Polystyrene nanoparticles may affect cell mitosis and compromise early embryo development in mammals.

A great interest surrounds the development of nanoparticles (NPs) for biomedical applications such as drug delivery and cancer therapy. However, the interplay between nanoscale materials and biological systems and the associated hazards have not been completely clarified yet. In this study, bovine oviductal epithelial cells (BOECs) and embryos were used as in vitro models to investigate whether cell mitosis and early mammalian embryo development could be affected by the exposure to polystyrene (PS) nanoparticles. Analysis of the karyotype performed on BOECs exposed to PS-NPs did not show chromosomal anomalies compared to the control, although more tetraploid metaphase plates were observed in the former. In vitro fertilization experiments designed to understand whether exposure to PS-NPs could affect pre-implantation development showed that incubation with PS-NPs decreased 8-cell embryo and blastocyst rate in dose-dependent fashion. The quality of the blastocysts in terms of mean cell percent blastomeres with fragmented DNA was the same in exposed blastocysts compared to controls. These results show that the exposure to PS-NPs may impair development. In turn, this may affect the rate of mitosis in embryos and yield a lower developmental competence to reach the blastocyst stage. This suggests that release in the environment and the subsequent accumulation of PS-NPs into living organisms should be carefully monitored to prevent cytotoxic effects that may compromise their reproduction rates.

Long-term viability and differentiation of bovine oviductal monolayers: bidimensional versus three-dimensional culture.

Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Gray's medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Gray's medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Gray's medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Gray's medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo.

Pyriform cell differentiation in Podarcis sicula is accompanied by the appearance of surface glycoproteins bearing alpha-galNAc terminated chains.

The present histochemical and cytochemical study using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, LCA, Con-A, DBA, MPA, BPA) has demonstrated that, in Podarcis sicula, the differentiation of small follicle cells into pyriform cells by means of intermediate cells is accompanied by the appearance of glycoproteins bearing alpha-GalNAc terminated O-linked side chains on the cell surface. The distribution of DBA- and MPA-binding sites over the follicular epithelium changed during the different stages of oocyte growth. DBA- and MPA-binding sites first appeared at the beginning of folliculogenesis within the zona pellucida (ZP) and on the surface of small cells, i.e., the stem cells of pyriform cells. Afterward, labeling was evident on the cell surfaces of intermediate cells and, later on, also of pyriform cells. On the other hand, no labeling was detected on the small cells located under the basal lamina, which, reportedly, do not differentiate into pyriform cells (Filosa et al. J. Embryol. Exp. Morphol., 1979; 15:297-316). Once pyriform cells were differentiated, the distribution of DBA- and MPA-binding sites over the follicular epithelium remained unchanged until intermediate and pyriform cells underwent apoptosis (Motta et al. J. Exp. Zool., 1996; 276:233-241) and the follicular epithelium transformed into a monolayer composed of small follicle cells only (Filosa Mon. Zool. Ital., 1973; 7:151-165). During this stage of oocyte growth, DBA and MPA labeling gradually decreased to completely disappear in the follicular epithelium of vitellogenic follicles. It is noteworthy that the observed changes in the distribution of DBA- and MPA-binding sites represent the first evidence recognized by lectins of a gradual modification of surface glycoprotein distribution over the follicular epithelium in the ovarian follicles of nonmammalian vertebrates so far studied. Finally, the zona pellucida (ZP), characterized by the presence of GalNAc, GluNAc, Man, and Gal, was demonstrated to be first synthetized by the oocyte and later on by the follicle cells.

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release.

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.

Malignant supratentorial glial-neuronal neoplasms: report of two cases and review of the literature.

