Granulocyte macrophage colony-stimulating activity production by cultured human thymic nonlymphoid cells is regulated by endogenous interleukin-1.

Supernatants of cultured human thymic nonlymphoid cells were assayed for granulopoietic factors using cultures of low density bone marrow mononuclear cells (LD-BMMC). Thymic nonlymphoid cell-conditioned medium (TNLC-CM) supported vigorous myeloid colony growth of LD-BMMC, and of LD-BMMC depleted of T lymphocytes and/or monocytes. Colony stimulating activity (CSA) in TNLC-CM was abrogated by a highly specific neutralizing antiserum against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). TNLC-CM also enhanced colony growth in LD-BMMC stimulated by colony stimulating activity from a giant cell tumor culture (GCT). The enhancing activity of TNLC-CM, unlike its CSA activity, required the presence of adherent cells in the marrow cell culture. The addition of anti-interleukin-1 (anti-IL-1) antibody to TNLC-CM inhibited the GCT-enhancing activity, but not the CSA. When the anti-IL-1 immunoglobulin was added directly to cultures of thymic nonlymphoid cells, GM-CSF production was completely inhibited, and the GCT enhancing activity was neutralized. We conclude that an intercellular regulatory network exists in cultured thymic explants in which GM-CSF expression is induced by IL-1. In this system, the granulopoietic effect of IL-1 derives not from a direct effect on myeloid progenitors, but from its ability to recruit CSA production by other cells.

Regulation of the immune response by sex hormones. I. In vitro effects of estradiol and testosterone on pokeweed mitogen-induced human B cell differentiation.

Sex hormones have been implicated in the pathogenesis of many autoimmune disorders, presumably through regulatory influences on the immune system. However, the mechanisms of sex steroid action on humoral and cellular immune responses are not precisely understood. In this study, the in vitro effects of physiologic concentrations of 17 beta-estradiol and testosterone on the Ag non-specific differentiation of human PBMC were examined using optimal and sub-optimal doses, respectively, of PWM. In cultures of PBMC from 14 normal donors (7 men and 7 women, aged 25 to 45 yr), 17 beta-estradiol (0.5 to 30 ng/ml) enhanced PWM-induced generation of PFC by 46% (p less than 0.01), whereas testosterone (10 to 300 ng/ml) inhibited PFC generation by a mean of 36% (p less than 0.001). The enhancing and suppressing effects of the sex steroids on PBMC occurred early inasmuch as estradiol and testosterone had to be added to the cultures at their initiation (6 and 24 h, respectively) in order to observe their influence. Moreover, deletion of the hormones from the cultures after as short a period as 12 h did not obviate their effects. There was no alteration of the kinetics of the response to PWM or an effect on the number of spontaneous PFC generated in vitro in the absence of PWM. In addition, there was no difference among men and women in response to either sex steroid, and within the female group, no variation was observed on different days of the menstrual cycle. These studies demonstrate direct immunoregulatory effects of specific sex steroids on human PBMC and support the idea that these hormones may have a role in the pathogenesis and treatment of some autoimmune disorders.

Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines.

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony-stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM-CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor.

A culture system has been developed that promotes growth of clonogenic lymphoma cells of some patients with intermediate and high-grade malignant lymphoma. The formation of colonies in bone marrow, lymph nodes, and peripheral blood samples is best supported by human plasma. Colony formation of some patients was dependent upon growth factors, which in this study were added in the form of medium conditioned by phytohemagglutinin (PHA)-stimulated leukocytes (PHA-LCM). Some gave rise to lymphoma colonies without PHA-LCM but improved their frequency with PHA-LCM; others were completely independent of PHA-LCM. Colonies grown in primary cultures were routinely recloned and propagated as Epstein-Barr virus (EBV)-negative cell lines with stable B cell phenotype. The cell lines showed the same immunoglobulin rearrangement pattern as that observed in the primary lymphoma sample. In addition, a significant clinical correlation was observed between culture data and clinical outcome. Survival of patients who formed lymphoma colonies at any time during their clinical course was significantly shorter than survival of patients who did not give rise to colonies (P = 0.0009). The same observation was made when the survival assessment was performed for the subset of patients studied at diagnosis (P = 0.0014).

