The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death.

The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.

Hypochlorite scavenging activity of polyphenols.

Chemiluminescence, generated by the mixture of sodium hypochlorite solution and luminol, was completely eliminated by polyphenols, such as natural lignins, phenylpropenoid monomers and polymers, and epigallocatechin gallate. On the other hand, hypochlorite scavenging activity of polysaccharides, such as PSK (Krestin) and Schizophyllan, was relatively weak. Human myelogenous leukemic cell lines (HL-60, ML-1) showed higher production of active oxygen(s) (detected by luminol chemiluminescence) and iodination capacity, than six other cultured cell lines. Since lignin did not completely eliminate the active oxygen production by HL-60 cells, possible stimulation of hypochlorite production by lignin was suggested.

Endonuclease activity and induction of DNA fragmentation in human myelogenous leukemic cell lines.

When four human myelogenous leukemic cell lines (HL-60, ML-1, U-937, THP-1) were exposed to either ascorbic acid, hydrogen peroxide, etoposide, tumor necrosis factor, hyperthermia or UV irradiation, their growth inhibition and oligonucleosome-size DNA fragmentation were induced. Non-myelogenous leukemic cell lines (MOLT-4, K-562) were similarly sensitive to ascorbic acid and hydrogen peroxide, but relatively resistant to etoposide, TNF, hyperthermia and UV irradiation. Furthermore, these treatments except for UV irradiation, did not induce any apparent DNA fragmentation in MOLT-4 and K-562 cells. An autodigestion experiment revealed that all of these six cell lines contained divalent cation-independent endonuclease activity as a major endonuclease. The ability of this endonuclease to produce oligonucleosome-size DNA fragmentation was stimulated at acidic, but not at neutral pH. Since this enzyme activity was not detected in the lysosomal enzyme-free nuclei, prepared from all six cell lines, the cytoplasmic localization of this enzyme was suggested. The results suggest that the endonuclease activity might be differently regulated between myelogenous and non-myelogenous leukemic cell lines.

Human HER-2/neu protein immunization circumvents tolerance to rat neu: a vaccine strategy for 'self' tumour antigens.

Many newly defined tumour antigens are 'self' proteins. Immunizing cancer patients against these antigens may be difficult due to tolerance. The HER-2/neu oncogenic protein is such a 'self' tumour antigen. Rat neu is homologous with human HER-2/neu and provides a model system for studying vaccination strategies. Rats are tolerant to rat neu. Vaccination with this 'self' protein elicits no detectable immune response. The current studies evaluated whether tolerance to rat neu can be circumvented by immunizing with the highly homologous foreign human HER-2/neu protein. Rats were immunized with human HER-2/neu intracellular domain (hICD) protein that is 92% homologous to rat neu ICD. Animals immunized with hICD developed significant antibody and T-cell responses that were specific for both human HER-2/neu and rat neu. Neu-specific antibodies were present in titres of greater than 1:200,000. Analysis of the specificity of the antibody response using synthetic peptides demonstrated substantial reactivity to an epitope with 100% homology between rat and human protein. Significant T-cell responses (stimulation index > 10) to hICD and rat neu protein (stimulation index > 4) were detected. The T cells also responded to both human and rat ICD. The results imply that immunization with foreign proteins, which are highly homologous to 'self' tumour antigens, may be an effective vaccine strategy for 'self' tumour antigens.

Lack of toxicity of acyclovir to granulocyte progenitor cells in vitro.

Granulocyte progenitor cells were grown with acyclovir to study potential marrow toxicity. Concentrations of up to 220 microM had little effect on progenitor cell growth.

Increased proliferative capacity of CD4+ and CD8+ T lymphocytes from mutant sphha/sphha mice is associated with increased IL-2 receptor expression.

