This study reports results from the first clinical tests in which 7 patients with myeloid leukaemia (5 acute and 2 chronic leukaemias in metamorphosis) were treated from 4 to 45 days with granulocyte chalone, the tissue-specific endogenous inhibitor of granulopoiesis. It was observed that i.v. injection of partially purified chalone inhibits proliferation of leukaemic, and presumably also normal granulocytic cells, leaving all other cell types unaffected. Inhibition of leukaemic growth was distinct in 6 of the 7 patients; in 5 cases the inhibition was followed by acutal regression of the leukaemia, lasting up to several months in the absence of any maintenance therapy, and in one case the treatment led to a complete remission. Chalone treatment also resulted in an enhancement of erythropoiesis and megakaryopoiesis, and in phenomena some of which were totally unexpected, such as immunostimulation and a remarkable resistance to bacterial infections in the presence of extreme granulocytopenia. This study shows that granulocyte chalone is biologically active against myeloid leukaemia in man, but not that the therapeutic value of the impure chalone is superior to modern cytostatic drugs. Long-term therapeutic trials were not possible with the partially purified preparations available, mainly because of side-effects which prevented adequate dosing.

We tested the effects of interleukin-2 (IL-2), human recombinant tumor necrosis factor alpha (TNF-α), Staphylococcus aureus Cowan I (SAC), TPA, and their combinations, using a standard thymidine incorporation assay, in order to identify an optimal mitogen combination (OMC) for 24 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL). The combination that induced the highest thymidine incorporation was chosen as the OMC for each patient. Among 14 mitogen combinations tested, there were six different OMCs, of which the most frequent was TNF-α + IL-2. It was the OMC in 9 of 24 cases. The other OMCs were TNF-α + TPA1 (5/24), SAC + IL-2 (5/24), TPA1 + IL-2 (3/24), TPA10 + IL-2 (1/24), and TNF-α + TPA10 + IL-1 (1/24). The mitogenic power of the selected OMC in each case was then evaluated both by the combination of immunophenotyping and molecular cytogenetic techniques known as MAC (Morphology, Antibody, Chromosomes) and standard chromosome analysis. After OMC stimulation, the levels of DNA synthesis and B-cell proliferation (mitotic index) were, on average, 10-fold higher than those observed after standard TPA stimulation (P < 0.0001). The proportion of mitotic B cells exceeded the proportion of mitotic T cells in 70.1% of the cases after OMC stimulation. After TPA stimulation, 7.7% ± 2.5% of all mitoses were B-cell mitoses, whereas after OMC stimulation this proportion rose to 57.9% ± 5.3%. The frequency of clonal chromosomal aberrations increased from 46% after TPA stimulation to 79% after OMC stimulation. The clonal aberrations del(6q), del(11q), and/or del(13q) were observed in 26%, 32%, and 42% of the patients with the respective clonal chromosomal aberrations, whereas the corresponding frequencies after TPA stimulation were only 4%, 21%, and 17%. When the lineage involvement of cells with clonal chromosomal aberrations from three patients was analyzed, the aberrations were found to be restricted to B cells only, and in one patient to a minor subset of B cells. The results demonstrate that an individually chosen OMC induces a high rate of proliferation in neoplastic B cells. We found deletions in 6q, 11q, and 13q at higher frequencies than reported previously, most probably as a result of an improved mitogenic response. The identification of an optimal mitogen stimulation for each patient, prior to chromosome analysis, can well be expected to reduce the rate of false-normal results in the future. This is essential for accurate evaluation of the prognostic significance of chromosomal aberrations in B-CLL.

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.

Part of young mouse radii were isolated and transplanted into an intraperitoneal Millipore diffusion chamber. The survival of the implants was followed for 22 days, using morphological criteria, and by autoradiography with tritiated thymidine. Periosteal cells and epiphyseal plate chondrocytes survived well for one to two weeks. DNA synthesis was noted up to the twenty-second day of culture. Osteogenetic cells proliferated well in transplants of shorter duration, but in the third week there was overgrowth by fibroblasts. The method seems well suited for the culture of osteogenetic cells as whole-bone pieces for at least one to two weeks.

