Wayne F. Green

The Beige/Chediak-Higashi Syndrome Gene Encodes a Widely Expressed Cytosolic Protein

The human autosomal recessive disorder Chediak-Higashi syndrome and its murine homologue beigeare associated with the formation of giant lysosomes that cluster near the perinuclear region of cells. We prepared a polyclonal antiserum against a glutathione S-transferase-Beige fusion protein and demonstrated by Western analysis that the beige gene encodes a protein of 400 kDa that is expressed in cultured murine fibroblasts as well as most mouse tissues. The protein was not detected in either cultured fibroblasts or mouse tissues from two differentbeige mutants. Cultured fibroblasts transformed with multiple copies of yeast artificial chromosomes that contain the full-length beige gene showed much higher levels of Beige protein than either wild type fibroblasts or mouse tissues. Subcellular fractionation experiments demonstrated that the Beige protein was cytosolic and, under the conditions of isolation, had no measurable membrane association. Cultured mouse fibroblasts in which the Beige protein was overexpressed had smaller than normal lysosomes that were more peripherally distributed than in control cells. These findings, coupled with earlier published results, suggest that the Beige protein regulates lysosomal fission.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Polycythaemia vera. III. Burst-forming units-erythroid (BFU-E) response to stem cell factor and c-kit receptor expression

We previously demonstrated that highly purified normal human blood burst‐forming units‐erythroid (BFU‐E) need the direct action of recombinant human stem cell factor (rSCF) in the presence of recombinant human erythropoietin (rEP) and recombinant human interleukin‐3 (rIL‐3) for further development in a serum‐free medium. To study the response of polycythaemia vera (PV) BFU‐E to rSCF, we performed dose–response experiments in a serum‐free medium using highly purified BFU‐E from PV patients. A marked increase in the number of PV bursts occurred with increasing concentrations of rSCF, compared to normal burst formation, when the cells were cultured in the presence of rIL‐3 at 1 U/ml. The percentage of maximum growth for normal BFU‐E was 31±11% while for PV it was 64·9% at the highest concentration of rSCF (P<0.01). Without rIL‐3, only 11% of maximum normal BFU‐E growth occurred as the rSCF concentration was increased and the size of the colonies was very small, but PV BFU‐E still expressed 48% of the maximum number of large erythroid bursts (P<0.001). This demonstrated an enhanced sensitivity of PV BFU‐E to rSCF, compared to normal BFU‐E. The pattern of 59Fe incorporation into haem after 8 d of cell culture indicated that PV BFU‐E had a time course of maturation and a degree of cellular maturity similar to normal BFU‐E. The percentage positivity and intensity of c‐kit receptors on PV erythroid cells were examined using immunofluorescence flow cytometry. When BFU‐E, CFU‐E, or erythroblasts were incubated with phycoerythrin‐conjugated SR‐1 anti‐c‐kit receptor monoclonal antibody, 90% of the PV and normal BFU‐E displayed c‐kit receptor at comparable intensities, as well as 80% of the PV and normal CFU‐E. A distinct loss of c‐kit expression occurred with erythroid differentiation beyond the CFU‐E stage, but at all stages no difference of c‐kit receptor expression was evident for PV erythroid precursors compared to normal precursors. These results indicate that the hypersensitivity to rSCF did not appear to be related to the number of c‐kit receptors. Since we have previously shown that highly purified PV BFU‐E are hypersensitive to rIL‐3 and rGM‐CSF, as well as rEP, it is now evident that PV BFU‐E are hypersensitive to each of the cytokines that have a prominent role in guiding their normal proliferation and differentiation. While the precise mechanism for the hypersensitivity is not known, it may be a key factor in the pathogenesis of PV.

Effect of prior chemotherapy on hematopoietic stem cell mobilization.

Summary:A number of studies have suggested that prior chemotherapy correlates negatively with the efficiency of hematopoietic stem cell mobilization. However, little data exist with regard to the relative effects of the specific chemotherapeutic drug classes. We retrospectively reviewed the records of 201 consecutive patients with nonmyeloid malignancies undergoing CD34+ cell mobilization with chemotherapy+granulocyte colony-stimulating factor (G-CSF). The number of prior chemotherapy courses correlated negatively with the peripheral CD34+ cell concentration (pCD34) on the first day of collection (P<0.001). No significant correlation was found for age, gender, tumor primary, mobilization chemotherapy regimen, disease status, marrow involvement, prior radiation therapy, or dose and timing of G-CSF administration. When the number of courses of individual classes of chemotherapeutic agents was correlated with pCD34, only exposures to platinum compounds (P=0.001) and alkylating agents (P=0.01) were found to be independent negative predictive factors for pCD34. Within classes, DNA crosslinking agents and etoposide appeared possibly more damaging than DNA methylating agents and doxorubicin, respectively. None of the drug classes showed evidence of recovery. We conclude that exposure to chemotherapy, especially platinum compounds and alkylating agents, should be minimized prior to mobilization.

Intracellular and Extracellular Cytokine Production by Human Mixed Mononuclear Cells in Response to Group B Streptococci

ABSTRACT Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-α) by human mononuclear cells. The present study was designed to measure the production of TNF-α as well as additional cytokines, including interleukin 1β (IL-1β), IL-6, IL-8, IL-12, and gamma interferon (IFN-γ) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 μg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-α, IL-1β, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-α but delayed appearance of IL-1β, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-γ and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-α, IFN-γ, and IL-12 in GBS pathogenesis and/or immunity.

Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor β

OBJECTIVE To investigate if cell cycle progression plays a role in modulating the engraftment potential of mouse hematopoietic stem and progenitor cells (HSPC). MATERIALS AND METHODS HSPC were isolated from adult mouse bone marrow, cultured in vitro under conditions promoting cell cycle arrest, and subsequently were evaluated for cell cycle status, clonogenic activity, and transplant potential. RESULTS In the presence of steel factor (STL) as a survival cytokine, transforming growth factor beta (TGF-beta) increased the G0/G1 fraction of cycling progenitor cells (Rh(high)) after a 20-hour culture. Clonogenic activity of quiescent long-term repopulating (Rh(low)) HSPC was unaffected by this culture, whereas clonogenic potential of Rh(high) cells decreased by about 30%. In competitive repopulation assays, Rh(low) cells cultured in STL + TGF-beta engrafted better than cells cultured in STL alone. However, culture in STL + TGF-beta did not overcome the failure of Rh(high) cells to engraft after transplant. We also utilized a two-stage culture system to first induce proliferation of Rh(low) HSPC by a 48-hour culture in STL + interleukin 6 + Flt-3 ligand, followed by shifting the culture to STL + TGF-beta for 24 hours to induce cycle arrest. A competitive repopulation assay demonstrated a relative decrease in repopulating potential in cultures that were cycle arrested compared to those that were not. CONCLUSION Cell cycle progression by itself cannot account for the decrease in repopulating potential that is observed after ex vivo expansion. Other determinants of engraftment must be identified to facilitate the transplantation of cultured HSPC.

Involvement of thyroid hormones in the expression of MHC class I antigens during ontogeny in Xenopus.

The major histocompatibility complex (MHC) is a cluster of genes encoding products central to all major functions of the vertebrate immune system. Evidence for an MHC can be found in all vertebrate groups that have been examined except the jawless fishes. Expression of MHC class I and class II antigens early in ontogeny is critically important for development of T lymphocytes capable of discriminating self from nonself. Because of this essential role in T-cell development, the ontogeny of MHC expression in the South African clawed frog, Xenopus laevis, was studied. Previous studies of MHC class I expression in Xenopus laevis suggested that class I antigens are virtually absent from tadpole tissues until climax of metamorphosis. We therefore examined the possible role of thyroid hormones (TH) in the induction of class I. By flow cytometry, a small amount of class I expression was detectable on splenocytes and erythrocytes in untreated frogs at prometamorphic stages 55-58, and the amount increased significantly at the conclusion of metamorphic climax. Thus, metamorphosis is associated with increased intensity of class I expression. Neither inhibition nor acceleration of metamorphosis altered the timing of onset of class I expression. However, inhibition of metamorphosis prevented the increase in class I expression characteristic of adult cell populations. Because expression was not accelerated in TH-treated frogs or delayed in metamorphosis-inhibited frogs, it is unlikely that TH are the direct developmental cues that induce expression, although they seem to be required for the upregulation of class I expression occurring at metamorphosis. Differences in the pattern of expression in different subpopulations of cells suggest a complex pattern of regulation of expression of class I antigens during ontogeny.

The effects of aprotinin on platelets in vitro using whole blood flow cytometry

We sought to evaluate the effects of aprotinin on the number and function of the platelet glycoprotein (GP) IIb–IIIa receptor and on the expression of P-selectin in vitro in order to gain insight into the potential mechanisms involved in the platelet-protective action of aprotinin during cardiopulmonary bypass. Aprotinin at 50 to 200 kallikrein inhibiting units/mL decreased the expression of activated GP IIb–IIIa complex in response to adenosine diphosphate or thrombin receptor activator peptide 6 in a dose-dependent manner in both citrated and heparinized whole blood experiments. Aprotinin inhibited adenosine diphosphate-induced platelet aggregation, but it exhibited no effect on the expression of GP IIIa and P-selectin. These results indicate that aprotinin interferes with the platelet fibrinogen receptor function during pharmacological activation. Reduced aggregability and platelet adhesion to fibrinogen adsorbed to synthetic surfaces in the presence of aprotinin may prevent platelet consumption during clinical cardiopulmonary bypass. This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated GP IIb–IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces. Implications This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated glycoprotein IIb–IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.

Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.

Intracellular and Extracellular Cytokine Production by Human Mixed Mononuclear Cells in Response to Group B Streptococci |[diams]| 31

Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-a) by human mononuclear cells. The present study was designed to measure the production of TNF-a as well as additional cytokines, including interleukin 1b (IL-1b), IL-6, IL-8, IL-12, and gamma interferon (IFN-g) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 mg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-a, IL-1b, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-a but delayed appearance of IL-1b, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-g and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-a, IFN-g, and IL-12 in GBS pathogenesis and/or immunity. Group B streptococci (GBS) are a major cause of severe and overwhelming infection in newborns, pregnant females, and other immunocompromised hosts (15). Patients in the neonatal period often present with early onset infection, which includes septicemia, shock, pneumonia, and respiratory failure. Late onset infection, in contrast, commonly is associated with meningitis. Interestingly, early in both types of infection, the