Interleukin 7-dependent B lymphocyte precursor cells are ultrasensitive to apoptosis.

We have compared the sensitivity of clonogenic interleukin 7 (IL-7)-dependent murine B cell precursors with that of clonogenic mature B cells and myeloid precursors to alpha-particles from plutonium-238 and X radiation. All three populations are relatively sensitive, but B cell precursors are ultrasensitive. This differential sensitivity is also observed with corticosteroid, etoposide, and cisplatin, all apoptosis-inducing drugs used in the treatment of leukemia and other cancers. Further, we show that x-rays and drugs induce the bulk of the B cell precursor population to undergo rapid apoptosis, despite the continued presence of IL-7. B cell precursors were found to express very low levels of BCL-2 protein compared with mature splenic B cells and their resistance to x-rays and corticosteroid could be enhanced by expression of a BCL-2 transgene. These data have important implications for normal lymphopoiesis and for the behavior of leukemic lymphoid precursor cells.

Clonal characteristics of acute lymphoblastic cells derived from BCR/ABL p190 transgenic mice.

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (JH) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL).

ts BCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.

Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming properties of BCR-ABL and are relevant to the clinical intransigence of Ph-positive leukaemias.

Absence of p53 permits propagation of mutant cells following genotoxic damage.

Much evidence has been gathered in support of a critical role for p53 in the cellular response to DNA damage. p53 dysfunction is associated with progression and poor prognosis of many human cancers and with a high incidence of tumours in p53 knockout mice. The absence of a p53-dependent G1 arrest that facilitates DNA repair or apoptosis might impact critically on clinical cancer in two ways. First, by abrogating the impact on therapy that operates via genotoxic damage and apoptosis; and second, by encouraging progression either by inducing genomic instability and DNA mis-repair or by permitting survival of mutants. However, experiments examining the relationship between p53 deficiency and mutation frequency have so far failed to confirm these predictions. The precise role played by p53 is therefore unclear. We now report use of a short term in vitro approach to assess the influence of p53 on radiation-induced mutations at the hprt locus in murine B cell precursors that are normally radiation ultrasensitive. We find a high number of hprt mutants among X-irradiated p53 null cells, which results from preferential survival as clonogenic mutants rather than from a p53-dependent increase in mutation rate. This result has important implications for genotoxic cancer therapy.

The beneficial effects of alpha-interferon in hairy cell leukemia are not attributable to NK cell-mediated cytotoxicity.

The natural killer (NK) activity of peripheral blood mononuclear cells against K562 targets was variable in 10 untreated patients with hairy cell leukemia and was inversely related to the number of hairy cells (HCs) present. After therapy with alpha-interferon (IFN-alpha) NK activity in vitro was equivalent to that of normal controls. It is suggested that the low activity often seen before treatment is attributable to dilution of NK cells by large numbers of inactive HCs and that this diluting effect is reduced as HCs disappear from the blood during IFN-alpha treatment. HCs was consistently resistant to NK lysis by normal or hairy cell leukemia allogeneic and autologous mononuclear cells, despite whether effector or target cells had been pretreated with IFN-alpha. Cold-target inhibition and direct binding experiments showed that HCs do not bind to NK effectors. It is therefore concluded that NK cells play no direct role in the progressive disappearance of HCs seen in patients receiving IFN-alpha.

Alpha-interferon and lymphokine-activated killer cells in hairy cell leukemia.

Lymphokine-activated killer (LAK) cell activity generated from peripheral blood was tested in 6 patients with typical hairy cell leukemia, 3 not on treatment with alpha-interferon (alpha-IFN) and 3 receiving therapy. In all cases, substantial killing of the LAK-sensitive target Daudi was observed, but hairy cells, whether or not they had been pretreated with alpha-IFN, were uniformly resistant to LAK lysis. The hairy cells were also resistant to LAK cell killing generated from normal peripheral blood mononuclear cells. alpha-IFN added at various times during LAK generation had little or no effect on LAK activity. It is concluded that LAK cells are not important in mediating the beneficial effects of alpha-IFN in hairy cell leukemia.

The effect of cytokines, including IL-1, IL-4, and IL-6, in hairy cell proliferation/differentiation.

