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Biomedical subjects

N G Testa

Publications and source records attributed to N G Testa.

At least 91 records · Page 5Linked to original sources
PubMed1991

In vitro biological activities of the haemopoietic growth factors: implications for their clinical use.

The recent availability of several haemopoietic growth factors in purified recombinant form suitable for clinical use has prompted their use in haematology and oncology. The knowledge of their various effects in vitro on cell proliferation, differentiation and function and of their target cell populations will help in the design of treatment protocols with regard to selection of the best growth factor (or combination of them) for specific desired effects.

Cell Differentiation↗
PubMed1991

Recovery of the proliferative and functional integrity of mouse bone marrow in long-term cultures established after whole-body irradiation at different doses and dose rates.

Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro. However, no evidence was found for persistent functional defects in the stroma after irradiation, using the present endpoints.

Animals↗
PubMed1991

Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video.

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.

Animals↗
PubMed1991

The radiosensitivity of populations of murine hemopoietic colony-forming cells that respond to combinations of growth factors.

Combinations of murine recombinant interleukin 3 (IL-3), purified murine macrophage colony-stimulating factor (M-CSF), and human recombinant interleukin 1 alpha (IL-1 alpha) were used to determine the effects of growth factors on the measured radiosensitivity of different populations of murine colony-forming cells (CFC). The data showed that combinations of growth factors resulted in different values of CFC radiosensitivity, being less than values observed when colony growth was stimulated using a single factor. For various combinations of growth factors, Do values ranged from 106 +/- 8 to 175 +/- 24 cGy for progenitor cells in normal bone marrow; 74 +/- 3 to 171 +/- 18 cGy for primitive multipotent CFC enriched using fluorescence-activated cell sorting; and from 46 +/- 4 to 131 +/- 10 cGy for more mature granulocyte-macrophage CFC, enriched by counterflow centrifugal elutriation. Only combinations of three factors produced the high values of Do reported in experiments using unpurified conditioned medium as a stimulus for colony formation.

Animals↗
PubMed1990

The hematopoietic defect in aplastic anemia assessed by long-term marrow culture.

Thirty-two patients with aplastic anemia (AA) have been studied using the long-term bone marrow culture (LTBMC) system. Of these patients, 26 had been treated with immunosuppressive therapy including antilymphocyte globulin (ALG) with or without androgens or high-dose methyl prednisolone. The remaining six patients either required no treatment or were studied before therapy was begun. Thirty-one of 32 patients (96%) had defective hematopoiesis in LTBMC with little or no evidence for the generation of primitive progenitor cells. The only exception was a patient with spontaneous recovery of aplasia in whom the defect was less marked. Crossover LTBMC experiments were performed in 23 cases by inoculating (1) patient marrow hematopoietic cells that had been depleted of adherent cells onto preformed, irradiated, normal stromas to assess the proliferative capacity of the hematopoietic cells, and (2) normal marrow hematopoietic cells that were depleted of adherent cells onto preformed, irradiated stromas from patients with AA to assess stromal function. Results of these experiments demonstrated a hematopoietic defect in all patients that was independent of the degree of hematologic recovery after ALG therapy. Only one patient had a probable stromal defect and this coexisted with a defect in the regenerative capacity of hematopoietic cells. We conclude that LTBMC is a sensitive method for detecting and defining the hematopoietic failure in AA. We suggest that the defective hematopoiesis present in all patients studied may be important in the pathogenesis of clonal evolution in AA.

Adolescent↗
PubMed1990

Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture.