We previously discovered that mutant anemic mice (sphha/sphha) show increased numbers of cycling lymph node T lymphocytes when analyzed by pulse and continuous infusion of tritiated thymidine. We have now further analyzed this in vivo phenomenon by evaluating the in vitro proliferative response of anti-CD3 activated lymphocytes from anemic mice using flow cytometric cell cycle analysis with 5'-bromodeoxyuridine and Hoechst dye. We determined that sorted CD4+ and CD8+ T lymphocytes from anemic mice have significantly greater proliferative capacity when compared with syngeneic control (+/+) mice (P < 0.001). In order to explain this increased growth capacity, we examined whether these cells exhibit differences in cell-surface phenotype (Pgp-1 and IL-2 receptor expression), activation state, or transmembrane signaling, or alterations in accessory cells or cytokines. Increased proliferation of T cells from anemic mice was associated with a larger percentage of T cells expressing IL-2R (p55 or CD25) at 24 and 48 hr after activation. Increased proliferative capacity was not associated with differences in activation state, Pgp-1 phenotype, transmembrane signaling, accessory cells, or cytokines. The mechanism for the abnormally high proliferative rate of T cells from anemic mice remains unclear, but we suggest that this mutant mouse may provide an important model for further studies on the molecular basis of T-cell replication.

Circulating granulocytic and erythroid progenitor cells in chronic granulocytic leukaemia.

We used a standard methyl cellulose method to assay erythroid progenitor cells in the blood of 35 patients with untreated CGL and of 18 normal controls. In 28 patients we simultaneously assayed granulocyte/monocyte committed progenitor cells (CFU-c) by an agar method. Circulating erythroid burst-forming units (BFU-e) in CGL were increased above normal by a factor of about 180; CFU-c were increased by a factor of about 9000. Both BFU-e and CFU-c numbers were linearly related to the total leucocyte count in individual patients but not to numbers of circulating blast cells. There was a positive correlation in individual patients between CFU-c and BFU-e numbers. Circulating BFU-e and erythroid colony-forming cells (CFU-e) were unable to proliferate in vitro in the absence of erythropoietin. We conclude that erythroid progenitor cells are involved in the 'clonal expansion' that characterizes CGL, but apparently to a lesser extent than are granulocyte/moonocyte progenitor cells.

Pathologic features associated with decreased longevity of mutant sphha/sphha mice with chronic hemolytic anemia: similarities to sequelae of sickle cell anemia in humans.

A colony of sphha/sphha mice with congenital hemolytic anemia and an abnormality in erythrocyte spectrin assembly was screened to determine the cause of premature death. Sphha/sphha mice have decreased life span, with 50% of animals dying by 6 months of age. The phenotype of these mutant mice includes moderate anemia (hematocrit: 21 to 28%), reticulocytosis, leukocytosis, lymphocytosis, extensive extramedullary hematopoiesis in spleen and liver, lymph node hyperplasia and membranoproliferative glomerulonephritis. With increased surveillance of this mouse colony, 20 clinically sick anemic mice were evaluated (complete blood counts and cultures of blood), euthanized and necropsied. Compared with anemic mice without clinical signs of disease, sick anemic mice had significantly higher white blood cell counts with only 4 (20%) of 20 animals being severely anemic (hematocrit: 4 to 8%). Blood from 11 (45%) of 20 animals was culture-positive for Pasteurella pneumotropica, Enterococcus, and/or Escherichia coli. In addition to the usual lesions in sphha/sphha mice, sick anemic mice had pneumonitis (95%) with thrombosis and infarction (80%) of one or more organs (spleen, myocardium, pancreas, liver, or bone marrow). The thrombotic tendency that accompanies the chronic hemolytic anemia in sphha/sphha mice, as well as the other clinicopathologic changes in these mutant mice, bears a striking resemblance to some poorly understood sequelae in human patients with sickle cell anemia. This mouse model may be useful in studying the pathophysiology of complications associated with sickle cell anemia in humans.

Toxicity of amphotericin B, miconazole, and ketoconazole to human granulocyte progenitor cells in vitro.

Granulocyte progenitor cells were grown in culture with amphotericin B, miconazole, and ketoconazole. Significant suppression of progenitor cell growth could be demonstrated with all three drugs at increasing concentrations. No additive suppression was seen when amphotericin B and ketoconazole were combined.

