Evidence for a qualitative hierarchy within the Hoechst-33342 'side population' (SP) of murine bone marrow cells.

In vitro cobblestone area (CA)-forming cell (CAFC) and in vivo (short-term and competitive repopulation) assays demonstrate that a qualitative hierarchy exists within the Hoechst-33342-defined side population (SP) in murine bone marrow (BM). Consistent with and extending previous studies, we demonstrate that (i) hematopoietic activity found in whole BM (WBM) is concentrated within the SP, rather than the non-SP (NSP); and (ii) within the SP, those cells that more strongly efflux the dye (lower SP, LSP) are qualitatively different from those that less strongly efflux the dye (upper SP, USP). Qualitative differences are highlighted by evidence that (i) CA derived from LSP CAFC persist in culture significantly longer than CA derived from USP CAFC; (ii) short-term, multilineage repopulation of lethally irradiated mice by LSP cells is more rapid than that in mice receiving USP, NSP, whole SP (WSP), or WBM cells and (iii) LSP cells out-compete USP cells in the multilineage hematopoietic repopulation of lethally irradiated recipients. These data suggest that LSP cells are of higher quality than USP cells and potentially provide a means by which qualitative changes in primitive hematopoietic progenitors occurring naturally with aging, or clinically as a consequence of therapeutic manipulation, can be assessed.

Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes.

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and beta-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0-200 micrograms/ml) for 24 h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 micrograms/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.

The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract.

Red grape seed extract containing proanthocyanidins and other antioxidants are being used as nutritional supplements by many health conscious individuals. The beneficial effects of grape seed proanthocyanidins (GSPE) have been reported, however, little is known about their mechanism(s) of action. One of the beneficial effects of GSPE is chemoprevention of cellular damage. The precise mechanism by which GSPE mediates, chemoprevention is not yet understood. This report addresses this issue. We investigated the mechanisms of actions of GSPE, which ameliorates chemotherapy-induced toxic effects of Idarubicin (Ida) and 4,-hydroxyperoxycyclophosphamide (4-HC) in normal human Chang liver cells. Exposure to GSPE resulted in a significant reduction in apoptosis in response to the cytotoxicity of chemotherapeutic agents. RT-PCR analysis showed a significant increase in the anti-apoptotic gene Bcl-2 and a decrease in the cell cycle associated and proapoptotic genes, c-myc and p53 in cells treated with GSPE. These results suggest that some of the chemopreventive effects of GSPE are mediated by upregulating Bcl-2 and down regulating c-myc and p53 genes.

VLA-4 mediated adhesion to bone marrow stromal cells confers chemoresistance to adherent lymphoma cells.

The mechanisms of maintenance of residual lymphoma in bone marrow during chemotherapy are currently not well understood. Previous studies have shown that primary lymphoma cells obtained from histologically negative bone marrow of non-Hodgkin's lymphoma (NHL) patients grew in long-term bone marrow cultures primarily in association with bone marrow stromal cells. Furthermore, the interaction of NHL patient cells with bone marrow stromal cells inhibited their spontaneous apoptosis. The current studies were designed to characterize the components of the heterotypic interaction between lymphoma cells and bone marrow stromal cells as well as to probe the consequences of this interaction as it pertains to the potential survival of minimal numbers of lymphoma cells during chemotherapy. Cellular adhesion assays performed in the presence of either neutralizing antibodies to VCAM- or the alpha and beta subunit of VLA-4 resulted in >95%, 82% and 35% inhibition of lymphoma cell line adhesion to the bone marrow stromal line MS-5, respectively. Modulation of VLA-4 affinity by the 8A2 antibody resulted in enhanced secondary adhesion at 24 and 72 hours to either cellular fibronectin (65% and 65%) or MS-5 cells (60% and 55%), superceding levels obtained using untreated lymphoma cells (<20%). The bone marrow stromal cells induced a chemoprotective effect for adherent lymphoma cells over a 3-log dose range of vincristine, resulting in a 2-log increase in the ED50 at day 6 of culture. The failure of glutaraldehyde fixed stromal cells to induce a chemoprotective effect demonstrated that viable bone marrow stromal cells were necessary. Similarly, lymphoma/stromal cell conditioned medium also failed to provide a survival advantage. These data demonstrated that viable bone marrow stromal cells possessed the ability to actively inhibit the apoptotic pathways of intimately adherent lymphoma cells and this potentially contributes to their survival during chemotherapy.

Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer.

In this study, in vitro and in vivo antitumor effects of mononuclear cells from human umbilical cord blood cells (UCBCs) and peripheral blood stem cells (PBCs) harvest obtained by leukapheresis were compared. Interleukin 2 (IL-2)-activated mononuclear cells from UCBCs showed increased cytotoxicity against K562 and Raji hematopoietic malignant cells compared with PBCs (P < 0.05). After IL-2 activation, both UCBCs and PBCs showed significant cytotoxicity against MDA-231 human breast cancer cells. The UCBC population involved in this antitumor activity appeared to be CD56+ natural killer precursors. The cytotoxicity of UCBCs was inhibited in the absence of Ca2+ (P < 0.05), supporting a perforin/granzyme-mediated target of cell lysis. In addition, antibodies to Fas ligand blocked cytotoxic activity, suggesting that some of the antitumor cytotoxicity was Fas ligand mediated. In vivo antitumor effects of UCBCs and PBCs were studied using a human leukemic cell-bearing severe combined immunodeficient mouse model. There was a significant increase in the survival of K562 leukemia-bearing mice that also received 5 million in vitro IL-2-activated UCBCs or PBCs i.v. on days 3 and day 5 after tumor transplantation compared with untreated mice (P < 0.01). Similar antitumor cytotoxicity of UCBCs and PBCs was also observed against MDA-231 human breast cancer grown in severe combined immunodeficient mice (P < 0.01). These studies suggest that IL-2-activated UCBCs may be a useful source of cellular therapy for patients with hematological malignancies and breast cancer.

Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

Chemopreventive effects of grape seed proanthocyanidin extract on Chang liver cells.

In an attempt to ameliorate the chemotherapy associated normal cell toxicity, in this study a known antioxidant, grape seed proanthocyanidin extract (GSPE) using Chang liver cells has been used. Chang liver cells were treated in vitro with idarubicin (Ida) (30 nM) and 4-hydroxyperoxycyclophosphamide (4-HC) (1 microg/ml) with or without proanthocyanidin (25 microg/ml). The cells were grown in vitro and the growth rate of the cells were determined using MTT assay. The results showed that the GSPE decreased growth inhibitory effects of Ida and 4-HC on Chang liver cells in vitro. Since these chemotherapeutic agents are known to induce apoptosis in the target cells, these cells were also analyzed for presence of apoptotic cells using flow cytometry. The GSPE decreased the number of apoptotic cell population induced by either chemotherapy. In an attempt to determine the mechanisms of ameliorating effects of proanthocyanidin, the expression of apoptosis/cell cycle/growth related genes, Bcl-2, p53 and c-myc was determined in the treated and control cells using Western blotting or reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. There was an increased expression of Bcl-2 in the cells treated with GSPE. However, there was a significant decrease in the expression of other cell cycle related genes such as p53 and c-myc in these cells following treatment with GSPE. Thus, these results indicate that proanthocyanidin can be a potential candidate to ameliorate the toxic effects associated with chemotherapeutic agents used in treatment of cancer.

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells.

Grape seed proanthocyanidins are natural antioxidants which possess a broad spectrum of chemoprotective properties against free radicals and oxidative stress. In this study, we have assessed the cytotoxicity of a novel IH636 grape seed proanthocyanidin extract (GSPE) against MCF-7 human breast cancer cells, A-427 human lung cancer cells, CRL-1739 human gastric adenocarcinoma cells and K562 chronic myelogenous leukemic cells at 25 and 50 mg/lit concentrations for 0-72 h using cytomorphology and MTT cytotoxicity assay. In addition, we compared the effects on normal human gastric mucosal cells and normal J774A.1 murine macrophage cells with the effects on the cancer cell lines. Concentration- and time-dependent cytotoxic effects of GSPE were observed on the MCF-7 breast cancer, A-427 lung cancer and gastric adenocarcinoma cells. Following incubation of the MCF-7 cells with 25 mg/lit of the GSPE approximately 6.5, 30 and 43% inhibitions in cell growth were observed at 24, 48 and 72 h of incubation, respectively, while incubation of the MCF-7 cells with 50 mg/lit of the GSPE resulted in 11, 35 and 47% inhibition in cell growth at these same points, respectively. Similar results were observed in the A-427 and gastric adenocarcinoma cells. GSPE exhibited no cytotoxicity toward the neoplastic K562 myelogenous leukemic cells. However, GSPE enhanced the growth and viability of the normal human gastric mucosal cells and J774A.1 murine macrophage cells. These data demonstrate that GSPE exhibited cytotoxicity towards some cancer cells, while enhancing the growth and viability of the normal cells which were examined.

Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes.

Tetracyclines have been used in the treatment of chronic inflammatory diseases associated with local infiltration of inflammatory cells and matrix destruction as observed in rheumatoid arthritis and periodontal disease. Fas/Fas ligand (FasL)-mediated apoptosis plays an important role in maintaining T lymphocyte homeostasis and modulating immune response. The present study demonstrates that doxycycline inhibits Jurkat T lymphocyte proliferation and induces apoptosis. The phytohemagglutinin (PHA)-activated Jurkat cells are more susceptible to doxycycline-induced apoptosis. Furthermore, doxycycline-induced apoptosis is associated with increased Fas/FasL expression in Jurkat cells. The increase of apoptosis in Jurkat cells treated with doxycycline is consistent with the increase of FasL expression. These results suggest that doxycycline may downregulate the inflammatory process in certain diseases by eliminating activated T lymphocytes through Fas/FasL-mediated apoptosis.

Amelioration of the cytotoxic effects of chemotherapeutic agents by grape seed proanthocyanidin extract.

Anticancer chemotherapeutic agents are effective in inhibiting growth of cancer cells in vitro and in vivo, however, toxicity to normal cells is a major problem. In this study, we assessed the effect of a novel IH636 grape seed proanthocyanidin extract (GSPE) to ameliorate chemotherapy-induced toxic effects in cultured Chang epithelial cells, established from nonmalignant human tissue. These cells were treated in vitro with idarubicin (Ida) (30 nM) or 4-hydroxyperoxycyclophosphamide (4HC) (1 microg/ml) with or without GSPE (25 microg/ml). The cells were grown in vitro and the growth rate of the cells was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue] assay. Our results showed that GSPE decreased the growth inhibitory and cytotoxic effects of Ida as well as 4HC on Chang epithelial cells in vitro. Because these chemotherapeutic agents are known to induce apoptosis in the target cells, we analyzed the Chang epithelial cells for apoptotic cell population by flow cytometry. There was a significant decrease in the number of cells undergoing apoptosis following treatment with GSPE. We also found increased expression of the anti-apoptotic protein Bcl-2 in GSPE-treated cells using western blot techniques. Thus, these results indicate that GSPE can be a potential candidate to ameliorate the toxic effects associated with chemotherapeutic agents and one of the mechanisms of action of GSPE includes upregulation of Bcl-2 expression.

Mechanism of gastroprotection by bismuth subsalicylate against chemically induced oxidative stress in cultured human gastric mucosal cells.

Reactive oxygen species (ROS) are implicated in the pathogenesis of chemically induced gastric mucosal injury. We have investigated the effects of ethanol, hydrochloric acid (HCl), and sodium hydroxide (NaOH) on: (1) enhanced production of ROS including superoxide anion and hydroxyl radicals, (2) modulation of intracellular oxidized states by laser scanning confocal microscopy, and (3) DNA fragmentation, indices of oxidative tissue, and DNA damage in a primary culture of normal human gastric mucosal cells (GC), which were isolated and cultured from Helicobacter pylori-negative endoscopic biopsies from human subjects. The induction of ROS and DNA damage in these cells following exposure to ethanol (15%), HCl (150 mM) and NaOH (150 mM) were assessed by cytochrome c reduction (superoxide anion production), HPLC detection for enhanced production of hydroxyl radicals, changes in intracellular oxidized states by laser scanning confocal microscopy, and DNA damage by quantitating DNA fragmentation. Furthermore, the protective ability of bismuth subsalicylate (BSS) was assessed at concentrations of 25, 50, and 100 mg/liter. Incubation of GC with ethanol, HCI, and NaOH increased superoxide anion production by approximately 8.0-, 6.1-and 7.1-fold and increased hydroxyl radical production by 13.3-, 9.6-, and 8.9-fold, respectively, compared to the untreated gastric cells. Incubation of GC with ethanol, HCl, and NaOH increased DNA fragmentation by approximately 6.7-, 4.3-, and 4.8-fold, respectively. Approximately 20.3-, 17.5-, and 13.1-fold increases in fluorescence intensities were observed following incubation of gastric cells with ethanol, HCl, and NaOH, respectively, demonstrating dramatic changes in the intracellular oxidized states of GC following exposure to these necrotizing agents. Preincubation of GC with 25, 50, and 100 mg/liter of BSS decreased ethanol-induced increases in intracellular oxidized states in these cells by 36%, 56%, and 66%, respectively, demonstrating a concentration-dependent protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion and hydroxyl radical production, and DNA damage. The present study demonstrates that ethanol, HCl, and NaOH induce oxidative stress and DNA damage in GC and that BSS can significantly attenuate gastric injury by scavenging these ROS.

