Relation between cell cycle stage and the activity of DNA-synthesizing enzymes in cultured human lymphoblasts: investigations on cell fractions enriched according to cell cycle stages by way of centrifugal elutriation.

Undifferentiated human lymphoblasts (culture LS-2) were separated according to cell size during their exponential growth phase by way of centrifugal elutriation. The cell fractions thus obtained were characterized in terms of different cell cycle stages by flow cytometric measurement of their deoxyribonucleic acid (DNA histogram), the [3H]thymidine labeling index, and by determining the rate of [3H]thymidine incorporation. In these cell fractions the activities of thymidine kinase, thymidylate synthase, DNA polymerase, dihydrofolate reductase, methionine synthase, and hexokinase were determined. The results showed that all the enzymes investigated exhibited activities in all cell fractions. With the exception of DNA polymerase, all of the enzymes exhibited the lowest level of activity in the fraction containing the highest proportion of G0 + G1 phase cells (fraction 2); the activity of thymidine kinase was particularly low. This would suggest that thymidine kinase is not active in G0 + G1 phase cells and that the activity measured in fraction 2 is perhaps attributable to contamination of this fraction by S and G2 + M phase cells.

Kinetics and cellularity of erythroblasts in the growing rat.

Parameters of recognizable erythroid cell proliferation were measured in four groups of normal rats weighing 50, 100, 150 and 300 g in order to provide a comprehensive set of data suitable for a re-investigation of erythropoietic models. Total erythroblast cellularity was measured by the 59Fe technique, DNA synthesis time by quantitative 14C-autoradiography, and the erythrocyte production rate was derived from the increase with time of the erythrocyte labelling index after repeated injections of 3H-leucine. Furthermore, the relative erythroblast density was determined in the various morphological compartments. From the total erythroid cell mass in DNA synthesis and the absolute erythrocyte production rate, figures were derived for the mean DNA synthesis time of erythroid cells and compared with the directly measured ones. The discrepancies in all weight groups between direct and indirect determination of DNA synthesis time were considerable. In a previous study re-evaluation of comparable data in literature had revealed comparable inconsistencies. Since a critical discussion of possible errors in the experimental techniques does not indicate data acquisition to be the principal source of disagreement it is concluded that the type of model applied to describe how cells pass the boundaries of morphological cell compartments is of high significance. Models based on a sequential flux of cells through the individual compartments are inadequate for evaluation of the presented set of data.

Characterization of a phosphoglycerate kinase deficiency variants not associated with hemolytic anemia.

The properties of a variant phosphoglycerate kinase (PGK) found in a large German clan were examined. The normal and variant enzymes, isolated by affinity chromatography, have the same molecular weight, specific activity, substrate affinity, and nearly identical pH-optima. Using immunoinactivation and immunodiffusion, the same specific activity for both forms was again determined. Since the enzymatic activity in older and younger erythrocytes varied only slightly, and since the specific activity of the variant was normal, the variant seems to be stable in vivo. This suggests that the decreased enzyme content is due to a decreased synthesis rate. The variant PGK described here is distinctly different from the known PGK variants and has been designated as "PGK München."

In vitro stimulation with mitogen and antigen in patients with monoclonal gammopathy.

Peripheral blood lymphocytes from 32 patients with defined paraproteinaemia (16 IgG, 9 IgA and 7 IgM) and from 15 healthy donors were studied for their in vitro response to various stimuli, including for unspecific mitogens such as Phytohaemagglutinin (PHA), Pokeweed mitogen (PWM) and Concanavalin A (ConA) as well as specific antigens such as purified Tuberculin, Candida, Varidase, Tetanus Toxoid, Vaccinia antigen and Vaccinia-control antigen. Mitogens and antigens were lyophilized in Microtiter plates. The lymphocytes of all tested patient-groups responded (measured by H3-Thymidin-uptake) significantly lower towards the unspecific mitogens than those of the control group. If the patients' lymphocytes were stimulated by the specific antigens, their in vitro response was significantly diminished to candida and vaccinia. Macroglobulinaemia showed significantly lower response to ConA if compared to myelomas of IgG- and IgA-type. No correlation was found between mitogen and antigen response and the serum concentration of the paraproteins or immunoglobulins. The results show that monoclonal gammopathy and especially macroglobulinaemia are associated with abnormalities of the cellular immunity which correlates with the clinical observation of increased fungal and viral infections.

Myeloid membrane markers. I. Expression of marker antigens specific for murine granulocytes and their precursor cells.

