Sensitivity of murine multipotential stem cell colony (CFU-GEMM) growth to interleukin-3, erythropoietin, and hemin.

The in vitro growth of murine marrow-derived CFU-GEMM in response to partially purified preparations of interleukin-3 and erythropoietin was assessed in the presence of hemin. Although CFU-GEMM exhibited a near-absolute requirement for interleukin-3, some colony growth was observed in the absence of exogenous erythropoietin. Erythropoietin was nevertheless capable of further stimulating CFU-GEMM growth in the presence of optimal concentrations of interleukin-3 and hemin. Optimal concentrations of all three factors allowed at least a 60% reduction in the serum concentration without an effect on colony numbers or their detection efficiency. Furthermore, nearly half the colonies continued to grow without the addition of serum, suggesting that hemin supplementation, along with interleukin-3 and erythropoietin, may provide the basis for a relatively simple serum-free culture system for murine CFU-GEMM.

Growth of murine multipotent stem cells in a simple "serum-free" culture system: role of interleukin-3, erythropoietin, and hemin.

The growth requirements of normal murine marrow-derived multipotent stem cells (CFU-GEMM) in a simple clonal cell culture system substantially devoid of exogenous serum proteins was assessed. The ability of murine interleukin-3 (Il-3), recombinant human erythropoietin (rEPO), and a crystalline preparation of the protoporphyrin hemin to support colony growth in "serum-free" cultures was examined by titration. The results suggest that both Il-3 and hemin are limiting for multipotential colony growth in "serum-free" cultures, but that EPO is not. In addition, the "sensitivity" of CFU-GEMM to each growth factor appeared to increase in the "serum-free" environment as evidenced by a "shift-to-the-left" in all the titration curves. Nearly half of the GEMM colonies grew to full maturity in the absence of exogenous EPO. Given the optimal concentration of each growth factor, high colony growth was consistently observed in the "serum-free" cultures, with a range from 65% to 119% of the serum control level. It is therefore concluded that supplementation of murine marrow cultures with Il-3 and hemin alone may provide the necessary setting for studying the factors that modulate the growth of multipotent stem cells in a serum-free environment.

Hemin acts synergistically with interleukin-3 to promote the growth of multipotent stem cells (CFU-GEMM) in "serum-free" cultures of normal murine bone marrow.

The role of hemin (iron protoporphyrin 9) in the enhancement of interleukin-3 (IL-3)-stimulated multipotent stem cell colony formation was assessed in both serum-containing as well as in "serum-free" marrow culture systems. A greater than 7-fold enhancement in colony number was observed when cultures were supplemented with both IL-3 and hemin compared with either factor alone. In addition, this effect was observed over a wide concentration range. Hemin by itself failed to promote CFU-GEMM in the "serum-free" marrow culture system. The results suggest that hemin acts synergistically with IL-3 to promote the growth of CFU-GEMM in a dose-dependent manner.

EBV binds to lymphocytes of transgenic mice that express the human CR2 gene.

Epstein Barr virus (EBV) is unable to bind to or infect normal mouse lymphocytes. A construct containing the human complement receptor type 2 (CR2) gene, the receptor for EBV, was placed under the control of the IgH/c-fos enhancer/promoter and microinjected into single cell embryos. A total of five transgenic mouse lines were established and four expressed hCR2 mRNA. Flow cytometry and immunostaining revealed that approximately 15-30% of the lymphocytes from the thymus, spleen and lymph nodes expressed hCR2 protein on their surface and bound EBV. Despite this binding, less than 1% of the cells showed evidence that the virus was internalized or replicated. Transgenic mouse lymphocytes, expressing hCR2, could not be immortalized with EBV. It is concluded that the simple expression of hCR2 receptor on mouse lymphocytes is not sufficient for efficient infection.

Disseminated intravascular coagulation in solid tumors: clinical and pathologic study.