Synergism between recombinant growth factors, GM-CSF and G-CSF, acting on the blast cells of acute myeloblastic leukemia.

The genes for the hemopoietic growth factors, GM colony-stimulating factor (CSF) and G-CSF have been cloned, and recombinant material is available for both. We tested these recombinant factors for their effects on the blast cells of acute myeloblastic leukemia (AML). Culture methods are available that support both colony formation by AML blasts and the growth of blast stem cells in suspension. Recombinant GM-CSF is active in both culture systems, although to a varying degree. We found that recombinant G-CSF was also effective; however, the two recombinant factors showed striking synergism for the stimulation of blast growth of cells from five of eight AML patients. In these cases, the combination was equivalent to the stimulating activity of supernatants from the continuous cell line 5637. This conditioned medium (HTB9-CM) is considered the standard for blast growth. Blasts from one of the patients grew without added factor. In another instance, recombinant GM-CSF alone was almost as effective as HTB9-CM. In the third case, both recombinant factors were active, but synergism was not observed and their combined effect was not equivalent to that of HTB9-CM. Both GM-CSF and G-CSF were active on normal bone marrow granulopoietic progenitors, but synergism was not observed. We conclude that the marked heterogeneity observed when AML blasts are examined by other criteria is also observed when their response to growth factors is evaluated.

An improved selection procedure for the rescue of hybridomas. A comparative study of methotrexate versus aminopterin.

Several of aminopterin's drawbacks such as photosensitivity and high toxicity prompted us to compare the ability of methotrexate to select for hybridomas. Assessing the effect of both drugs on X63/Ag 8.653 myeloma cells and hybrid cells secreting monoclonal antibodies by tritiated thymidine incorporation and percentage of viable cells, we have shown that methotrexate could be used in place of aminopterin for the rescue of hybrid cells in selective medium. In addition, methotrexate hybrids develop more rapidly and can therefore be processed earlier. The stability and low toxicity of methotrexate also favor its use in the selection of hybridomas.

Functional comparison of bone marrow-derived macrophages obtained by cultivation in serum-free or serum-supplemented medium.

Bone marrow-derived macrophages obtained by cultivation in a serum-free or in a serum-supplemented medium were compared in terms of the activation of the respiratory burst and the activation of tumor cytotoxicity. Serum-free-cultured macrophages responded to interferon-gamma (IFN-gamma) and to lipopolysaccharide (LPS) by an enhancement of the respiratory burst. Macrophages obtained in a serum-supplemented medium are characterized by a diminished capacity to release O2-. These cells did not respond to IFN-gamma unless the stimulation was performed in a serum-containing medium. In terms of activation of tumor cell cytotoxicity, serum-supplemented macrophage cultures seem to be primed by unknown serum constituents because they only need one signal (IFN-gamma or LPS) to become fully cytotoxic. Serum-free cultivated macrophages can be rendered cytotoxic only after exposure to combinations of IFN-gamma and LPS.

Biological profile of a bioactive aortic substance (BAS), released by vessel rings from indomethacin-treated rats.

The biological activity of a stable unknown material(s), generated by aortic rings (bioactive aortic substance = BAS) isolated from rats injected with a high dose of indomethacin, was explored on contractions of several smooth muscle preparations from normal rats and its effects compared with those elicited by prostacyclin (PGI2) or by 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha). The BAS evoked, as did PGI2 or 6-k-PGF1 alpha, positive inotropism in strips from rat stomach, ileum and urinary bladder, but failed to influence uterine contractions as did prostacyclin or its non-enzymatic metabolite. When tested in rat aortic strips both, PGI2 and the BAS produced relaxation, whereas 6-k-PGF1 alpha was not active. Moreover, lipid substances present in the incubates of aortic rings, were extracted and explored for effects on contractions of rat aortic strips and on arachidonate-evoked human platelet aggregation. These extracts were devoid of influence on both parameters. On the contrary, dried aqueous residues, after the lipid extraction of the supernatants of aortic ring incubates, exhibited human platelet antiaggregatory capacity as well as the ability to evoke positive and negative inotropism similar to those triggered by the BAS in different smooth muscle preparations. Experiments with BAS were also performed employing smooth muscle strips exposed to indomethacin, atropine, propranolol, phentolamine and cyproheptadine. The presence of these antagonists of several neuromodulators and of indomethacin failed to alter de BAS-induced inotropic capacity observed in controls. The findings suggest that the effects attributable to the BAS are not subserved by prostacyclin or other prostanoids, nor by acetylcholine, norepinephrine, histamine or 6-OH-tryptamine.