Immune function in offspring of nonhuman primates (Macaca nemestrina) exposed weekly to 1.8 g/kg ethanol during pregnancy: preliminary observations.

A preliminary investigation of immune host response was conducted in a group of fetal alcohol-exposed nonhuman primates (Macaca nemestrina) who were part of a broader ongoing study of ethanol teratogenicity. The mothers of the offspring received weekly oral doses of ethanol (1.8 g/kg) for the first 3 or 6 or the entire 24 weeks of gestation. A control group received sucrose solution weekly throughout pregnancy. Four of the 18 ethanol-exposed animals (22%) died or were euthanized after infectious disease or failure to thrive during the first year of life; none of the seven control animals died. This imbalance in survival prompted the present review of immune function in the remaining offspring. Parameters assessed included: (1) white blood cell count (WBC), (2) peripheral blood leucocyte subsets (CD4+, CD8+, CD20+, and CD11c+), (3) T-cell proliferation after activation with phytohemagglutinin (PHA), staphylococcus enterotoxin B (SEB), and tetanus toxoid (TT), (4) phagocytic activity of monocytes, and (5) serum immunoglobulin levels and serum antibody titers after TT vaccination. Mean T-cell proliferation to TT was significantly decreased (p = 0.01) in all ethanol-exposed animals relative to controls, with near-significant decreases (p = 0.06) in response to SEB in the ethanol-exposed animals. Lymphocyte proliferation in response to PHA was not altered. Ethanol-exposed animals had significantly lower TT titers than controls after initial vaccination and booster. WBC, leukocyte subsets, serum immunoglobulins, and monocyte phagocytic activity were not significantly different from control values. These preliminary observations suggest that T-cell proliferation and antigen-specific memory responses may be altered in offspring exposed to weekly doses of ethanol in utero and warrant further evaluation for confirmation.

Granulocyte-macrophage colony-stimulating factor: an effective adjuvant for protein and peptide-based vaccines.

The current studies evaluate granulocyte-macrophage colony-stimulating factor (GM-CSF) as a vaccine adjuvant. An important issue for developing vaccine therapy for human malignancy is identifying adjuvants that can elicit T-cell responses to proteins and peptides derived from "self" tumor antigens. GM-CSF, in vitro, stimulates the growth of antigen-presenting cells such as dendritic cells and macrophages. Initial experiments examined whether GM-CSF injected into the skin of rats could affect the number or character of antigen presenting cells, measured as class II major histocompatability complex expressing cells, in lymph nodes draining the injection site. Intradermal (id) inoculation of GM-CSF every 24 hours for a total of five inoculations resulted in an increase of class II+ fluorescing cells that peaked at the fourth inoculation. Subcutaneous (sq) inoculation resulted in an increase of class II+ fluorescing cells that peaked following the second inoculation, then decreased over time. Using this schema for "conditioning" the inoculation site, GM-CSF was administered id or sq for five injections and a foreign antigen, tetanus toxoid (tt), was given at the beginning or the end of the immunization cycle. Id immunization was more effective than sq at eliciting tt specific immunity. In addition, GM-CSF id, administered as a single dose with antigen, compared favorably with complete Freund's adjuvant (CFA) and alum in eliciting tt specific antibody and cellular immunity. We have shown that immunity to rat neu (c-erbB-2) protein, an oncogenic self protein, can be generated in rats by immunization with peptides derived from the normal rat neu sequence plus CFA. The current study demonstrates that rat neu peptides inoculated with GM-CSF could elicit a strong delayed type hypersensitivity reaction (DTH) response, whereas peptides alone were non-immunogenic. GM-CSF was as effective as CFA in generating rat neu specific DTH responses after immunization with a neu peptide based vaccine. Soluble GM-CSF is a potent adjuvant for the generation of immune responses to foreign proteins as well as peptides derived from a self tumor antigen.

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.