Disseminated intravascular coagulation (DIC) is a well known hemostatic complication of solid tumors. We evaluated the occurrence of DIC in 1117 patients with solid tumors. Of these patients, 76 (6.8%) were diagnosed with DIC. There were a total of 145 bleeding and clotting episodes reported in the 76 patients. Thrombocytopenia, hypofibrinogemia, elevated D-dimer and fibrinogen degradation products were the most common coagulation abnormalities encountered in patients with DIC. In multivariate analysis, older age (p = .0001), male gender (p = .009), advanced malignancies (p = .027), breast cancer (p = .038) and the presence of necrosis in the tumor specimen (p = .004), emerged as independent factors significantly related to the occurrence of DIC in patients with solid tumors. Of the 76 patients, 25 (33%) achieved response to treatment of DIC as defined in the study. Patients with early stage and advanced malignancies who developed DIC had inferior survival when compared with their counterparts without DIC (p = .039 and p = .005, respectively). Taken together, this study indicates that certain clinical and laboratory features are more common in patients with solid tumors who developed DIC. The occurrence of DIC appears to have an independent effect on survival of patients with cancer. Cooperative studies are encouraged to better address the usefulness and optimal prophylactic heparin regimen in patients at risk for DIC.

Transgenic marrow transplantation: a new in vivo and in vitro system for experimental hemopoiesis and radiobiology which employs sequential molecular monitoring of multiple genetic markers.

Efforts to understand the in vivo regulation of hemopoiesis have been greatly impeded by an inability to trace and quantify the fate of multiple hemopoietic stem cell (HSC) cohorts within a single animal simultaneously. We report here the development of a molecular marker system which overcomes this deficiency and can be readily extended to a wide variety of applications in experimental hematology and radiobiology. Mixtures of HSC from multiple strains of transgenic mice, derived on a common genetic background, were used as donors to reconstitute lethally irradiated wild-type mice. Using molecular hybridization and the polymerase chain reaction, we documented the presence and quantified the amount of various transgenic markers in the blood of individual recipient mice after transplantation. The transgenic markers were also detected in spleen, thymus, peritoneal exudates, bone marrow, spleen colonies (CFU-S), and in vitro colonies. The transgenic transplantation marker system thus permits repeated analysis of multiple stem cell cohorts over the entire spectrum of hemopoiesis including HSC, intermediate precursors, and functionally mature cells. Therefore, the transgenic markers should facilitate the in vivo analysis of stem cell development, gene transfer, leukemogenesis, recovery from radiation or drug treatment, and the influence of hormones/growth factors on these processes.

Synergism of hemopoietic growth factors on endothelial cell proliferation.

Endothelial cells, which are nonhemopoietic cells, express and/or produce most of the known hemopoietic receptors and cytokines. The biological role of these factors, and their respective receptors, on endothelial cells is still unknown. In this study, the authors assessed the effect of different hemopoietic growth factors, ie, interleukin-3 (IL-3), erythropoietin (EPO), macrophage-colony stimulating factor (M-CSF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), singly or in conjunction with others, on proliferation and chemotaxis of human umbilical vein endothelial cells (HUVECs). They found growth stimulatory activity with IL-3, EPO, and GM-CSF and potent synergism between EPO and IL-3, less with IL-3 and GM-CSF, and none with EPO and either GM-CSF or G-CSF. All the singly tested hemopoietic growth factors stimulated the migration of HUVECs, but in conjunction with other factors, they did not show any additive or synergistic effect.

Radioprotective potential of primitive hematopoietic precursors forming colonies in diffusion chambers in mice.

The purpose of this study was to determine the radioprotective ability of primitive hematopoietic precursors which form colonies in diffusion chambers in mice (CFU-D). Thirty-two lethally irradiated female ICR mice were injected with 5 to 7 male ICR mouse bone marrow-derived CFU-D colonies each. Fourteen of these mice survived over 30 days and were sacrificed at intervals up to a year. As a control, 20 lethally irradiated female ICR mice received cells from intercolony areas. All of these mice died before day 20. DNA samples obtained from hematopoietic organs and liver from 8 sacrificed mice were analyzed for the presence of CFU-D colony-derived cells. Only in 1 ICR mouse was CFU-D colony origin DNA detected by Southern analysis in all hematopoietic organs: bone marrow, spleen, thymus and lymph nodes. In 6 mice, only selected hematopoietic organs were repopulated by CFU-D colony-derived cells as judged by Southern analysis. In some of these mice, the remaining hematopoietic organs contained small CDU-D-derived cell populations which could be detected by more sensitive polymerase chain reaction (PCR). In 1 mouse, the presence of CFU-D-derived cells in all hematopoietic organs was only demonstrated by PCR. These findings suggest that lethally irradiated mice can be rescued by CFU-D-derived daughter cells. They appear to have the potential to give rise to clones containing lymphoid and myeloid cells in all hematopoietic organs, at least temporarily. Thus, it can be concluded that CFU-D represents a very primitive hematopoietic precursor cell with radioprotective capability.