We have studied the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), hG macrophage-CSF (hGM-CSF), and gibbon interleukin-3 (gIL-3) on cell proliferation and differentiation in human long-term bone marrow culture (LTBMC). hG-CSF induced a maximal increase of 2.3-fold in both total nonadherent cells and GM cluster-forming cells, but only an increase of 1.7-fold in GM-colony-forming cell (GM-CFC) numbers, influencing mainly neutrophil differentiation. Cultures treated with hGM-CSF demonstrated a peak of 12.8-, 21- and 3.2-fold elevations in total nonadherent cells, cluster, and GM-CFC, respectively, and influenced differentiation of neutrophils, monocytes, eosinophils, and lymphocytes. Cultures treated with gIL-3 demonstrated the largest expansion in the GM-CFC population, reaching a maximum of 5.3-fold in relation to that of unstimulated controls. IL-3 treatment also increased the numbers of GM clusters and mature cells (including all myeloid cells and lymphocytes) 7.8- and 4.8-fold, respectively. Similar quantitative and qualitative changes were induced by G-CSF, GM-CSF, and IL-3 in LTBMCs of patients in remission after treatment for acute lymphoblastic leukemia or Hodgkin's lymphoma. Overall, the expansion of GM progenitor cells in cultures treated with growth factors was larger in the adherent cell layer than in the nonadherent cell fraction. In addition, hGM-CSF, gIL-3, and hG-CSF to a less extent, increased the cycling rates of GM-CFC progenitors located in the adherent layer. These results indicate that hG-CSF is a much less potent stimulus of hematopoiesis in LTBMC than the other CSFs assayed, and that the increases in cell production after treatment with G-CSF, GM-CSF, or IL-3 may be achieved by primary expansion of different cell populations within the hierarchy of the hematopoietic system. The effects of the growth factors were transient and the longevity of hematopoiesis in the cultures was not altered, suggesting that treatment with IL-3, GM-CSF, or G-CSF had not compromised the ability of primitive cells to give rise to mature cells. This indicates that the stromal microenvironment in LTBMC can override potential differentiation-inducing activities of the CSFs.

Animals↗
PubMed1990

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias.

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.

Bone Marrow↗
PubMed1990

The long-term effects of MVPP chemotherapy for Hodgkin's disease on bone marrow function.

Using in vitro techniques, bone marrow (BM) function has been studied in 25 patients in complete remission and at least one year after the completion of MVPP chemotherapy for Hodgkin's disease. The numbers of granulocyte/macrophage (GM-CFC) and fibroblastoid (CFU-F) progenitors were significantly lower than controls and there was no evidence of any improvement with time (median months off treatment was 30 for GM-CFC and 34 for CFU-F). In long-term BM culture production of haemopoietic cells were strikingly lower in the post-MVPP group and the development of adherent stromal cell populations was also significantly less. In addition, the yield of GM-CFC in adherent layers after four weeks of culture was significantly lower than in controls. We conclude that following MVPP chemotherapy and in apparently disease free and haematologically normal individuals there is evidence of impaired BM function up to nine years after the completion of treatment. These abnormalities may be relevant to the known increased risk of acute non-lymphocytic leukaemias in this group of patients and are likely to render the BM less able to withstand subsequent insults such as further chemotherapy or infection. The eventual development of BM failure is also a possibility and long-term follow-up of these patients is essential.

Adolescent↗
PubMed1990

Functional studies of bone marrow haemopoietic and stromal cells in the myelodysplastic syndrome (MDS).

Long-term bone marrow culture (LTBMC) was used to investigate the proliferative behaviour of marrow cells from a spectrum of cases of the myelodysplastic syndrome (MDS), and the results compared with those obtained in the conventional short-term clonal assay. Two broad patterns of growth were revealed in LTBMC. In one group the incidence of haemopoietic progenitor cells steadily declined to abnormally low levels at 4 weeks, while in a second group they were maintained near normal levels for periods of up to 7 weeks. These growth patterns, which were not predictable from clonogenic assays on the marrow cells prior to LTBMC, or from the morphology of the bone marrow, may reflect the stage of evolution of the disease. Further studies of clonality are required to establish whether or not patients exhibiting the second pattern have a potentiality to harbour residual normal haemopoiesis. LTBMC was also used to study the function of MDS marrow stroma in terms of its ability to sustain the growth of normal haemopoietic progenitor cells. Although the phenotype of the cultured adherent cell layer, obtained from some patients, was atypical, no consistent functional defect of MDS stroma could be identified by studying the level of haemopoiesis reached by normal cells seeded into MDS stroma.