IL-1, IL-4, and IL-6 had no effect on hairy cell (HC) proliferation in vitro. Anti-mu and low molecular weight B cell growth factor (LBCGF) and tumor necrosis factor alpha (TNF-alpha) stimulated the proliferation of a minor subpopulation of HCs detected by double immunocytochemical staining. None of these cytokines had any effect on HC differentiation as measured by immunoglobulin secretion. It is concluded that none of the above growth factors are central to HC proliferation in-vivo. Since alpha-interferon IFN-alpha, but not IFN-gamma, consistently inhibited any proliferation observed, it seems likely that this monokine has a direct antiproliferative effect in vivo.

Monocytes/Macrophages Stimulate Hairy-Cell Proliferation.

During a study of the effect of B-cell growth factors on the growth of hairy cells (HCs), proliferation of a sub-population of HCs was observed in the absence of added growth factors in 1 (WB) of 9 patients with hairy-cell leukaemia (HCL). Double staining analysis using monoclonal antibodies detecting the Ki-67 nuclear proliferation antigen and a range of surface/cytoplasmic antigens, confirmed that the HCs were proliferating and that these cells broadly resembled the majority non-proliferative population. The HC proliferation of patient WB was shown to be dependent on the presence of autologous monocytes, but not of T cells. Investigation of other cases of HCL showed that allogeneic monocytes/macrophages from various sources stimulated HC proliferation in 3/4 cases. Neither supernatants from HC-monocyte/macrophage cultures nor tumour necrosis factor (TNF-α) could replace the proliferative effect induced by monocytes/macrophages. The proliferative effect was dependent on monocyte viability and on cell contact between monocytes/macrophages and HCs. This proliferation was markedly inhibited by alpha interferon (α-IFN). In view of the known tissue association in the bone marrow between HCs and macrophages, it is suggested that these observations are of pathophysiological significance in vivo.

Lethality and mutagenesis of B lymphocyte progenitor cells following exposure to alpha-particles and X-rays.

B lymphocyte precursor cells are the target cells for the major subtype of paediatric cancer, acute lymphoblastic leukaemia. Using a murine IL-7-dependent clonogenic assay for normal B cell precursors as a model, we have investigated the sensitivity of these cells versus other normal and leukaemic haemopoietic cells to alpha-particle radiation. We find that B cell precursors are remarkably susceptible to the lethal effects of alpha-particles and have a very low probability of surviving a single alpha-track. B cell precursors are also very sensitive to the lethal effects of low LET X-rays. The mutation frequency in a marker gene (HPRT) does not, however, appear to be greater in B cell precursors that survive X-radiation than in other haemopoietic cells.

A discussion of the environmental influences on asthma and the role of Environmental Health Officers in combating it.

Asthma is a multifactorial disease and environmental influences have long been associated with its everyday symptoms. In recent years it has been proposed that environmental factors can also influence the actual development of the disease. Underlying genetic factors may interact with these common environmental factors. Thus, the environment may be involved in both the aetiology and the symptomatic manifestations of the disease. The discussion is completed by looking at the role of local authority Environmental Health Officers (EHOs) in combating asthma.

Activation of the beta-globin locus control region precedes commitment to the erythroid lineage.

The beta-globin locus control region (LCR) is characterized by erythroid-specific DNase I hypersensitive sites and is involved in the chromatin organization, transcriptional potentiation, developmental regulation, and replication timing of the entire beta-globin gene cluster. When and how the LCR is first activated during erythropoiesis is not known. Here we analyze the chromatin structure of the LCR during early hematopoietic differentiation using nontransformed, multipotential, growth factor-dependent, murine hematopoietic progenitor cells. We show that LCR hypersensitive sites characteristic of erythroid cells are present in three independent multilineage progenitors [FDCP (factor-dependent cell, Paterson)-mix A4, B6SUtA, and LyD9] under conditions of self-renewal. Induction of differentiation down a nonerythroid pathway causes a progressive loss of hypersensitivity in the LCR. These results show that the beta-globin LCR is in an active chromatin configuration prior to erythroid commitment and indicate a significant role for selective gene repression in lineage specification.