OBJECTIVE: Malignant neoplasms exhibiting mixed populations of neuronal and glial cells occurring in the cerebral hemispheres of young adults and children are well recognized, but rare. A confusing array of diagnostic terms has arisen. We describe two patients with such tumors and review the literature concerning these interesting cases. PATIENTS: A 21-year-old man and a 5-year-old girl presented with large, cystic, intracerebral lesions on magnetic resonance images, which proved to be composite neoplasms exhibiting malignant neurons and astrocytes. RESULTS: The 21-year-old man had a frontal lobe mass with enhancing and nonenhancing regions, which corresponded to cerebral neuroblastoma and anaplastic astrocytoma, respectively. The presence of occasional microtubules and rare primitive presynaptic processes, accompanied by antisynaptophysin immunoreactivity, established the neuronal nature of the cells in the enhancing region. The nonenhancing region was composed of a moderately cellular neoplasm of fibrillar astrocytes that were mitotically active. The 5-year-old girl presented with a left parietal lobe neoplasm, which histologically was composed of lobular proliferations of neuroblasts and glia. The neuroblastic populations exhibited evidence of maturation with small anaplastic cells, spindle-shaped cells, and large dysmorphic ganglion cells. The glial tumor showed both well-differentiated fibrillary astrocytes with microcysts and anaplastic populations with central necrosis and pseudopalisading. CONCLUSIONS: Present classification systems devised to describe mixed neuronal and glial tumors do not adequately encompass the diversity of morphologies presented by these two cases. We conclude that the terms cerebral neuroblastoma-anaplastic astrocytoma for case 1 and cerebral ganglioneuroblastoma-glioblastoma for case 2 are preferred because they convey useful clinical information by reflecting concepts already encompassed by the World Health Organization's classification system of tumors of the central nervous system.

Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro.

The mammalian zona pellucida contains several glycoproteins whose oligosaccharide moieties are known to play a key role in the interaction with spermatozoa. Since zona pellucida defects may represent one of the most likely causes of failed fertilization in human in-vitro reproduction, we have studied the carbohydrate composition and distribution over the human zona pellucida by means of lectins. Donated, not inseminated cumulus-oocyte complexes, from cohorts with high fertilization rates, and fertilization-failed oocytes from cohorts inseminated with proven fertile donor semen, were analysed using 11 fluorescein-labelled lectins, on deplasticized semi-thin epoxy sections. Results showed that wheat germ agglutinin (WGA), Maclura pomifera (MPA) and Pisum sativum (PSA) bound to the extracellular matrix bordering the zona pellucida-corona radiata interface of cumulus-oocytes complexes, while the zona pellucida was labelled by WGA, Concanavalin A (ConA) and PSA. WGA labelling and correlative electron microscopy on the cumulus-oocyte complexes demonstrated that this lectin is a useful tool to trace the cortical granule distribution in the human oocyte. Surprisingly, in the failed-fertilized oocytes the zona pellucida was also labelled by MPA and showed three different patterns: (i) labelling of the zona pellucida outer surface; (ii) uniform labelling; (iii) labelling of an outer zona pellucida layer with variable thickness. Comparative analysis of WGA and MPA labelling on single failed-fertilized oocytes demonstrated that MPA zona pellucida patterns are not related to the cortical reaction. The nature and meaning of the MPA pattern of failed-fertilized oocytes were discussed in the light of zona pellucida defects impairing sperm receptivity.

A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia.

BACKGROUND: Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS: To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS: Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS: Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

Agranulocytosis associated with etodolac.

OBJECTIVE: To report a case of probable etodolac-induced agranulocytosis. CASE SUMMARY: A 72-year-old woman who had been taking etodolac 300 mg bid for approximately six weeks presented to the emergency department with symptoms of urosepsis. She was found to be profoundly granulocytopenic. Etodolac was discontinued and broad-spectrum intravenous antibiotic therapy was administered for the next 17 days. Results of bone marrow biopsy revealed marked hypocellularity consistent with drug-induced agranulocytosis. Following etodolac withdrawal, the total white blood cell count reached a low value of 0.9 x 10(9)/L and then returned to a pre-etodolac baseline after 15 days. Her hemoglobin concentration also decreased significantly during hospitalization. DISCUSSION: Agranulocytosis has rarely been reported in association with nonsteroidal antiinflammatory drugs (NSAIDs), and there are no literature reports associating etodolac with agranulocytosis. This case involving etodolac is consistent with the pattern described with other NSAIDs. Factors correlating etodolac as the causative agent are identified. Details of patient history, treatment, follow-up, and assessment are discussed. CONCLUSIONS: Detailed case assessment demonstrated probable etodolac-induced agranulocytosis in our patient. Clinicians should be aware that etodolac, like other NSAIDs, has potential to cause agranulocytosis.

