The marrow colony forming cell and serum colony stimulating factor of the chicken.

Marrow cells of the chicken produced colonies in semisolid media. Developing colonies consisted of granulocytes, macrophages or a mixture of these two cell types. The granulocyte-macrophage CFC was nonadherent. An adherent 'CFC' was also present and it differed in several ways from the nonadherent CFC: (a) clones contained only macrophages, (b) they contained a core of nonrefractile cells, (c) their appearance was delayed 1-2 weeks, (d) they were unaffected by the presence of erythrocytes and (e) the efficiency of cloning was increased but the percentage of clones able to produce 50 or more cells was markedly decreased, i.e., the cluster/colony ratio was increased. The growth of both colony types was strictly dependent on the presence of CSF. Data obtained from dose-response studies on unfractionated marrow indicated that clusters and colonies were derived from single cells. The CSF of chicken serum yielded sigmoid dose-response curves when tested on marrow cells. Calf serum could not support cluster or colony formation when tested alone but it did have an enhancing effect on the CSF of chicken serum. Levels of serum CSF were increased by injecting chickens with bacterial endotoxin. This phenomenon occurred with five chicken lines tested, but certain chickens of the Kimber line did not respond to endotoxin with elevated levels of CSF.

Levels of colony-stimulating and inhibiting activities in chicks with myeloblastic leukemia are related to disease progression.

Chicks with myeloblastic leukemia induced by avian myeloblastosis virus (AMV) exhibited increased levels of plasma granulocyte/monocyte (GM) colony-stimulating activity (CSA). Chromatography of plasma from AMV-infected chicks revealed that this CSA eluted in the first protein peak from a column of Sephadex G-200. A second protein peak from the same column contained an inhibitor of GM colony-formation. The presence of the inhibitor and the increased CSA was studied during the development of myeloblastic leukemia. The disease course was separated into two distinct phases: 1) an early phase characterized by the onset of viremia, the induction of leukemic colony-forming cells (CFC) in the marrow, and moderately increased marrow cellularity, 2) a late phase characterized by marked increases in the early parameters, dramatic increases in the number of myeloblasts and decreases in the number of erythrocytic cells. Induction of the GM-CFC-inhibitor occurred during the initial stages of the first phase. Increased GM-CSA occurred later in this phase. We conclude that induction of the inhibitor and increased CSA were associated with disease progression rather than ancillary characteristics of frank disease.

Characterization of an inhibitor of granulocyte/monocyte colony formation in leukemic chicken plasma.

The plasma of chicks with myeloblastic leukemia (induced by avian myeloblastosis virus, AMV) contained an inhibitor which blocked colony formation in vitro by marrow cells. It eluted in the second protein peak obtained by Sephadex G-200 gel filtration and was found in the diafiltrate following defiltration through a UM 10 membrane under acidic conditions. It was not extractable with chloroform, was heat-stable (65 degrees C, 30 min), pronase-sensitive and had a molecular weight less than or equal to 5000 daltons as determined by high performance liquid chromatography. Thus, it appears to be a small, acid-soluble, heat-stable peptide. It had no interferon activity. It was not present or present only at very low levels in normal plasma. Furthermore, it was not elevated in chicks infected only with the nonleukemogenic helper virus of AMV, and was, therefore, associated with leukemia rather than virus-replication. Leukemic myeloblasts, purified by passage in suspension culture, released the inhibitor. It acted directly on the colony-forming cell and inhibited normal cells much more than leukemic cells. In normal marrow, macrophage and granulocyte progenitors were affected. A similar inhibitor in normal plasma inhibited only granulocyte progenitors.

Properties of colony-stimulating and inhibiting activities of chicken serum.

Serum obtained from young chicks stimulated with endotoxin contained elevated levels of colony-stimulating activity (CSA). This CSA was partially characterized. Incubation at 65 degrees C (60 min) had no effect on the ability of the CSA to support colony formation by nonadherent cells but reduced 3-4 fold its ability to support colony formation by unfractionated marrow cells. These data suggest that most of the serum activity is due to a heat-labile, adherent cell-dependent factor which does not directly affect the nonadherent granulocyte/monocyte colony-forming cell (GM-CFC). The ability of serum to support colony formation was increased by chloroform extraction. This was evidently due to the presence of inhibitory lipoproteins. The lipoprotein fraction of pigeon serum was found to directly block colony formation. The CSA for nonadherent cells migrated as one peak in the postalbumin region during zone electrophoresis. When electrofocused, heterogeneity was detected but all activity was associated with molecules with isoelectric points between pH 4 and 5. Following sequential fractionation by chloroform extraction, ultrafiltration, zone electrophoresis, isoelectric focusing and gel filtration (Ultrogel AcA 44), the activity was separated from most of the remaining protein. One peak of activity was obtained at the excluded volume.

Avian monocytic leukemia cells release fibroblast growth factor: implications to associated myelofibrosis.

The marrow of chicks with leukemia induced by avian 'myeloblastosis' virus (AMV) exhibited a 5-10-fold increase in the number of fibroblast colony-forming cells (CFU-F). The increased CFU-F correlated with a mild fibrosis which can be seen in the marrow of these animals. Fibroblast proliferation likely was not simply due to the presence of leukemic cells because addition of formaldehyde-fixed peripheral leukemic cells failed to initiate CFU-F growth. Conditioned medium (CM) from day-4 cultures of peripheral leukemic cells was markedly stimulatory to CFU-F growth. The stimulatory activity was not due to virus released from the leukemic cells as, (1) removal of virus by pelleting had no effect on the CM activity and (2) direct inoculation of CFU-F cultures with virus failed to stimulate CFU-F growth. Normal avian marrow macrophage monolayers also released high levels of a fibroblast growth factor and both the macrophage-derived and leukemic cell-derived factors were heat-labile (65 degrees, 30 min).

