence of divalent cations. Rabbit immunoglobulin G antibodies directed against C. albicans inhibited the binding of EAC1423bi to C. albicans but not to human CR3. These inhibiting IgG antibodies recognized antigens expressed on the surface of pseudohyphae but not those of yeast cells. OKM-1, a monoclonal antibody to human CR3 inhibited the attachment of EAC1423bi to CR3 and also to C. albicans. OKM-1 precipitated a 130-kilodalton band from solubilized 125I-labeled C. albicans. We conclude that the complement receptors on C. albicans and human CR3 were antigenically related but not identical and that they differed in their functional characteristics.

Animals↗

PubMed1988

Structural and functional analysis of CR2/EBV receptor by means of monoclonal antibodies and limited tryptic digestion.

The receptor for the C3d fragment of the third component of complement, CR2, has recently been shown also to act as the receptor for the Epstein-Barr virus (EBV) and to be involved in the control of B-cell proliferation. In order to define functionally important domains on this molecule, we produced monoclonal antibodies to several distinct epitopes. CR2 was purified from a NP-40 lysate of human tonsils by a new method involving sequential chromatography on lentil lectin Sepharose 4B and DEAE-Sephadex and used to immunize mice. After fusion we obtained four stable hybridoma lines producing antibody to CR2. Specificity of these antibodies for CR2 was ascertained by immunofluorescence analysis on a panel of various cells known to possess CR2, by their reactivity in a recently described ELISA for C3 receptors, by Western blotting with purified CR2 and immunoprecipitation from 125I-labelled Raji cells. These four antibodies were found to recognize three distinct epitopes localized on the same fragments of 95,000, 72,000, 50,000, 32,000 and 28,000 MW obtained after mild tryptic digestion of CR2. The 72,000 MW fragment contains the binding site for C3d. Two monoclonal antibodies recognizing the same epitope did not inhibit the binding of C3d-coated sheep erythrocytes to Raji cells, whereas the other two antibodies against distinct epitopes did inhibit in the presence of a second antibody. All four monoclonal antibodies stimulated the proliferation of human peripheral blood B cells.

Antibodies, Monoclonal↗

PubMed1988

Cell cycle control of a Burkitt lymphoma cell line: responsiveness to growth signals engaging the C3D/EBV receptor.

CR2, the receptor for the C3d fragment of the third complement component and for Epstein-Barr virus (EBV) has been shown, on mouse B cells, to be involved in the control of B-cell proliferation by acting as a receptor for macrophage-derived growth factors. We examined whether the growth of a Burkitt lymphoma cell line, RAJI, could be influenced by ligands of human CR2. In serum-free culture, purified human C3d, as well as three monoclonal antibodies to distinct epitopes on human CR2, were capable of enhancing the growth rate of RAJI cells two to five-fold. This effect could not be observed if even trace amounts of serum were present in the culture medium. Simultaneous addition of pairs of antibodies did not enhance the growth rate, suggesting that a particular engagement of CR2 may be critical in order to induce a stimulatory effect. These results indicate that in a homologous serum-free human B-cell system human C3d as well as monoclonal antibodies to human CR2 can induce B-cell proliferation and that CR2-mediated triggering of B cells can be induced via epitopes other than the C3d-binding site. In addition we conclude that--unlike normal human B cells--at least some human B-lymphoma cells respond to CR2-mediated stimuli in the absence of any T-cell derived factors. Therefore the control mechanisms exerted through CR2 must still be intact on these autonomously growing cells.

Antibodies, Monoclonal↗