Bone marrow cells were investigated by immunoelectromicroscopy and by quantitative photometric immunoradioautography with a rabbit antiserum against murine bone marrow cells. The serum was absorbed with murine spleen, liver and thymus cells until it no longer reacted with thymocytes and lymph node cells in a quantitative complement fixation test. The antiserum stained granulopoietic but not erythropoietic or lymphopoietic cells. The density of the myeloid antigen on single cells increased with the differentiation from immature to mature granulopoietic cells. While the increase of label was statistically not significant at the level of differentiation from promyelocyte to myelocyte and metamyelocyte to band neutrophil, there was a remarkable gain of label from myeloblast to promyelocyte and from band neutrophil to segmented neutrophil. This was evident under the electron microscopy using peroxidase-labeled antibodies and could be measured quantitatively with photometric immunoradioautography using 125I-labeled antibodies.

Population data on two new HLA-D determinants, EI and RE.

Homozygous typing cells (HTC) for two new HLA-D determinants, EI and RE, defined by family studies, are described. The HLA-D typing experiments among more than 300 unrelated individuals showed phenotype frequencies of 0.045 for EI and 0.098 for RE. Since the tested population was also typed for its HLA-A and -B alleles, linkage disequilibrium parameters could be calculated: HLA-D type EI was statistically significantly associated with HLA-B13 and Bw17, HLA-D type RE with HLA-Bw40. These data support the working hypothesis that both EI and RE are new alleles of the HLA-D series.

[Case report on the problem of individual glycoside requirements].

A report is given on a patient with ischaemic heart disease, whose recompensation in tachyarrhythmia absoluta was for some times possible only by means of unusually high doses of digitoxin (fully effective dose to 5.72 mg, maintenance dose to 0.4 mg). The patient survived a severe decompensation with pulmonary oedema, which appeared under "normal" applications of glycosides, 2 1/2 years under this therapy. Thus it must again be referred to an individual glycoside treatment.

[Electronmicroscopic and immunohistochemical studies on human lymphocytes]. / Elektronenmikroskopische und immunhistochemische Untersuchungen an menschlichen Lymphozyten

Lymphocytes from the blood of healthy individuals and of patients suffering from CLL were investigated by electron microscopy and peroxidase-immunohistochemistry. B-lymphocytes were labelled by heterologous, peroxidase-conjugated antisera directed against the Id-determinants of their membranes. T-lymphocytes were labelled by an indirect method: specific incubation with a specific anti-T-cell-globulin from the rabbit; labelling-incubation with a peroxidase-conjugated anti-rabbit-IgG-globulin from the sheep. In addition, T-lymphocytes were identified by their ability to form rosettes with sheep erythrocytes spontaneously. The quantitative results were: about 80% T-lymphocytes and about 24% B-lymphocytes in normal persons, the opposite results in CLL. T- and B-lymphocytes were photographed electron microscopically; the number of organelles in the single cells was evaluated: lysosomes in the average are more numerous in T-lymphocytes, ergastoplasm in B-lymphocytes, mitochondria are equally distributed in both groups of cells. There is so much overlapping, however, that the single cell only with the aid of immunochemistry or rosette formation can be identified as a B- or T-cell. In both, the T- and the B-cell-series, different forms of lymphocytes can be distinguished according to the degree of cell differentiation. Some further problems, as specificity of the antisera and labelling of the cells by means of their Fc-receptor are discussed.

Quantitative immunoautoradiography at the cellular level. II. Absolute measurements using labeled standard cells as a source of reference.

A quantitative autoradiographic method is presented for determining absolute amounts of 125I-labeled compounds on the surface of individual cells. Autoradiographic evaluation of single cell radioactivity is accomplished by comparing the silver grain densities over the specimen and a radioactive standard being exposed simultaneously. In order to obtain a reference source of comparable physical properties, surface-radioiodinated human erythrocytes are used, the radioactivity of which is determined in a crystal counter. A simple enzymatic method for preparing such standard erythrocytes of very uniform label density is described. Numerous experimental advantages derived from the use of the standard are discussed and demonstrated by examples employing various exposure times and different radioactive standards. Hereby, very similar results were obtained when the number of A-antigenic sites was quantified on single erythrocytes in different experiments. The quantification of membrane-bound IgM on single human lymphocytes is shown as another application of this scheme.

Bone marrow transplantation in children with aplastic anemia and acute lymphatic leukemia.

A boy with severe Aplastic Anemia (AA) and a girl with Acute Lymphoblastic Leukemia (ALL) in relapse have been grafted with marrow from HL-A identical, mixed leukocyte culture (MLC) negative siblings after appropriate immunosuppressive and antileukemic therapy. Both of them are well 7 and 2 months after transplantation respectively. Bone marrow transplantation should be considered in children with AA and ALL in relapse, if HL-A identical, MLC negative siblings are available.