The role of monocyte-derived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a rare pediatric malignancy characterized by marked hepatosplenomegaly, leukocytosis with prominent monocytosis, elevated fetal hemoglobin, no Philadelphia chromosome, and generally a poor prognosis. In vitro, JCML peripheral blood granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) demonstrate the unique characteristic of "spontaneous" proliferation at very low cell densities in the absence of exogenous growth factors. The "spontaneous" CFU-GM proliferation can be abolished by prior adherent cell (monocyte) depletion, suggesting a paracrine mode of cellular proliferation. Although previous studies using a [3H]thymidine ([3H]TdR) incorporation assay suggested an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in JCML, many non-growth factor-related reasons for [3H]TdR incorporation and the relatively low level of inhibition of [3H]TdR uptake left those conclusions open to question. Therefore, we performed clonal CFU-GM assays, which more specifically reflect cytokine effects on CFU-GM, using JCML peripheral blood mononuclear cells (PBMNC) and neutralizing antibodies against GM-CSF, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating (M-CSF), interleukin 3 (IL-3), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 4 (IL-4), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma). Cultures containing anti-GM-CSF alone inhibited "spontaneous" JCML CFU-GM by 87% +/- 9% (mean +/- standard error of the mean [SEM]). No other anti-cytokine antibody produced a significant inhibition of CFU-GM growth. Various combinations of antibodies, excluding anti-GM-CSF, failed to demonstrate any synergistic inhibitory effects upon CFU-GM. Because this apparent paracrine cellular stimulation could be due to excessive cytokine production, by monocytes or other accessory cells, we examined cytokine levels in conditioned media from various JCML cell populations using enzyme-linked immunosorbent assays (ELISAs). Monocytes from only a minority of JCML patients produced higher than normal quantities of GM-CSF, G-CSF, IL-1 beta, IL-6, and/or TNF alpha, but no obvious pattern could be discerned. Further, only 7 of 15 JCML monocyte-conditioned media (MCM) had elevated GM-CSF, and 6 of 15 JCML patients had normal levels of all nine cytokines tested. The monocyte depletion experiments and the inhibition experiments with anti-cytokine antibodies taken together demonstrate clearly that the "spontaneous" growth of JCML CFU-GM in vitro critically depends on at least one monocyte-derived growth factor, GM-CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Urticaria pigmentosa and preleukemia: evidence for reactive mast cell proliferation.

A 64-year-old man had urticaria pigmentosa and myelodysplasia (refractory anemia with excess blast cells; partial trisomy 8 syndrome) without increased numbers of marrow mast cells. Clonal marrow assays in agar demonstrated normal to increased colony-forming units of granulocytes/macrophages. In long-term liquid cultures containing mast cell growth factor (interleukin 3), his marrow cells proliferated after 3 weeks to produce abnormal myeloid precursors similar to those in the corresponding marrow aspirate specimen. Cells with basophilic-staining granules were less abundant in comparison with normal marrow specimens cultured similarly. These results suggest that the mast cells in this patient are not of the same clone as the preleukemic marrow cells, although the possible marrow-cell origin of urticaria pigmentosa mast cells cannot be excluded. Previous reports suggest that urticaria pigmentosa without systemic mastocytosis occurs as a nonspecific abnormality in a variety of myeloid, lymphoid, and nonhematologic malignancies. Our data also support this hypothesis that urticaria pigmentosa is a reactive process rather than a manifestation of clonal proliferation of the primary malignancy.

Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors.

Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this "spontaneous" myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose-response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM-CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease.

