The in vitro culture of human bone marrow and peripheral blood cells in the study of haemolymphopoiesis. A historical review.
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Biomedical subjects
Publications and source records attributed to G Astaldi.
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We report on the main topics discussed at the "Human T-Lymphocytes in Normal and Leukemia Patients" meeting, including the "Second International Workshop on T-Cell Colonies" (Reggio Calabria, September 16-19, 1984). Improved knowledge in the field of T-lymphocyte biology has contributed greatly to a better clinical definition of T-cell-derived lymphoproliferative disorders. The T-cell colony assay seems to represent a useful tool for studying the early steps of T-lymphocyte maturation and for characterizing accessory cells and soluble mediators that regulate T-cell proliferation.
T-lymphocyte colony formation in agar culture was investigated in 20 untreated B-cell Chronic Lymphocytic Leukemia (CLL) patients in stage O. As compared to the controls, colony growth was very poor when unseparated peripheral-blood lymphocytes from CLL-patients were seeded. The number of colonies was greatly higher when T-cell enriched fractions from CLL were plated; however, they failed to reach the normal range even in this experimental condition. The role of adherent cells in the colony growth was also investigated. Depletion of these cells resulted in impaired colony generation either in normals or in CLL-patients, which was subsequently restored by the addition of the same adherent cells. In the patients investigated in this study, an imbalance of T-subsets was found with increase of OKT8-positive cells (T-suppressors). When the number of colonies and the percentage of OKT8-positive cells were plotted, an inverse correlation was found. Conversely, a linear relationship was detected between the percentage of OKT4-positive cells (T-helpers) and the values of colonies. On the basis of the present experiments, it is suggested that the defective colony growth of T-cell fractions in B-cell CLL may be related to the low number of OKT4-positive cells plated, which are known to be mainly responsible for the colony generation in agar culture.
Peripheral blood from 4 patients with chronic granulocytic leukemia during blast crisis has been investigated. One case has been studied before and after treatment with Arabinosyl-Cytosine and 6-Thioguanine. The nucleated blood cells have been separated by a discontinuous density gradient. Cells were obtained from six different gradient fractions (F1-F6: density ranging from 1.052 to 1.078). 0.5 X 10(5) cells from each density fraction have been cultured in agar culture system to evaluate the granulocyte-monocyte committed stem cells (Colony Forming Units-granulocyte monocyte: CFU-GM). Cells recovered from the same density fractions have been studied by electron microscopy to evaluate the number of less differentiated cells. A quantitative correlation between plating efficiency and blast cells number was carried out. The results indicate that the highest recovery of both CFU-GM and blast cells is present in light density fractions (specific density below 1.063). However a discrepancy between blast cell frequency and granulocyte-monocyte colony formation in the same density fractions appears to be evident. In the patient studied before and after treatment it appears that only one out of two light density fractions (F1) responds to the antiblastic treatment.
Natural Killer (NK) activity is investigated in 20 patients with non-Hodgkin's Lymphoma (NHL) and in 20 healthy subjects. Peripheral blood lymphocytes are used as effector cells, and 51Cr labelled K562 myeloid cells as targets. Experiments are carried out either at effector/target ratio of 20:1 or 40:1, in 4 hrs Cr released assay. As compared to controls, a reduced NK activity is found in NHL patients, but that difference is statistically significant only for the patients in active disease. Thus, patients in clinical remission display a better preservation of NK function. It is suggested that the study of NK function might offer a helpful tool in following the clinical course and the efficacy of therapy in NHL.