dl-alpha-tocopherol induces apoptosis in erythroleukemia, prostate, and breast cancer cells.

Vitamin E, best known as a potent antioxidant, has been shown to have other functions that are not mediated by this activity. Recent reports have suggested that vitamin E may inhibit smooth muscle cell and also cancer cell growth. We have studied the effect of dl-alpha-tocopherol (vitamin E) on a series of well-established cancer cell lines that included two erythroleukemia cell lines and a hormone-responsive breast and prostate cancer cell line. Cell proliferation was examined in these cell lines, which were maintained at optimal growth conditions. A dose-dependent inhibition of cell growth was found in all cell lines examined, with the MCF-7 breast and CRL-1740 prostate cancer cell lines showing potent suppression of growth at 0.1 mM vitamin E, whereas the erythroleukemia cell lines, HEL and OCIM-1, responded only at > 0.25 mM vitamin E with inhibition of proliferation. Studies of [3H]thymidine incorporation showed that vitamin E supplementation reduced DNA synthesis in all cell lines. Analysis of high-molecular-weight DNA revealed extensive fragmentation, indicating apoptosis of all cell lines supplemented with vitamin E. Our studies thus give evidence of a general inhibition of cell proliferation by dl-alpha-tocopherol, with breast and prostate cancer cells distinctly more sensitive than erythroleukemia cells.

Factors affecting the proliferation and differentiation of clonogenic hematopoietic stem cells in vitro.

The characteristics of hematopoietic factors modulating the growth of clonogenic pluripotent stem cells (PSC) in culture (i.e., CFU-GEMM) are examined. The chemical and biologic properties of "burst-promoting activity" (BPA) are compared with those of the lymphokine interleukin-3 (Il-3). The preponderance of the data available suggests that the active moiety of each of these growth factors may be chemically identical since both BPA and purified Il-3 are capable of supporting the growth of PSC in vitro. The effects of hemin on the growth of CFU-GEMM are also examined experimentally. The results show conclusively that, in the absence of exogenous (BPA), hemin is also capable of supporting the growth of CFU-GEMM in culture and to a level that is consistently higher than that of BPA alone. It is therefore suggested that hemin is capable of augmenting the growth of GEMM colonies in vitro in conjunction with BPA/Il-3. However, the in situ role of each of these "candidate" regulators of hemopoiesis remains largely unknown.

S-allylmercaptocysteine inhibits cell proliferation and reduces the viability of erythroleukemia, breast, and prostate cancer cell lines.

Organosulfur compounds are the biologically active components of allium vegetables. Many health benefits have been ascribed to them, including inhibition of carcinogenesis. Inasmuch as several of these thioallyl compounds are quite unstable and others are rapidly inactivated in the body, we have investigated one of the stable components present in aged garlic extract, S-allylmercaptocysteine (SAMC), in an effort to determine whether it can inhibit proliferation of cancer cells. Proliferation and viability of two erythroleukemia cell lines, HEL and OCIM-1, two hormone-responsive breast and prostate cancer cell lines, MCF-7 and CRL-1740, respectively, and normal human umbilical vein endothelial cells in response to different concentrations of SAMC were studied for up to two weeks. There were variations in sensitivity to this organosulfur compound in the different cell lines examined, but the two hormone-responsive cancer cell lines of breast and prostate clearly were far more susceptible to the growth-inhibitory influence of the thioallyl compound. The antiproliferative effect of SAMC was limited to actively growing cells. Human umbilical vein endothelial cells that had reached confluence escaped the reduction in viability so noticeable in the cancer cell lines tested. Our studies thus give evidence of a direct effect of SAMC on established cancer cells.