Adolescent↗
PubMed1990

Long-term bone marrow damage after chemotherapy for acute myeloid leukaemia does not improve with time.

Thirteen patients with acute myeloid leukaemia (AML) who had been in unmaintained complete remission for at least 5 years following successful chemotherapy, were studied by marrow culture techniques regularly for periods of up to 5 years. In 10 patients, short-term clonal assay revealed the incidence of granulocyte-macrophage progenitor cells (GM-CFC) to be at the lower limit of the normal range; but three showed wide fluctuations in GM-CFC numbers. Moreover the generation of GM-CFC in long-term bone marrow culture (LTBMC) was impaired in eight of nine evaluable patients. These results suggest that, despite normal blood counts, impairment of haemopoiesis persists in patients who are long-term survivors of AML, and this does not improve with the passage of time.

Adult↗
PubMed1990

Cell lineages in haemopoiesis: comments on their regulation.

The regulation of cell production and the determination of cell lineage in haemopoiesis are the result of multiple processes involving cell-cell interactions and the action of specific haemopoietic growth factors as well as other cytokines. Additive and synergistic interactions between these factors, and also with those involved in the regulation of lymphopoiesis have been described, which involve modulation of the growth and differentiation of haematopoietic precursors. However, the mechanisms which determine the self-renewal and the differentiation of the pluripotent lymphohaemopoietic stem cell remain largely undefined.

Animals↗
PubMed1990

Clinical application of hematopoietic growth factors.

Although many questions remain about the clinical use of hemopoietic growth factors, it is remarkable how much information has been gained in such a short time. There is enormous interest in these agents among physicians from a variety of specialties--all providing new ideas for potential studies. As the supplies increase, many new trials can be opened. It is important to design these studies with due regard to the results of in vitro and in vivo preclinical experiments--preferably after discussion with experimental hematologists--or vital information will be missed. There can have been few more exciting developments in clinical medicine in the recent past than the availability of the hemopoietic growth factors and, hopefully, it will not be long before the preliminary trials are completed and these agents are accepted for routine use to the benefit of a large number of patients.

Animals↗
PubMed1990

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors.

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.

Animals↗
PubMed1990

The use of cultured bone marrow cells in autologous transplantation.

The feasibility of ex vivo purging with long-term bone marrow cultures (LTBMC) for autologous transplantation in leukemia has been established. The procedure has been applied to patients with acute myeloid leukemia (in relapse or remission) and recently in one patient with chronic myeloid leukemia. The results in first remission AML are very encouraging with 4 out of 6 patients well and apparently disease free greater than 1 to 4 years post autograft. In patients transplanted with active disease, remissions of 6 to 8 months duration were seen in two patients transplanted in florid relapse. In a CML patient with 87% of his bone marrow cells Ph1 positive, there was a marked decline of karyotypically abnormal cells in culture. Reinfusion of the cultured cells into the patient resulted in engraftment with exclusively Ph1 negative cells. The therapeutic implications of LTBMC purging require further evaluation.

Bone Marrow Cells↗
PubMed1989

Residual haemopoietic damage in the mouse after fractionated gamma-irradiation, down to 0.1 Gy per fraction.

Residual damage in haemopoietic progenitor cell populations, spleen and granulocyte-macrophage colony-forming cells (CFU-S and GM-CFC) was detected in mice after 15 daily fractions where the dose per fraction was as low as 0.1 Gy. The injury was dose-dependent and after higher total fractionated doses of 7.5-10 Gy the CFU-S population recovered to about 50% of control between 2 and 12 months after irradiation. Residual damage was also detected in the stroma, in the form of reduced numbers of fibroblastoid colony-forming cells and of CFU-S in ossicles under the kidney capsule. The response to a second course of 15 fractions, given 3 weeks after the end of the first course, was similar and additive to the response to the first course in the short term. However, in the long term, recovery levels were similar after either one or two courses.

Animals↗