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In recent years, a large body of information has been accumulated on the origin and differentiation of lymphocytes. According to the present state of knowledge, their ontogeny can be outlined as follows: the pluripotent stem cell originates the lymphatic progenitors, which undergo a series of differentiation events within the microenvironment of the primary lymphatic organs (bone marrow and thymus). Thus lymphatic precursors appear, which finally give rise to mature T- and B-lymphocytes. These events are accompanied by distinct changes in cell markers, mostly surface markers, which have been clearly documented. Some controversy does exist as to whether T and B lineages stem from a common lymphatic progenitor, since recent evidence suggests that B lymphocytes, but not the T, might share a common progenitor with myeloid lineages (hemocytoblast). T-cell maturation starts in the thymic cortex, and under the influence of the soluble thymic factor(s) it progresses in the several post-thymic locations, until final maturation. This process goes from immature (To) to semi-mature (T1) and mature (T2) lymphocytes, these latter mostly characterized by the absence of TdT and by the property to form E-rosettes with SRBC and respond to mitogens. Circulating mature T-lymphocytes display a considerable degree of heterogeneity in terms of functional properties; in this way, various T-subpopulations have been identified on the ground of recognized different properties. The sequence of maturational events of B-lineage takes place within the bursa-equivalent sites. This includes an antigen-independent stage with early B precursors (B0 - B1 - B2), and an antigen-driven maturational phase which actually produces the plasma cells. Among the early B-precursors (B-virgin population), memory cells arise following the antigenic stimulation. Also at that stage, B-cells are highly susceptible to tolerance induction. B-circulating lymphocytes are mainly characterized by S-Ig, Fc receptors, and complement receptors.
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The effect of in vitro irradiation with increasing in logarythmic progress X-ray doses on lymphocyte viability and on T and B lymphocyte populations was studied in normal adults, patients with myasthenia gravis and in patients undergoing long-term steroid therapy. Decrease in numbers of lymphocytes carrying T or B lymphocyte surface markers was higher than viable cell loss. The decrease showed no linear correlation with X-ray doses applied, which might reflect the existence of radioresistant T and B lymphocytes. A higher so called early radiosensitivity of B lymphocytes was demonstrated. In patients with myasthenia gravis early radioresistance of T lymphocytes was detected. In patients undergoing long-term steroid therapy, an increase in numbers of cells lacking markers of any of lymphocyte populations was found in parallel with a decrease in T lymphocyte number which, in these patients, showed a higher radiosensitivity.
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In 24 men aged 32 to 58 years with precancerous states of the larynx, i.e., leukoplakia, papillomas and pachydermia the peripheral blood lymphocytes were cytochemically stained for N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid phosphatase and glycogen; and the neutrophils were stained for alkaline phosphatase, myeloperoxidase and lipids. The results were expressed in terms of the absolute counts of reaction-positive cells and of the activity index score. The serum immunoglobulins IgG, IgA and IgM were also determined by Mancini's method. The results obtained were compared with those in 20 healthy men aged 20 to 30 years. The patients exhibited elevated numbers of N-acetyl-beta-glucosaminidase and beta-glucuronidase-positive lymphocytes. A characteristic feature was an increase in the absolute counts of lymphocytes with diffuse and granular-diffuse types of cytochemical reaction for all enzymes studied. The number of cells with the granular type of enzymatic reaction (intact enzyme-positive lysosomes) was significantly diminished. These cytochemical alterations were accompanied by a significant increase in the serum IgA level. These results are discussed with reference to the lymphoid system response to tissues of precancerous lesions of the larynx. So far as the neutrophils are concerned the patients exhibited significant intracellular deficiency of beta-glucuronidase and decrease in the lipid content as well as an elevated alkaline phosphatase activity. The possible significance of the beta-glucuronidase deficiency in neutrophils for the diminished cytotoxic response of these cells against the tumor and precancerous lesion cells is discussed.
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The Authors studied the effect of the human serum thymic factor on the peripheral-blood leukocytes from ALL patients, by testing the frequency of the E+cells by means of the e rosette assay before and after cell incubation with the above mentioned factor. In 3 out of 24 patients tested, the thymic factor incubation of peripheral-blood mononuclears increased the number of E+cells. The shift from R to E+cells caused by the human serum thymic factor occurred among null cells (E-, SM-Ig-). The possible value of the test in investigating whether a T0 leukemia might really exists is pointed out, as well as its prospective importance in monitoring the variations of the immature T cell counts in relationship to the patient's treatment and his disease course.
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In acute leukemia, the frequency of the E+ cells among the blood mononuclears before and after their incubation with human serum thymic factor was investigated by means of the E rosette assay. In 4 of 24 patients tested, the thymic factor incubation of their peripheral blood cells increased the number of the E+ cells among the mononuclears. Prospective importance of the test either in investigating whether a T0 leukemia might really exist, or in monitoring possible variations of the immature T cell counts in relationship to the patient's treatment and his disease course, are discussed.