Half-life of polyreactive antibodies.

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The half-life of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.

Polyreactive IgM antibodies in the circulation are masked by antigen binding.

Human plasma containing IgM showed only minimal, if any, reactivity with a panel of antigens as measured by ELISA. In contrast, affinity-purified IgM showed many times more reactivity with the same panel of antigens. When plasma was added back to the affinity-purified IgM, the reactivity of the IgM with antigens was completely inhibited by undiluted plasma and by as much as 40% with as little as a 1:100 dilution of plasma. When the affinity-purified IgM was affinity-purified a second time by passage through antigen-specific columns (e.g., insulin or Fc or beta-galactosidase), the eluted antibodies bound not only to the antigen used for purification, but also to a panel of unrelated antigens, indicating that the antibodies were polyreactive. It is concluded that polyreactive IgM antibodies are present in the circulation but are masked by binding to circulating antigens.

Human polyreactive and monoreactive antibodies: effect of glycosylation on antigen binding.

The present experiments were initiated to determine whether the carbohydrate portions of antibody molecules contribute to polyreactivity. Cell lines making human monoclonal polyreactive or monoreactive antibodies of the immunoglobulin (Ig) M, IgG and IgA isotypes were treated with tunicamycin to block N-linked glycosylation of the proteins. Analysis of the secreted native and non-glycosylated proteins revealed a > 95% inhibition of [3H]mannose incorporation. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels of the proteins from tunicamycin-treated cells showed increased mobility and the absence of [3H]mannose incorporation of the immunoglobulin heavy chains, consistent with the lack of glycosylation. The native and non-glycosylated antibodies were then tested for their ability to bind different antigens. Despite the lack of glycosylation, both polyreactive and monoreactive antibodies bound to antigens with little if any loss of reactivity or specificity. It is concluded that the carbohydrate moieties do not contribute significantly to polyreactivity.

dl-alpha-tocopherol, a potent inhibitor of phorbol ester induced shape change of erythro- and megakaryoblastic leukemia cells.

Synthetic vitamin E, dl-alpha-tocopherol, added to a human erythroleukemia HEL and a megakaryoblastic leukemia, Meg-01, cell culture produced potent dose-dependent inhibition of phorbol ester-induced adhesion and of the morphologic changes accompanying it. The inhibition was reversible by withdrawal of supplemental vitamin E from the medium. dl-alpha-Tocopherol also inhibited protein kinase C activity both at baseline and after phorbol ester stimulation. Arachidonic acid stimulated protein kinase C activity of erythroleukemia cells and promoted their adhesion, an effect that was also inhibited by dl-alpha-tocopherol. Introduction of a protein kinase C-neutralizing antibody or a protein kinase C-inhibitor substrate into permeabilized HEL cells inhibited phorbol ester-induced adhesion and shape change. dl-alpha-Tocopherol also affected the cellular distribution of protein kinase C, shifting the major portion of the enzyme to the cytosol fraction and reducing phorbol ester-induced membrane association of the enzyme. Thus, protein kinase C appears to mediate shape change and adhesion, both of which are strongly inhibited by dl-alpha-tocopherol.

S-allylmercaptocysteine, a stable thioallyl compound, induces apoptosis in erythroleukemia cell lines.

The antiproliferative potential of S-allylmercaptocysteine (SAMC), a stable organosulfur compound of aged garlic extract, has been investigated using two erythroleukemia cell lines, HEL and OCIM-1. It induces a dose-dependent inhibition of cell growth with a 50% lethal dose of 0.046 mM for OCIM-1 cells and 0.093 mM for HEL cells. [3H]thymidine incorporation was reduced in cells treated with this thioallyl compound, and analysis of high-molecular-weight DNA showed fragmentation compatible with apoptosis. Flow cytometric analyses of DNA revealed an abnormal cell cycle progression in both types of erythroleukemia cells, with the major portion of the unsynchronized cells in the G2/M phase. Measurement of acid-soluble free sulfhydryl groups showed an initial increase in response to SAMC followed by a progressive dose-dependent decrease with extended incubation of cells. We conclude from these studies that SAMC is an effective antiproliferative agent against erythroleukemia cells that induces cell death by apoptosis.