Accumulation of DNA fragments in aging Paramecium tetraurelia in axenic and nonaxenic media.

The accumulation of DNA fragments in aging Paramecium tetraurelia after 15 days (or approximately 60 generations) of clonal growth in both axenic and nonaxenic media was assayed by alkaline elution assay. This sensitive technique permits measurement of single-strand breaks in double-stranded DNA. The results obtained indicate that P. tetraurelia aged in axenic medium accumulate more apurinic/apyrimidinic lesions, strand breaks, or single-strand gaps than do P. tetraurelia aged in nonaxenic medium. This implies that cells grown in axenic medium have a shorter lifespan than do cells grown in nonaxenic medium. The accumulation of DNA fragments may be the basic mechanism of aging in P. tetraurelia.

[First results of in vitro cultivation of stem cells from bone marrow and peripheral blood in an autologous test system of patients with hematological diseases]. / Erste Ergebnisse der In-vitro-Kultivierung von Stammzellen aus Knochenmark und peripherem Blut im autologen Testsystem bei Patienten mit hämatologischen Erkrankungen.

In 17 patients with haematological diseases an autologous test system was elaborated for determining the percentage of unipotent myeloic stem cells in the bone-marrow and peripheral blood and implemented by experiments. In comparing the results obtained by means of the traditional heterologous culture method, differences (diminutions, increase of aggregate numbers) could be found which allow certain conclusions to be drawn on the patient's real bone marrow function. For reasons of standardization, cultivation in the heterologous system cannot be abandoned. The investigations are continued.

The effect of conditioned medium on fetal human liver cells in primary culture.

A medium conditioned by rat embryo cultures (RCM) promoted the adhesion of liver cells from human fetuses to plastic dish. Colonies were also formed in primary cultures of the same cells in the presence of RCM. The majority of the colonies formed were composed of large polygonal cells with a few colonies composed of both clear epithelial-like cells and fibroblast-like cells. RCM was superior to the rat embryo feeder layer for promotion of colony formation of cells. A number of colonies were formed from fetal human livers when a conditioned medium from human hepatoma cells (HCM) was used, but most of the colonies formed were composed of fibroblast-like cells. The cells derived from the polygonal cell colonies, which were formed in the presence of RCM, have been passaged four times and they are still growing with albumin-producing capacity. The effect of RCM was reduced by various physico-chemical treatments.

[Establishment and characterization of the pig aortic endothelial cell line BSEz-3 in a serum-reduced medium]. / Etablierung und Charakterisierung der Schweineaorten-Endothelzellinie BSEz-3 in einem serumreduzierten Nähr-medium.

The cell line BSEz-3 was established in a serum-reduced medium MEMPAS. The cells were isolated and cultivated under the same conditions as the calf aortic endothelial cell line BKEz-7. With regard to cell isolation and cell density in the stationary phase, BSEz-3-cells show characteristic differences from BKEz-7-cells. For the isolation of BSEz-3-cells an enzymatic-mechanical method was more successful than a mechanical technique only. The low saturation density in the stationary phase with an average number of 50,000 cells/cm2 of glass surface area is considered as a strong contact inhibition of cell proliferation. As a cell type specific marker the BSEz-3-cells contain factor-VIII-antigen and possess angiotensin-converting enzyme activity. For a practical use of the BSEz-3-cells in drug research we give recommandations regarding cell inoculum density for subcultivation and cryo-conservation in ampoules.