Quantitative analysis of the effect of colony-stimulating factors on human marrow progenitor growth in liquid-suspension cultures: application of limiting dilution assay.

Proliferation of human marrow progenitors in liquid cultures can be quantitated by limiting dilution clonal analysis (LDA) of progenitors in microwells. In this study, we have used LDA to study the effect of purified native or recombinant granulocyte colony-stimulating factors (G-CSFs) and recombinant granulocyte-macrophage CSF (GM-CSF) on progenitor growth. These results were compared to those of simultaneous cultures in methylcellulose. In LDA, single-hit kinetics were obtained with up to 500 U/ml of the recombinant preparation. In LDA with recombinant GM-CSF, progenitor growth conformed to single-hit kinetics from 100 to 2000 U/ml with maximum progenitor frequency at 500 U/ml. In simultaneous methylcellulose cultures with recombinant GM-CSF, colony formation reached a plateau at 100 U/ml. In LDA, purified native G-CSF was shown to support progenitor growth with single-hit kinetics at 100 U/ml, but at greater concentrations (greater than 150 U/ml), it suppressed progenitor growth with almost complete inhibition at a concentration of 200 U/ml. However, this dose-response effect was not observed in either simultaneous methylcellulose culture with G-CSF or in LDA with a purified recombinant preparation of the corresponding G-CSF. In methylcellulose cultures, colony formation reached a maximum at 100 U/ml and maintained a plateau up to 1000 U/ml. Hence, liquid culture allowed detection of contaminating suppressive activity in the G-CSF preparation that was not detected by methylcellulose assay. LDA may be more sensitive than methylcellulose culture for screening factors regulating human hematopoietic cell growth.

Tetramer Bence Jones protein in the immunoproliferative diseases. Angioimmunoblastic lymphadenopathy, primary amyloidosis, and multiple myeloma.

The authors report the clinical features and the results of immunochemical studies of patients with angioimmunoblastic lymphadenopathy, primary amyloidosis, and multiple myeloma, each of whom had in the serum and urine multiple forms of Bence Jones protein (BJP). The BJPs were isolated and purified and were shown by electrophoretic, gel filtration, and ultracentrifugal analyses to exist as tetramers and dimers. The components in two cases were kappa type and in one lambda type. The kappa tetramers consisted of two covalent dimers and the lambda tetramer two noncovalently bound dimers present in both serum and urine.

Suppression of hematopoiesis by activated T-cells in infectious mononucleosis associated with pancytopenia.

Two patients with primary Epstein-Barr virus (EBV) infection followed by pancytopenia were studied. They showed increased numbers of DR-positive, activated T-cells and serological evidence of persistent EBV infection over a 12 and 18 week period. Bone marrow granulocyte-macrophage colony formation (CFU-GM) was investigated by limiting dilution assay (LDA) and methylcellulose assay. CFU-GM of bone marrow mononuclear cells (BMMNC) was markedly suppressed in both patients during the pancytopenic phase. Removal of bone marrow T-cells by E-rosetting resulted in a significant increase of CFU-GM to normal levels in BMMNC of both patients, while no significant increase was observed in the BMMNC of normal subjects. CFU-GM in BMMNC from Patient 1 in the recovery phase was normal when DR-positive T-cells were within normal levels, irrespective of the presence or absence of T-cells. These results suggest that pancytopenia due to infectious mononucleosis in these patients was due to bone marrow suppression by activated T-cells. In vitro studies with Con A activated T-cells and their culture medium showed that suppression of CFU-GM by T-cells was mediated by a lymphokine.

Inhibition of fibrin monomer polymerization by Bence Jones protein in a patient with primary amyloidosis.

The mechanism of inhibition of fibrin monomer polymerization was studied in a patient with primary amyloidosis. Thrombin and reptilase times of the patient's purified fibrinogen (Fbg) were remarkably prolonged, and polymerization of the patient's fibrin monomer was disturbed. Fbg-Bence Jones protein (BJP) complex was demonstrated by immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the patient's purified Fbg. The patient's BJP not only prolonged the thrombin time of normal Fbg but also inhibited the polymerization of normal fibrin monomer. These results suggested that in this patient fibrin monomer polymerization was inhibited by binding of BJP to Fbg.

A cluster of human T-lymphotropic virus type-I carriers found in the southern district of Tokushima Prefecture.

Since we had experience of eight patients with adult T-cell leukemia lymphoma from 130 cases of non-Hodgkin's lymphoma between 1978 and 1986, a sero-epidemiological survey of anti human lymphotropic virus type-I (HTLV-I) antibody was performed using a gelatin particle agglutination test, an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay for 2,190 adults (768 men and 1,422 women) aged between 21 and 86 years, in the southern district of Tokushima Prefecture. The inconclusive data obtained using the above mentioned methods have been re-examined by western blotting analysis using enzyme-labelled anti IgM and anti IgG. There was an overall prevalence rate of 6.0% (132/2, 190) but higher rate were found in village C (10.9%) and village D (14.2%). These two villages together with town K (6.1% seropositive) form one community unit with a small population in a mountain area, which could be prone to producing a cluster of HTLV-I carriers. Town G with 8.0% sero-positivity is on the Pacific Ocean coast neighboring one of the endemic areas in the eastern section of Kohchi Prefecture. Interestingly, only IgM antibody was detected in 17 of 137 carriers (13%), all females who had never had a blood transfusion, suggesting them to h ave been in a sort of immunodeficient state, as reported in cases of HTLV-I infection. In 13 of 29 HTLV-I carriers (44.8%) from villages C and D, the viral antigen was detected in 1-9% (0.41 +/- 0.19%) of the peripheral blood mononuclear cells after being cultured with phytohemagglutinin for three days. The data indicate that those carriers had evidently been infected with the HTLV-I virus.

Cell-mediated suppression of human hematopoiesis: evaluation by limiting-dilution analysis of hematopoietic progenitors.

We have used limiting-dilution clonal analysis (LDA) in microwells to study the inhibitory effects of T lymphocytes (T-cells) or natural killer (NK) cells on human marrow progenitor cell growth. In four subjects with normal hematopoiesis, the growth of progenitors showed single-hit kinetics both before and after T-cell removal, indicating that, in the presence of colony-stimulating activity, T-cell have no effect on progenitor growth. In a patient with marrow hypoplasia associated with thymoma, hypogammaglobulinemia, and an increased number of suppressor T-cells (Good's syndrome), the progenitor growth deviated from linearity, demonstrating the presence of cells with suppressor activity. After T-cells were removed from this sample, the progenitor growth showed single-hit kinetics. The suppressive action of E-rosette-positive cells with NK or cytotoxic activities was also suggested in a patient with severe combined immune deficiency and in a patient with T gamma lymphocytosis. Poor progenitor-cell growth in three other patients with aplastic anemia was not significantly altered by T-cell removal. Thus, LDA of human hematopoietic progenitors is useful for evaluating cell-mediated interactions affecting hematopoiesis. This method may facilitate elucidation of mechanisms of myelosuppression in clinical settings.