Antitumor activity of human umbilical cord blood cells: A comparative analysis with peripheral blood and bone marrow cells.

Although the hematopoietic reconstituting ability of human umbilical cord blood cells (UCBC) is well documented, their antitumor cytotoxic potential has not been well studied. Therefore, UCBC were compared to normal peripheral blood stem cells (PBSC) and bone marrow (BM) stem cell harvests for cytomorphology, antitumor cytotoxic activity before and after ex vivo cytokine manipulation, response to T and B cell mitogens, expression of adhesion molecules and immunophenotypes using flow cytometry, cytokine production and in vivo antitumor activity. BM and PBSC, but not UCBC, did not form cellular clusters in culture. More cytotoxic granules were present in the cytoplasm of UCBC than PBSC following activation in vitro. Ex vivo manipulation of UCBC with cytokines produced more cytotoxicity to K562 and Raji tumor cells than PBSC or BM (p<0.001). Most cytotoxic cells in UCBC cultures were T lymphocytes, and a correlation existed between the number of CD56+ cells and cytotoxicity levels, particularly after in vitro activation with interleukin-2. No significant difference in adhesion molecule expression was noted among UCBC, PBSC and BM cells. However, there was a significantly decreased expression of CD54 molecules (ICAM) on UCBC compared to PBSC (p<0.05). IL-2 activated UCBC showed significant antitumor effects against K562 leukemic cells grown in SCID mice. Thus UCBC contained more antitumor effector cells and precursors than cells from marrow or peripheral blood cells which might be capable of providing a therapeutic effect.

Identification of metallothionein receptors in human astrocytes.

Metallothionein (MT) isoforms are low molecular weight (6000-7000 Da) zinc binding proteins containing 60-68 amino acid residues, 25-30% cysteine, no aromatic amino acids, and binding between 5-7 g zinc/mol of protein. Since the synthesis of MT is induced by endotoxin, cytokines, and glucocorticoids, MT is now considered to be an acute phase protein protecting against oxygen radicals and oxidative damages caused by inflammation, tissue injury, and stress to the central nervous system. By postulating that a specific mechanism must exist to foster the induction of MTs I and II by numerous and diversified factors, we searched for and identified for the first time, MT receptors on U373MG cell membrane preparations, by using fluoresceinated MT I isoform probe; and by employing cysteine, glutathione, and four MT isoforms to determine high affinity and specific binding. MT receptors revealed a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein. Moreover, MT receptors were found in greater density on the surface of aggregated astrocytes. We postulate that conditions or agents generating reactive oxygen species may influence the expression of MT receptors.

Cell adhesion molecules and insulin-like growth factor-1 in vascular disease.