Granulocyte-macrophage colony-stimulating factor is an endogenous regulator of cell proliferation in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood that is clinically and cytogenically distinct from the well-recognized adult type of chronic myeloid leukemia. Unlike the adult disease, growth of hematopoietic progenitors from peripheral blood (PB) occurs in the absence of exogenous stimulus even at low cell densities. This so-called "spontaneous" growth can be abrogated by adherent cell depletion and appears to depend on production of endogenous growth factors. We studied seven children with JCML to determine the nature of endogenous stimulators. With isolated PB mononuclear cells (PBMNCs) and a 3H-thymidine (3H-TdR) incorporation assay, JCML cells were shown to incorporate high levels of 3H-TdR when cultured in the absence of stimulus even at low cell densities. When neutralizing antisera prepared against each of the four known colony-stimulating factors (CSFs), GM-CSF, G-CSF, M-CSF, and interleukin-3 (IL-3), as well as antisera against interleukin-1 (alpha and beta) and tumor necrosis factor (TNF) were added to these cultures, only the antisera against recombinant human GM-CSF (rhGM-CSF) consistently resulted in significant inhibition of cell proliferation, achieving up to 72% inhibition of 3H-TdR incorporation in one case. Monoclonal antibodies (MoAbs) against rhGM-CSF resulted in a similar and highly significant degree of inhibition. A marked inhibitory effect of rhGM-CSF antiserum on "spontaneous" growth of PB CFU-GM derived colonies in semisolid medium was also demonstrated in four of five patients studied (87% to 90% inhibition). Production of growth factors by highly enriched JCML monocytes was variable. When initially studied in five of the seven patients, the monocytes from three of the patients revealed increased release of IL-1-like activities; two patients had levels similar to those of controls. One patient with normal levels when initially studied was later shown to have markedly increased amounts of IL-1-like activities in a second preparation of monocyte-conditioned medium (MCM). High levels of GM-CSF were detected in the initial MCM from one patient, but this may have indirectly reflected elevated IL-1-like activities present in the MCM. IL-3 and M-CSF levels were either low or undetectable in the patients studied as compared with MCM prepared with normal adult monocytes. These results clearly implicate GM-CSF as the primary endogenous regulator of JCML cell proliferation in culture and suggest that this malignant myeloproliferative disease may in part result from paracrine stimulation of marrow progenitor cells by growth factors/cytokines secreted by the malignant monocytes.

Cell culture studies and oncogene expression in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) may be distinguished from adult CML based upon in vitro cell growth characteristics. We studied four untreated children with JCML and report additional unique findings. Peripheral blood (PB) and bone marrow (BM) cells were grown in soft agar. Without exogenous colony-stimulating activity (CSA) there was exuberant "spontaneous" colony formation in both PB and BM cultures. In the absence of exogenous stimulus, PB colony morphology was predominantly, but not exclusively, monocyte/macrophage. When PB was depleted of adherent cells, "spontaneous" colony formation was nearly completely abrogated. Cultures were also performed in the presence of various sources of CSA including giant cell tumor-conditioned medium (GCT-CM), a melanoma cell line-CM (LD1-CM), human placenta-CM (HPCM), and normal PB mononuclear cell (PBMC) feeder layers. Colony formation was typically increased with HPCM and PBMC, whereas in two patients GCT-CM and LD1-CM failed to stimulate additional colony growth when compared to cultures without exogenous CSA and, in fact, appeared to inhibit baseline "spontaneous" growth. The morphology of colonies in the presence of exogenous stimuli was highly variable. Because of the recent association between the c-fms protooncogene product and the receptor for the monocyte growth factor CSF-1, we analyzed the PB cells from two JCML patients for c-fms expression. Although expressed, c-fms levels were less than that in an adult with Ph1-positive CML in chronic phase. These studies indicate that in JCML, there are dramatic increases in both PB and BM colony-forming cells and that "spontaneous" growth is dependent on an accessory adherent cell fraction. Furthermore, patterns of responsiveness to various sources of CSA suggest that the colony-forming cells may not be a uniform population of malignant cells.

Differential staining of neutrophils and monocytes: surface and cytoplasmic iron-binding proteins.

Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and alpha-naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.

Consequences of extremely high doses of irradiation on bone marrow stromal cells and the release of hematopoietic growth factors.