Interaction of interleukin 7 (IL-7) with glycosaminoglycans and its biological relevance.

We have investigated the binding of interleukin 7 (IL-7) to sulfated glycosaminoglycans and evaluated its biological consequences. IL-7 binds to heparin and heparan sulfate, to a lesser extent to dermatan sulfate and does not bind to chondroitin sulfate. It was eluted from heparin by 0.3-0.6 M NaCl and from heparan sulfate by < 0.3 M NaCl. We also measured the affinity of IL-7 for heparin using an affinity co-electrophoresis method and found an affinity of 25 nM. In spite of these findings, IL-7 does not bind to the S17 cell line which supports lymphopoiesis. However, addition of heparin to cultures of an IL-7-dependent pre-B cell line (2E8) inhibited IL-7-stimulated proliferation and IL-7 complexed with heparin was more resistant than free IL-7 to protease treatment. Taken together, these results suggest that heparin may act as a carrier for IL-7, blocking its interaction with target cells and protecting it from degradation during transit.

Purine analogues kill resting lymphocytes by p53-dependent and -independent mechanisms.

To resolve the controversy concerning the role of p53 in the killing of resting lymphocytes by purine nucleoside analogues, we examined the cytotoxic effects of chlorodeoxyadenosine, fludarabine and deoxycoformycin (plus deoxyadenosine) on unstimulated spleen cells from p53-knockout versus wild-type mice. p53-knockout cells were more resistant to all three nucleosides than were wild-type cells. However, substantial killing still occurred in the absence of p53, indicating that purine analogues can kill resting lymphocytes by both p53-dependent and -independent mechanisms. We suggest that these results are relevant to chronic lymphoid malignancies, and that characterization of the p53-independent component of nucleoside action may indicate potential ways of overcoming therapeutic resistance.

Granulocyte-macrophage colony-stimulating factor as an autocrine survival factor for mature normal and malignant B lymphocytes.

The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.

Cryptosporidiosis: an outbreak associated with drinking water despite state-of-the-art water treatment.

OBJECTIVE: To determine the magnitude and source of an outbreak of cryptosporidiosis among persons with human immunodeficiency virus (HIV) infection and to determine whether the outbreak extended into the immunocompetent population. DESIGN: Matched case-control study and environmental investigation. SETTING: Clark County, Nevada. PARTICIPANTS: Adults with HIV infection (36 case-patients with laboratory-confirmed Cryptosporidium parvum infection and 107 controls), matched by physician or clinic and by CD4+ cell count category. MEASUREMENTS: Potential risk factors for infection, death rates, and data on water quality. RESULTS: Review of surveillance and microbiology records identified 3 cases of cryptosporidiosis in 1992 (the first year that cryptosporidiosis was reportable in Nevada), 23 cases in 1993, and 78 cases in the first quarter of 1994. Of the 78 laboratory-confirmed cases in the first quarter of 1994, 61 (78.2%) were in HIV-infected adults. Of these 61 adults, 32 (52.5%) had died by 30 June 1994; at least 20 of the 32 (62.5%) had cryptosporidiosis listed on their death certificates. In the case-control study, persons who drank any unboiled tap water were four times more likely than persons who drank only bottled water to have had cryptosporidiosis (odds ratio, 4.22 [95% Cl, 1.22 to 14.65]; P = 0.02). For persons with CD4+ cell counts less than 100 cells/mm3, the association between tap water and cryptosporidiosis was even stronger (odds ratio, 13.52 [Cl, 1.78 to 102.92]; P = 0.01). Additional data indicate that this outbreak also affected persons who were not infected with HIV. No elevated turbidity values or coliform counts and no Cryptosporidium oocysts were found in testing of source (Lake Mead) or finished (treated) water during the study period, but so-called presumptive oocysts were intermittently found after the investigation in samples of source water, filter backwash, and finished water. CONCLUSIONS: A cryptosporidiosis outbreak was associated with municipal drinking water, despite state-of-the-art water treatment and water quality better than that required by current federal standards. This outbreak highlights the importance of surveillance for cryptosporidiosis and the need for guidelines for the prevention of water-borne-Cryptosporidium infection among HIV-infected persons.