PURPOSE: Recent advances in the understanding of the biologic mechanisms of vascular diseases suggest that multifactorial stimulation of the endothelial cell and its subsequent adhesion to leukocytes is a prerequisite to the formation of atherosclerotic and restenotic lesions. As leukocyte-endothelial cell interaction is coordinated by a variety of cell adhesion molecules (CAMs), we hypothesized that the expression of certain CAMs is up-regulated in the vasculature of patients who have peripheral vascular disease. In addition, we proposed that insulin-like growth factor-1 (IGF-1) increases monocyte-endothelial adhesion by means of upregulation of these CAMs. METHODS: Using immunohistochemical techniques, the expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin was examined in human vascular disease specimens. Normal aortas obtained from the organ retrieval system were studied as control specimens. Adhesion studies between human umbilical vein endothelial cells (HUVECs) incubated with IGF-1 and purified human blood monocytes labeled with 51chromium were completed. Western blotting and flow cytometry were performed to show CAM expression on IGF-1-treated HUVECs. RESULTS: Of the CAMs, ICAM-1, P-selectin, and E-selectin were distinctly increased in diseased specimens when compared with control specimens (p < 0.05). Adhesion studies showed an increase in monocyte-endothelial cell adhesion of as much as 40% to 45% (p < 0.01) over baseline, with peak adherence occurring 4 hours after treatment with IGF-1. IGF-1 increased adherence in a dose- and time-dependent manner. The threshold concentration of IGF-1 that induced increased adhesion was 20 ng/ml, with a maximum effect occurring at 150 ng/ml. This increased adhesion was attenuated by pretreatment with IGF-I receptor antibody, as well as with genistein and herbimycin-A, which are potent and selective tyrosine kinase inhibitors. Increased adhesion correlated with an increase in the expression of CAMs on the surface of the HUVECs. An additive effect on adhesion was observed between IGF-1 and tumor necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1). Finally, immunohistochemical analysis of human vascular disease specimens revealed an increased expression of IGF-1 receptors as compared with control specimens (p < 0.05). CONCLUSIONS: These results suggest that IGF-1 may be important in the pathogenesis of peripheral vascular disease by increasing endothelial cell-monocyte adhesion by means of an increase in the expression of ICAM-1 and VCAM-1.

Comparison of molecular cytokeratin 19 reverse transcriptase polymerase chain reaction (CK19 RT-PCR) and immunocytochemical detection of micrometastatic breast cancer cells in hematopoietic harvests.

Detection of small numbers of breast cancer cells in patient blood, aphereses, and bone marrow has become increasingly important as data have accumulated showing immunocytochemically (ICC) positive tumor cells in up to 50% of women with stage I and II breast cancer, who were initially thought to be cured of their disease but later relapsed. The ability to rule out the presence of micrometastatic disease at any stage of the clinical management protocol, whether before, during, or after therapy, would provide a useful monitoring and diagnostic tool for both the clinician and the scientist. Monitoring for the presence of minimal residual disease (MRD) is traditionally performed using ICC. A more recently established RT-PCR technique uses a molecular marker (the presence of the cytokeratin 19, CK19, transcript) to identify MRD in patient samples, with a level of sensitivity reported to be one tumor cell in 10(6) nucleated cells. This level of sensitivity is generally higher than that claimed for ICC. Based on the discriminating results of this first study, a number of laboratories have evaluated this technique for its diagnostic potential. Results from several laboratories showed a higher than expected false positive rate due to a variety of identified and unidentified sources. Therefore, the current study was designed to achieve two aims: to establish the level of sensitivity and specificity of the RT-PCR technique and to dissect out the possible variables that may contribute to a false positive result using this molecular approach. To accomplish the first goal, two simulation strategies were used, limited dilution of tumor cells into apheresis harvests and semi-quantitative PCR using stepwise dilutions of extracted RNA from tumor cells in apheresis harvests. The second goal was accomplished by performing sequential blood drawings with variably timed sample processing to identify some of the more common variables (time, anticoagulant, sample sequence) that may contribute to false positive results. Of three variables investigated, including type of blood preservative, sequence of blood tube collection, and time point of sample processing, each may contribute to a false positive result. In addition to these problems, known events involving illegitimate transcription of specific genes nonspecifically in tissue is also a potential source of false positive results. These issues may be further compounded by lack of attention to the more common methodologic problems encountered in any laboratory using the PCR technique. However, recommendations can be developed for the effective application of this technique, whose greatest strength is the demonstration of tumor negativity of the sample.

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation.