Analysis of factors related to the occurrence of chronic disseminated candidiasis in patients with acute leukemia in a non-bone marrow transplant setting: a follow-up study.

BACKGROUND: Chronic disseminated candidiasis (CDC) is a serious complication of treatment in patients with acute leukemia. Although some general risk factors are known to predispose to systemic fungal infections, few studies have addressed the relevance of certain clinical and laboratory features in patients with CDC. PATIENTS AND METHODS: To define a subset of patients at high risk for CDC, the authors evaluated the demographics and clinical and laboratory characteristics of 423 patients with acute leukemia. Patients who had bone marrow transplant before the diagnosis of CDC were excluded from the analysis. The diagnosis of CDC was based on blood cultures, liver biopsy, and imaging studies. The authors conducted 2 separate regression analyses on 3 subsets of patients: patients without documented candidiasis (n = 374), patients with CDC (n = 23), and patients with candidemia (n = 26). RESULTS: According to multivariate analysis, younger age (P = 0.009; odds ratio [OR], 1.96; 95% confidence interval [CI], 1.72-2.99), duration of neutropenia of 15 days or longer (P = 0.0003; OR, 11.7; 95% CI, 3.04-45.1), and use of prophylactic quinolone antibiotics (P = 0.039; OR, 3.85; 95% CI, 1.11-13.4) emerged as independent factors related to the development of CDC in patients with acute leukemia. The presence of severe mucositis, colonization with Candida, and administration of high-dose ara-C were statistically significant parameters in univariate analysis only (P = 0.0001, P = 0.003, and P = 0.058, respectively). CONCLUSIONS: On the basis of the results of this investigation, it is possible to define a subset of patients with acute leukemia at very high risk for CDC. Because of the morbidity and mortality of this infection, a targeted prophylactic approach may be more effective and less costly than the random administration of antifungal agents.

Autoimmune hemolytic anemia in patients with non-Hodgkin's lymphoma: characteristics and significance.

BACKGROUND: The occurrence of autoimmune hemolytic anemia (AIHA) in patients with non-Hodgkin's lymphoma (NHL) is well known. However, there is lack of information in the literature in terms of the significance and impact of such phenomenon on the clinical course of these patients. PATIENTS AND METHODS: We analyzed the clinical and laboratory features, course and response of 16 patients with non-Hodgkin's lymphoma (NHL) and autoimmune hemolytic anemia (AIHA). Patients with small lymphocytic lymphoma and angioimmunoblastic lymphadenopathy with dysproteinemia were excluded from the analysis. The significance of certain parameters, such as cell type (B- vs. T-cell), stage of NHL and presence of serum monoclonal immunoglobulin were examined. The cohort consisted of 501 patients with NHL evaluated during the study period. RESULTS: The response rate for the group of patients with NHL/AIHA and for the cohort was 44% and 62%, respectively; P = 0.0138. T-cell histology was overrepresented in the patients with AIHA/NHL (33% vs. 14%; P = 0.048). The occurrence of AIHA was not statistically significant among the four stages of NHL (P = 0.722), while a significantly higher proportion of patients with AIHA had serum monoclonol gammopathy when compared to the cohort (25% vs. 8%; P = 0.03). The patients with NHL who did not develop AIHA had better overall survival and median survival compared to the NHL/AIHA group (P = 0.006 and P = 0.0001, respectively). CONCLUSIONS: The study provides for the first time a descriptive clinicopathologic analysis of patients with AIHA and NHL. Certain pathologic and laboratory features were more likely to be associated with the occurrence of AIHA in patients with NHL. Most importantly, was the adverse impact of AIHA on the survival of patients with NHL. Therefore, this finding should be taken in consideration when risk-stratifying patients with NHL.