Efecto del tratamiento sobre lo crecimiento de colonias gránulo-monocitarias en la médula ósea de pacientes con policitemia vera / Effect of treatment on the growth of monocyte-granulocytic colonies in bone marrow of patients with polycythemia vera

Se cultivó en agar semisólido la médula ósea de 15 pacientes con policitemia vera (PV) con el propósito de conocer la acción del tratamiento sobre el crecimiento de las colonias gránulo-monocitarias. Tres de los enfermos no habían recibido ningún tipo de tratamiento previo al estudio, 6 habían tenido como única terapéutica flebotomías seriadas y 6 solamente P a la 32. Se observó que en los enfermos sin tratamiento previo, el número de colonias fue similar al control. En los pacientes tratados con flebotomías el número de colonias fue menor que el control, pero sin diferencia significativa. Los cultivos de médula ósea en los casos tratados con P a la 32 mostraron una reducción marcada en el número de colonias que fue significativa con respecto al control (p < 0,005). Ninguno de los enfermos presentaban leucocitosis, ni hiperplasia granulopoyética. Se consideró que la disminución de colonias en los pacientes tratados con flebotomías refleja un tránsito de los progenitores granulopoyéticos de médula ósea hacia la sangre periférica, por el estrés que se produce en la hemopoyesis como consecuencia de las flebotomías. En cuanto a los enfermos tratados con P a la 32, todo parece indicar que el radionúclido inhibe la población de progenitores granulopoyéticos, sin que se produzca una disminución en el compartimiento de las células precursoras

Modulation of human erythropoiesis by hydrocortisone in vitro.

The effect of hydrocortisone on the formation of erythroid colonies was studied in vitro in cultures of normal human bone marrow in an agar system. A range of concentrations of hydrocortisone (10(-10) to 10(-3) mol/l) showed significant effects on erythroid burst formation, in terms of the number of colonies, on d 10 and 14 of culture. At subphysiological concentrations (10(-10) to 10(-8) mol/l), no effect was seen, but at both physiological (10(-7) mol/l) and pharmacological (10(-6) and 10(-5) mol/l) concentrations stimulation of erythroid burst formation was noted. At 10(-4) mol/l hydrocortisone inhibited erythroid colony formation and 10(-3) mol/l was uniformly lethal. In the concentration range of 10(-7) to 10(-5) mol/l hydrocortisone also appeared to increase erythroid colony size. Thus hydrocortisone (10(-7) to 10(-5) mol/l) stimulates erythroid colony growth and it is suggested that the hormone may play a role in the physiological regulation of human erythropoiesis.

In-vitro granulopoiesis in adult acute lymphoblastic leukemia at various phases of the disease.

We used the in-vitro agar culture technique to monitor the granulopoiesis in 68 adult patients with ALL during the course of their disease. Bone marrow cells were cultured from 42 patients at diagnosis, 26 patients in relapse, 36 patients in early remission and 31 patients in full remission. The results of culture growth were characterized by sparse or no growth at diagnosis. No inhibition of normal CFU-C by leukemic cells was demonstrated by co-culture experiments. In relapsed marrows with blasts exceeding 60%, the culture results were identical to those at first presentation. The colonies grown in ALL cultures showed normal morphology with a normal granulocytic and monocytic differentiation. The colony-forming potential gradually increased following induction therapy, but there was no relationship between the CFU-C number and the percentage of blasts. The impaired granulopoiesis usually recovered once a remission was obtained and remained normal throughout the remission period. In some instances, cultures were performed within a short period prior to relapse or carried out more than one occasion during stable remission, wide fluctuations in CFU-C incidences were observed. Our study indicates that the CFU-C assay in ALL is useful for monitoring the in-vitro granulopoietic activity at various phases of the disease, but is of limited value in predicting the response to treatment as well as in determining the remission-relapse status.

In vivo study of lactoferrin and murine rebound myelopoiesis.

Lactoferrin has been reported to inhibit the production of colony-stimulating factor (CSF); thus we sought to study its possible effects on myelopoiesis in vivo. The characteristics of rebound myelopoiesis in C57BL mice injected with a sublethal dose of cyclophosphamide (CY) were used to test lactoferrin for any granulopoietic activity. An experimental group received daily injections of 50 micrograms of human lactoferrin beginning 24 hr after CY injections. By measuring the total nucleated cellularity of the femoral marrow, the peripheral blood count, and the incorporation of tritiated thymidine by the marrow in six replicate experiments, no statistically significant difference was noted between the lactoferrin injected groups and the control groups. Neither the route of lactoferrin administration (i.v. or i.p.) nor the sex of the animal altered the myelopoietic recovery. Lactoferrin had no stimulatory or inhibitory effect on murine rebound myelopoiesis in vivo contrary to the reported in vitro results.

Characterization of erythroid and granulocyte monocyte progenitors in human cord blood.

Some characteristics of both erythroid and granulocyte monocyte progenitors in human cord blood were compared to those in adult blood and bone marrow. The number of progenitors in cord blood was higher than that in adult blood and bone marrow. Most colonies in cord blood culture were monocyte-macrophage, whereas those from adult blood were largely eosinophilic. Cord blood progenitors had a slower sedimentation velocity than that reported for marrow, but sedimented faster than that for adult blood. A significant proportion of progenitors in cord blood as well as adult marrow was found to be in the DNA synthetic phase of the cell cycle whereas progenitors in adult blood were not. Cord blood BFU-E were more resistant than adult blood BFU-E but cord blood CFU-GM were not different from adult blood CFU-GM with regard to radiation sensitivity. Cord blood CFU-GM appeared to be more radio-resistant than adult marrow GFU-GM. From these results is seems clear that progenitors in cord blood differ in some aspects from those in adult blood and bone marrow.