Although hematopoietic growth factors have previously been difficult to demonstrate in long-term murine bone marrow cultures, it is possible to demonstrate release of growth factors from adherent cells of these long-term cultures following modest doses of irradiation. The present studies were undertaken to determine the maximally tolerated dose of irradiation for growth factor-producing stromal cells and to characterize the growth factor activities. It was discovered that stromal cells could survive extremely high doses of irradiation (500 Gy) and continue to elaborate hematopoietic growth factors. Using escalating doses of irradiation, a dose-dependent increment in detectable hematopoietic growth factors was detectable in unconcentrated conditioned medium. Conditioned medium from long-term cultures exposed to 500 Gy stimulated both fresh murine bone marrow cells (15 +/- 2 to 81 +/- 5 CFU-C/5 X 10(4) target cells) and the interleukin-3/GM-CSF-responsive cell line FDC-P1. In the CFU-C assay, this activity appeared to be predominantly monocyte/macrophage differentiating activity (M-CSF), based upon colony morphology. However, following stimulation of these irradiated stromal cells with endotoxin, there was a significant increase in FDC-P1 growth-promoting activity, and in the CFU-C assay there was increased production of granulocyte, megakaryocyte, and blast-cell type colonies indicating the increased release of a multilineage growth factor. The stromal cells surviving these extremely high doses of irradiation represent a heterogeneous population as demonstrated by morphologic, histochemical, and functional characterization. The two predominant cell populations included a macrophage-like cell and a large flat cell previously referred to as a "blanket" cell.

Identification of the hematopoietic growth factors elaborated by bone marrow stromal cells using antibody neutralization analysis.

These studies were undertaken to characterize the subclasses of hematopoietic growth factors produced by stromal cells in long-term murine bone marrow cultures. Exposure of these cultures to extremely high doses of irradiation (500 Gy), followed by endotoxin stimulation, permitted detection and characterization of various growth factor activities in the unconcentrated conditioned medium. To determine the nature of these activities, neutralization studies were performed using antisera against the following subclasses of purified colony-stimulating factors (CSFs): purified L-cell CSF-1, recombinant granulocyte-macrophage CSF (rGM-CSF), and recombinant interleukin 3 (rIL3). The antiserum against CSF-1 consistently abrogated 100% of the CSF bioactivity in irradiated stromal cell-conditioned medium (CM) but was only capable of neutralizing 62%-91% of the bioactivity in endotoxin-stimulated, irradiated stromal cell-CM. Antisera against rGM-CSF and rIL3 demonstrated variable effects. When the antisera were used in combinations, only the mixture of anti-CSF-1 + anti-GM-CSF resulted in 100% neutralization of the activities in endotoxin-stimulated, irradiated stromal cell-CM. This CM stimulated the IL3/GM-CSF-responsive cell line FDC-P1 but not the IL3-responsive (GM-CSF-unresponsive) cell line 32D cl-23. The FDC-P1 growth-promoting activity was inhibited only by the antiserum against GM-CSF and not by antiserum against IL3. These experiments indicate that stromal cells from long-term bone marrow cultures can produce and release CSF-1 and GM-CSF while the production of IL3 in this system, if there is any, could not be demonstrated.

Effect of adenine nucleotides on granulopoiesis and lithium-induced granulocytosis in long-term bone marrow cultures.

Studies were undertaken to evaluate the role of adenine nucleotides in regulating hematopoiesis using a long-term liquid culture system. In contrast to early investigations using clonogenic stem cell assays, where inhibitory effects were observed, adenosine and adenosine-5'-monophosphate (AMP) were found to stimulate myelopoiesis whereas the dibutyryl derivative of cyclic adenosine-3',5'-monophosphate (dcAMP) had either a modest inhibitory effect or no effect on long-term hematopoiesis. Dose effects for AMP enhancement of hematopoiesis were relatively narrow. When cultures were exposed to a broad range of concentrations (10 mM-10 nM), stimulation was only seen at a molar concentration of 1 X 10(-4) M. Stem cell assays revealed stimulation of multipotent stem cells (CFU-S), as well as committed progenitor cells (CFU-C). Lithium chloride has been shown to cause granulocytosis both in vivo and in vitro. Reductions in intracellular cAMP levels resulting from adenylate cyclase inhibition is a proposed mechanism for this stimulatory effect. However, lithium-induced granulocytosis in long-term cultures could not be blocked by the addition of dcAMP. Measurement of nucleotide levels on spent medium revealed rapid utilization and/or degradation of these reagents. This suggests that failure to abrogate the lithium effect with dcAMP may have been related to the inability to maintain constant intracellular concentrations. The varied observations regarding adenine nucleotide effects on hematopoiesis, as well as the reproducible stimulation by lithium, may be explained by our current appreciation of the complex adenylate cyclase system, which contains both inhibitory and stimulatory subunits for nucleotides and monovalent cations.