Bestatin (ubenimex), an inhibitor of aminopeptidase, is an oral immunomodulator that binds to CD13 (aminopeptidase N) on macrophages/monocytes. To examine its immunomodulatory effect after high-dose therapy and autologous bone marrow transplantation (BMT), a dose-finding phase Ib trial was conducted with 30 Hodgkin's disease and non-Hodgkin's lymphoma patients who received no drug (control), 10 and 30 mg (low dose), or 90 and 180 mg (high dose) of bestatin daily for 60 days following autologous BMT. Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on 2 consecutive days. The serum neopterin levels, an indicator of monocyte/macrophage activation, increased in the high-dose group compared to the control group (not significantly) and the low-dose group (significantly). Similarly, the colony-stimulating activity in the sera was significantly increased in the high-dose group compared to the control and low-dose groups. We also examined the expression of cell-surface markers on monocytes in these patients by fluorescent cytometry analysis. There was no significant difference either in the frequency or absolute number of monocytes (CD14+) among the three groups at any time. However, a significant increase in the frequency of CD16(FcgRIII)-positive monocytes (a marker of activation) was observed in the high-dose group compared to controls from day 14 to day 60 after the start of bestatin administration. Further, the frequency of HLA-DR+ monocytes (another marker of activation) was significantly increased in the high-dose group. These results indicate that bestatin at higher doses (90 and 180 mg daily), but not lower doses, activates macrophages/monocytes, as demonstrated by phenotypic marker (HLA-DR and CD16) up-regulation, and this provides augmentation of neopterin and colony-stimulating activity in the serum of patients following autologous BMT.

Fibroblasts restrict tissue factor from vesicles which form in response to low concentrations of detergent.

Studies of tissue factor activity on fibroblasts have found that manifestation of the otherwise cryptic activity is evoked by Triton X-100 or octyl glucoside at concentrations that lyse the cells. Even though sublytic concentrations of the detergents extract membrane lipids into the soluble phase, they were without effect on tissue factor activity. Those experiments led us to conclude that either the fibroblasts maintain plasma membrane lipid asymmetry even as lipids are extracted by the detergents, up to the onset of lysis, or additional mechanisms for regulation of tissue factor specific activity were operative. Using phase contrast and immunofluorescent microscopy, we now show that at least one additional regulatory mechanism is indeed operative. In response to sublytic concentrations of octyl glucoside or Triton X-100, the cells release vesicles from which tissue factor antigen is excluded. Lytic concentrations of the detergents preclude this segregation, leaving only low amounts of tissue factor antigen associated with the adherent cytoskeletons. Two-color staining reveals marked tissue factor-actin filament co-localization, which implies the potential for cytoskeletal participation in the observed tissue factor segregation. We propose that tissue factor activity is indeed regulated by the phospholipids with which it is associated and the degree to which phosphatidylserine is available on the membrane surface, but the cells possess additional mechanisms by which the association of tissue factor with potentially procoagulant membrane domains is controlled.

Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation.

The immunologic attributes of cytokine mobilized peripheral blood stem cell (PSC) products (n = 52) and the resulting reconstitution of the hematopoietic and immunologic system following autologous transplantation were examined in a consecutive population of non-Hodgkin lymphoma (NHL), or solid tumor patients at the University of Nebraska Medical Center. Granulocyte-monocyte colony stimulating factor (GM-CSF)-mobilized PSC products had a high frequency of monocytes (31%) and bands (15%) as compared to normal peripheral blood (PB) cells. The phenotypic analysis of the mobilized PSC product revealed that they had normal levels of CD4+ cells, an increased frequency of CD8+ cells and a corresponding decrease in the CD4+:CD8+ cell ratio as compared to the peripheral blood leukocytes (PBL) of normal individuals. PSC products also had an increase in CD34+ cells as compared to PB. Natural killer (NK) and T cell activity in the PSC products were also lower than that observed in PB. Post-transplantation there was an accelerated reconstitution of NK-cell function in the PB as compared to T cell function (PHA (phytohemagglutinin) mitogenesis) which did not return to normal by day 100 post-transplantation. We also report for the first time high levels of an irradiation resistant suppressor cell activity in the PSC product and in the PB post-transplantation. There was also a concomitant increase in CD4-, CD8-, TCR alpha/beta+ cells (phenotypic homolog of 'natural suppressor' (NS) cells) in the PB post-transplantation. The number of months of prior chemotherapy correlated with PHA response but the NS activity and frequency of CD4-, CD8- and TCR alpha/beta+ cells did not. Further, cytokine mobilization and apheresis appears to contribute to the loss of PHA responsiveness and the increased levels of suppressor cell activity.