Serum-free conditions for the growth of avian granulocyte and monocyte clones and primary leukemic cells induced by AMV, and the apparent conversion of granulocytic progenitors into monocytic cells by a factor in chicken serum.

The supportive activity of chicken serum for the soft-agar growth of chicken granulocyte and monocyte clones could be replaced with defined ingredients [bovine serum albumin (BSA), conalbumin, selenium, linoleic acid and for routine work, a liquid soy lecithin preparation (59% phospholipids, 39% linoleic acid, and less than 2% of inositol and choline)]. The lecithin preparation could be replaced with L-alpha-phosphatidylcholine. The source of colony-stimulating factor was medium conditioned by fibroblasts cultured under protein-free conditions. AMV-induced leukemic cells could be cloned under identical conditions. In the presence of both chicken serum (10%) and the replacement ingredients, most of the proliferative clones produced were monocytic (84%). In the presence of serum alone, all of them were monocytic. Under serum-free conditions, all the clones produced were granulocytic when a day-3 conditioned medium (CM) was used; monocytic ones were also present when a day-6 CM was used. When the serum was serially diluted in the presence of the replacement ingredients, the number of proliferative monocytic clones progressively decreased while the number of proliferative granulocytic clones progressively increased and the kinetics of each were essentially the same, only opposite in direction. Moreover, the total number of proliferative clones did not change more than 33% at any dilution (or in the absence of serum). We postulate the existence in the chick system of a serum macrophage differentiation factor (M-DF) which converts early granulocytic progenitors (or exclusively diverts a common progenitor) into monocytic cells.

Differential regulation of spleen cell-mediated eosinophil and neutrophil-macrophage production.

Nonadherent spleen cells of mice infected with Trichinella spiralis released growth stimulatory factors (GSFs) in vitro when challenged with excretory/secretory products of muscle stage larvae. The assay of GSF was based on proliferation of normal, nonadherent syngeneic marrow cells in liquid tube cultures. Media conditioned for 1 day by challenged spleen cells stimulated eosinophil production but failed to stimulate production of other cell types. In contrast, media conditioned for 5 days supported eosinophil, neutrophil, and macrophage production. The kinetics of cell production were also different. Eosinophil production started within 1 day, reached a peak at day 2, and was down to control levels by day 4. In contrast, neutrophil/macrophage production began between 2 and 4 days and reached a peak at 6--8 days. The short duration of eosinophil production was evidently due to depletion of growth-factor-responsive cells.

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.

Antigen-mediated release of eosinophil growth stimulating factor from Trichinella spiralis sensitized spleen cells: a comparison of T. spiralis stage-specific antigen preparations.

When non-adherent, Trichinella spiralis-sensitized mouse spleen cells were challenged in vitro with T. spiralis antigens, an eosinophil growth factor (Eo-GSF) was released into the culture medium. This factor was assayed by its ability to initiate eosinophil production in liquid cultures of syngeneic, non-adherent marrow cells obtained from unsensitized mice. Extracts of each parasite stage as well as excretory-secretory (ES) products of adult and muscle larva stages were compared for their ability to stimulate spleen cells to release Eo-GSF. All stages and ES products had this ability but most of the preparations had unique dose-optima and there was a very wide range with regard to the optimum dose (in microgram protein/ml): (i) preadult stage, 1 x 10(-5); (ii) muscle stage ES products, 1 x 10(-3); (iii) muscle stage, 1 x 10(-2); (iv) adult stage, 1 x 10(-2); (v) adult ES products, 1 x 10(-1); and (vi) newborn stage, 1.0. When the Eo-GSF-containing conditioned media derived from spleen cell cultures exposed to the optimum dosages were tested on the same population of marrow cells, three potency groups were identified. The rank order of potency was: muscle stage ES products greater than preadult, newborn and adult stages greater than muscles stage and adult ES products. Preliminary experiments revealed that this ranking was not maintained with regard to the release of neutrophil and macrophage growth factors by these preparations.

Stimulation of eosinophil production in vitro by eosinophilopoietin and spleen-cell-derived eosinophil growth-stimulating factor.

Eosinophilopoietin (EPP) was previously characterized by the ability to stimulate eosinophil production in vivo, but these studies could not ascertain whether EPP had a direct effect on the bone marrow or acted indirectly by causing release of eosinophilopoietic activity by other tissues. The present studies demonstrate that EPP stimulates eosinophil growth in liquid culture of mouse bone marrow in vitro. The timing of stimulation by EPP in vivo and in vitro were parallel, with maximal eosinophil growth after 48 hr. Moreover, EPP appears similar to, and possible identical with, the eosinophil growth-stimulating substance (EO-GSF) released by antigenic stimulation of immune nonadherent spleen cells. Both EPP and EO-GSF are of low molecular weight, both produce stimulation of eosinophil growth with identical kinetics, and both produced similar dose-response curves in the liquid culture system.

Processing forms used in the bone marrow transplantation laboratory: toward standardization.

We developed a set of one-page data forms for each procedure that is used in the different types of bone marrow transplant processing performed in our laboratory. In this paper we duplicate this set of forms and illustrate how they can be "mixed and matched" to accommodate the type of processing that is being carried out on a particular day. Finally, we describe the benefits that we have gained from using this system and suggest interlab advantages to adopting such a system.