Autoimmune hemolytic anemia associated with a hypernephroma.

This report describes a case of warm-antibody-mediated hemolytic anemia associated with a hypernephroma. Only four patients with renal cell carcinoma and Coombs'-positive hemolytic anemia have been reported previously. Although rare, the occurrence of autoimmune hemolytic anemia in association with a hypernephroma should be considered one of the possible hematologic complications of this tumor.

Bone marrow adherent cell hemopoietic growth factor production.

These data suggest that two types of radioresistant adherent stromal cells are adequate for maintenance of long term hemopoiesis in the Dexter culture system. One cell type appears to be a typical adherent macrophage while the other is an alkaline phosphatase positive epitheloid cell possibly representative of the adventitial reticular cell of the bone marrow. These two cell types seem to be clearly defined by the in-vivo irradiation studies and less clearly defined in the in-vitro irradiation experiments. These data do not exclude other cell types as playing important roles in modulating hemopoiesis but do suggest that these major types are probably playing important roles in maintaining stem cells in long term liquid cultures. In addition, data suggests that the epitheloid cell directly nurtures hemopoietic cells on its surface while the macrophages may serve a separate function. A number of growth factors are produced by these two cell types which appear to include CSF-1, a granulocyte CSA separate from CSF-1 and a megakaryocyte CSA separate from both the GM-CSA and CSF-1 (Table 5). Thus the present data suggest that there are at least three (formula; see text) separate hemopoietic growth factors produced. The FDC-P1 activity produced by irradiated stroma would appear most likely to be GM-CSA-II. The fact that lectins enhanced production of these activities is intriguing but the cell type on which the lectins are acting has not as yet been defined. It appears that lithium also acts upon adherent marrow stromal cells to induce production of myeloid regulatory growth factors. Lithium appears to stimulate both normal and irradiated stroma to produce hemopoietic maintenance and growth factors and the present data suggests that factors active on pre IL-3 cells, HPP-CFC, CFU-D, CFU-meg, and CFU-S are all induced from these stromal cells by lithium. Whether the lithium induced factors and the factors derived from irradiated stroma represent a number of different growth factors or one or two critical regulatory molecules is at present unclear. The synergistic activity derived from the TC-1 cell line is of particular interest in this regard. This CSF-1 dependent activity is capable of acting on a very primitive stem cell to induce impressive proliferation and differentiation. In addition an activity in TC-1 conditioned media which may be synergistic activity appears capable of inducing adherent marrow cell lines which then can subsequently produce more of the same factor, a classic autocrine system.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the regulation of hemopoiesis.

Normal hemopoietic cell differentiation and proliferation is critically dependent upon marrow stromal elements. Long-term liquid culture of marrow provides a model for the study of stromal function. We evaluated the effects of radiation and 5-fluorouracil (5-FU) on various aspects of long-term murine hemopoietic cell growth and stromal function. Exposure of C57BL/6J murine adherent cells from long-term marrow cultures to varying doses of irradiation (0-1000 rad) in vitro resulted in the elaboration of growth factors stimulating granulocyte, macrophage, megakaryocyte, mixed megakaryocyte-granulocyte macrophage, and blast colonies. This increased production of growth factors appears to be related to the ablation of normal granulocyte production in the culture system since addition of normal stroma to irradiated stroma blocks growth factor production. Two cell types appear to be mediating stromal factor production and support of liquid culture hemopoiesis: a macrophagelike cell and an alkaline-phosphatase-positive epithelioid cell. Exposure of these two cell types to pokeweed mitogen results in marked enhancement of growth factor production. Furthermore, a cell line isolated from normal murine stroma produced an activity capable of acting at an early hemopoietic stem cell level and of inducing secondary marrow cell lines. The establishment of cultures from 5-FU-treated animals revealed that chemotherapy-depleted marrow was capable of establishing adequate stromal function and that the residual surviving stem cells had a higher than normal proliferative rate. In addition, the function of granulocytes derived from this post-5-FU marrow was normal. Thus, it appears that both chemotherapy and radiation exposure of marrow results in an enhanced capacity of stromal elements to produce growth factors and support hemopoiesis and that post-5-FU marrow represents an enriched source of high proliferative potential bone marrow stem cells.