The effect of increased bone mass on osteogenesis was studied using the diffusion chamber culture of mouse distal radius as an experimental model. Two distal thirds of a mouse's radii were cultured in separate diffusion chambers in a recipient mouse; one chamber contained only a distal radius, but the other contained a distal radius plus two proximal radii and two ulnae. The DNA synthesis of osteogenetic cells was significantly inhibited in cultures with excess bone mass. The mean inhibition in labelling indices of epiphyseal plate chondrocytes was 27% and of periosteal cells 40%. The nature of this inhibition seems to be specific.

Non-pyrogenic preparations of granulocytic chalone have been administered to patient with acute myeloid leukaemia (A.M.L.) who had relapsed after chalone-induced complete remission of the disease. Another regression of the leukaemia was obtained. Normal and leukaemic granulocytes were inhibited without effect on other cell types. Survival was prolonged in 4 other patients with A.M.L. Further investigations are needed to elucidate the therapeutic role of granulocytic chalone.

Growth of haematopoietic cells from fetal mouse liver of 14- to 16- day gestation was studied with the diffusion chamber technique. The culture period varied from 1 to 8 days. Cell proliferation and haemoglobin synthesis were seen in erythroblasts during the first days of culture. However, the nearly pure erythroblastic population of the primary inoculum changed to vigorously growing granulocytic cells and macrophages during the 8-day assay period. It seems likely that end-product inhibition is operative within the monocyte-macrophage and granulocyte cell lineages, respectively, when cells grow in diffusion chambers in vivo.

This study showed that growth of granulocytes, both normal and leukaemic, is inhibited in a specific manner in diffusion chamber cultures carried by chloroma-bearing host rats (→ elevated granulocyte chalone content in the body). This inhibition could be demonstrated directly by a reduced formation of granulocytes and by measuring the 3H-thymidine uptake in the cells. Non-granulocytic cell lines (mastocytoma and HeLa cells) were not detectably inhibited when grown in identical conditions. The inhibition of granulocytic cells did not depend on a selective immunological reaction developed against granulocytic cells in the chloroma-bearing host rats.

Morphological and cytochemical characteristics of cells obtained from Shay chloroleukaemia tumour growing subcutaneously in the rat and after 0.5–16 d of diffusion chamber culturing were assayed using seven different cytochemical procedures. Further, the cytochemical characteristics of the recent subline of Shay chloroma were compared with those reported in 1962 by other workers.The chloroleukaemia cells did undergo some maturation in diffusion chamber cultures, but full differentiation to polymorphonuclear cells was not seen. The most striking change in the cytochemical pattern of chloroleukaemia cells during the diffusion chamber culturing was a significant increase in alkaline phosphatase activity. There were some cytological differences in the recent chloroma tumour compared with that described 10 years ago.The observations made in the present work show that all properties of cells after a neoplastic transformation are not irreversible: many characteristics of malignant chloroleukaemia cells can alter when these cells are maintained in different host animals or in the diffusion chamber milieu.

ABSTRACT The proliferation kinetics of Shay chloroleukaemia cells was studied by the labelled mitoses technique and some other methods. Detailed estimates of the phase durations of the cell cycle for proliferating cells were obtained using young, exponentially growing diffusion chamber cultures; estimates of kinetic parameters were also obtained for ‘old’ cultures and for local subcutaneous chloroma tumours. The results enabled determination of the growth fraction and the cell loss factor. Cell loss was found to be the dominant factor determining the growth rate of chloroleukaemia cell populations. The mechanism of cell loss was cell death. Some implications of these findings are discussed.

Tissue eosinophils were systematically determined from 36 organs or tissues of three cows and three pigs. The number of eosinophils ranged from 0 to 1,500 per mm2. Their densities were highest in the intestinal region, especially the small intestine. Moderate numbers (50–200 per mm2) were noted in the stomach, spleen, lymph nodes, thymus, dermis, and conjunctiva. No eosinophils were seen in 12 tissues. The quantitative presence of tissue eosinophils in the different organs suggests that they are part of the local, cellular defence mechanism of the organism.