Granulocytic colonies on macrophage-coated cellulose acetate membranes (CAM) in S1/S1dmice.

The kinetics of formation of granulocytic colonies on macrophage-coated cellulose acetate membranes (CAM) were investigated in Sl/Sld mice, which have a genetic defect in their hematopoietic microenvironment. CAM were left in the peritoneal cavity of mice for 7 days to become coated with peritoneal cells. The mice were then sublethally irradiated to suppress endogenously derived granulocytic colonies, and bone marrow cells were injected i.p. within 1--2 hr irradiation. During the next 7 days, peroxidase-positive granulocytic colonies appeared on CAM. The number of colonies on CAM in Sl/Sld mice at 7 days was consistently less than that from littermate +/+ mice. Since it appeared that the CAM in the Sl/Sld provided an inferior microenvironment to that of the +/+, the effect of raising a CAM in one genotype and transferring it to another was investigated. CAM raised in +/+ or in Sl/Sld were transferred to an irradiated +/+ or Sl/Sld following which cells were injected into the secondary host and colonies determined subsequently. The number of granulocytic colonies on CAM was influenced primarily by the type of secondary host. Colony number was consistently less in the secondary Sl/Sld host than in the +/+ host whether the primary host was a Sl/Sld or a +/+. These observations confirm the concept of a microenvironmental defect in Sl/Sld mice. In addition, the studies indicate that the microenvironment on a CAM can be modified by a secondary host and suggest that "remodeling" of CAM may be a continuous, kinetic process.

Human T cell leukemia-lymphoma cell lines and granulocyte-macrophage colony stimulating activity.

Thirteen human T cell leukemia-lymphoma cell lines were examined to determine whether or not they released a product into the media in which they were growing which could stimulate granulocyte-macrophage colony formation by human hemopoietic cells. None of the cell lines studied released colony stimulating activity.

Glucocorticoid therapy in the preleukemic syndrome (hemopoietic dysplasia): identification of responsive patients using in-vitro techniques.

Bone marrow cells from 54 patients with the preleukemic syndrome were cultured in agar (granulocyte colony forming units) in the presence and absence of cortisol. Twenty-four patients were given trials of prednisone therapy after the initial culture was performed. Cortisol (in vitro) failed to enhance colony growth in 29 of these 34 cases, and none of the 29 patients responded to prednisone therapy. Cortisol enhanced colony growth in five patients and three of these responded favorably to prednisone therapy. The correlation of in-vivo with in-vitro events is significant (P less than 0.005). Glucocorticoid therapy is of value in the management of a small number of patients with the preleukemic syndrome but is hazardous in those who fail to respond. These preliminary observations suggest that bone marrow cell culture techniques may aid in the identification of those patients who will and those who will not respond favorably to such therapy.

Effect of glucan on granulopoiesis and macrophage genesis in mice.

Glucan, a potent reticuloendothelial stimulant, is a glucopyranose polysaccharide derived from zymosan. Because of glucan's potential as an immunotherapeutic agent, we performed studies in order to determine its effect on granulopoiesis and macrophage production in mice. One week after the i.p. injection of 4 mg of glucan, there was a tenfold increase in colony-forming cells in the spleen and approximately a twofold increment of cells in the bone marrow and the peritoneal cavity capable of colony formation in vitro. There was a relative and absolute increase in the number of pure macrophage colonies from bone marrow and spleen. The total macrophage content in spleen, peritoneal cavity, and bone marrow as also increased in the treated mice. Serum from glucan-injected mice had high colony-stimulating activity levels, and the peritoneal macrophages elaborated increased colony-stimulating activity in vitro as compared to controls. Peripheral white blood cell counts were two times greater than those of control in the glucan-treated mice. These studies indicate that glucan administration results in increased granulocyte and macrophage production. The enhanced leukopoiesis is probably mediated in part by augmented release of colony-stimulating activity from macrophages. These observations suggest that the use of glucan as an immunotherapeutic agent can result in an increased number of available effector cells.

Inhibition of agar colony formation by partially purified granulocyte extracts (chalone).

Granulocytic extracts (GE) of different sources, presumably containing the granulocytic chalone, were prepared in different laboratories and purified to some extent. They specifically inhibited the formation of granulocyte and macrophage colonies in agar. The effect was however most pronounced on granulocyte and mixed granulocyte-macrophage colonies, and less on macrophage types. Addition of GE to bone marrow cells at the time of plating in agar, as well as short incubation of the cells together with GE prior to plating, inhibited subsequent colony formation. The inhibitory effect could easily be reversed by washing the cells with an excess of medium prior to plating during the first hour of preincubation, but not after five hours. Increasing the doses of colony stimulating activity (CSA) (at low doses of GE) released the inhibitory effect, but not at high doses of GE. The inhibitory effect of GE on colony formation was dose dependent down to almost 100% inhibition. No apparent cytotoxic effect of GE on bone marrow cells could be found and lymphoblastic cells were not inhibited. Extracts containing a specific inhibitor of erythropoiesis (EIF) stimulated myelopoietic colony formation in agar.