Fractionated irradiation and haematopoiesis. III. Effect of time interval between fractions.

The effect of total single fractionated irradiation with short time interval on haematopoietic regeneration of the bone marrow and spleen was investigated. Also, the importance of first dose, when dose of 600 R was divided in two unequal fractions with time interval of 300s was studied. The investigation was performed on 25 day old rats. The dose of 600 R (X-rays) was divided on: 500 + 100, 400 + 200, 300 + 300, 200 + 400 or 100 + 500 R with time interval of 150, 300 or 600s. Ten days after irradiation the changes in blood, bone marrow and spleen were observed. After unequal fractionated dose with interval of 600s slight effect was found. The results after intervals of 600s and 300s were significant, when the total dose was divided in two equal doses. The first dose has no promoting role in haematopoietic regeneration when total dose was unequally fractionated.

Abscopal effects of local fractionated X-irradiation of face and jaw region.

The abscopal effects of high doses of X-irradiation, 6000 R given in 12 fractions locally on rat face and jaw region, upon the haematopoietic and lymphatic tissues, were studied. Haematological and pathophysiological studies were performed. Peripheral blood samples were taken after the 7th, 17th and 27th day during the course of the irradiation. 24 hours after the last irradiation the smears of femur bone-marrow were done. In the smears, the cells of erythrocyte and leukocyte series, as well as their mitotic index were determined. 24 hours after the irradiation the pieces of lymph nodes and salivary glands were taken for histological analysis. A significant depression of peripheral leukocyte counts was found in all observation intervals as well as the change in mitotic index of granulocyte series in bone marrow. No significant histological changes on the lymph nodes and salivary gland section were found 24 hours after the last irradiation.

Fractionated irradiation and haematopoiesis. II. The effect of bone-marrow cell transplants in conditions of split dose of irradiation.

Haemoregeneration after the irradiation with 600 R was studied using two different fractions given before and after the transplantation of bone-marrow cells. The dose of 600 R was divided in two uneven fractions: 500 + 100 R, 400 + 200 R and 300 + 300 R. During the free interval between the two doses (5 min) transplantation of bone-marrow cells was performed. Recolonization of bone-marrow and spleen was analysed on the 10th day after treatment. For analysis, samples of blood, bone-marrow and spleen were used. Maximal effect was found in the experimental group of animals irradiated with 500 R before and with 100 R after marrow-cell transplantation. Minimal haematopoietic response was in the group irradiated with 300 R before and after transplantation. This points at the importance of the primary dose for acceptance of the transplants and their activation.

Fractionated irradiation and haematopoiesis. I. The effect of a differently fractionated radiation dose of 600 R.

The effect of the same dose of irradiation (600 R) divided into two different fractions (500 + 100 R; 400 + 200 R; 300 + 300 R) upon the haematopoietic system of young rats (25 days old) was investigated. Free interval between the first and the second dose was 5 minutes. Changes were observed in bone-marrow, spleen and blood in all the experimental groups of 10 days after the irradiation. Spontaneous regeneration of haematopoiesis after a single dose of 600 R is higher than after division of this dose into two fractions, excluding the group where the dose was divided into two equal fractions. The most intensive haematopoietic repopulation was found in the group of rats irradiated with 300 + 300 R, where the primary dose was the smallest. In fractional conditions, the first dose is of primary importance for the repopulating capacity of the haematopoietic system regardless of the short free interval.

The effect of X-irradiation on phytohemagglutinin (PHA)-stimulated rat lymphocytes.

The effect of X-irradiation on PHA-stimulated rat lymphocytes has been investigated. The animals were exposed to doses of 100 to 800 R. Blood obtained by cardiac puncture was defibrinated and the lymphocytes sedimented by Dextraven. Lymphocytes were separated from supernatant and cultivated for three days in standard medium supplemented with rat serum and PHA. It was found that irradiation reduces the percentage of blast cells in function of the doses of irradiation. This reduction is accompained by an increased number of macrophage-like cells. The labelling index with tritiated thymidine in PHA-transformed lymphocytes was also reduced following irradiation.

Experimental and clinical investigations on stem cell take and colony formation.

First, lymphocyte transplantation into total body-irradiated rats is discussed. The effect on spleen colony formation caused by the transplantation of untreated lymphocytes, as well as of lymphocytes previously incubated with PHA, or with PHA plus L-asparaginase, or with lymphokines, was studied. Then the effect of the urinary colony-stimulating factor in vitro, and the in vitro feeder-layer activity of leucocytes on colony formation of human and mice bone marrow cells in haematological diseases is dealth with. The injection of rat lymphocytes previously incubated for 24 hours with PHA resulted in a higher number and a larger size of colonies in the spleen of the recipient rats. Lymphocytes preincubated with lymphokines gave rise to the formation of spleen colonies which were larger than those developing after the injection of untreated lymphocytes. When the lymphocytes were previously incubated with PHA plus L-asparaginase, PHA failed to stimulate colony formation in the spleen. The phenomenon is explained by assuming that PHA, as an aspecific stimulator of cell division, initiated the division of CFUs, thus the CFUs content of the preincubated samples increased, resulting in an increase in the number of colonies formed after the transplantation of lymphocytes pretreated with PHA. Another possible explanation is that CFUs division, or their spleen take is enhanced by the immunocompetent lymphocytes activated by PHA, either directly or via soluble mediators produced or released by immunocompetent lymphocytes such as lymphokines. The study of colony-forming cells and colony-stimulating activity in primary myelofibrosis (PM) showed an increase in the number of circulating CFUc in this condition, and an abnormal density of these cells reaching a peak below 1.062. The lowering of CSA in the first two peripheral blood gradient fractions agrees with the observation in the same fractions of a high percentage of CFUc at the expense of the CSC population. Thus, double cell population seems to exist in PM. One is greatly abnormal with a low specific density and high plating efficiency, whereas the other population is almost normal, showing a higher specific density and a lower plating efficiency.

Administration of peripheral-blood rat-lymphocytes stimulated "in vitro" with human lymphokines and spleen colonies in the total body irradiated rat.

It was previously reported that PHA-incubation of a pool of peripheral-blood lymphocytes before their i.v. injection into rats submitted to total body irradiation, stimulates spleen colony formation and growth. On the same model, the present study has investigated the effect of lymphocyte preincubation with supernates obtained in short term cultures from human lymphocytes prestimulated with PHA (lymphokines). The "in vitro" culture of rat lymphocytes in a medium supplemented with human lymphokines showed either a marked increase of lymphocyte survival rate, or significant stimulation of lymphocyte blastogenesis, but at an extent much lower than that caused by PHA, also because in this particular experiment the culture medium was supplemented with too little amount of lymphokines. These data support the interpretation that human lymphokines may act across the species barriers. Peripheral-blood rat lymphocytes prestimulated with human lymphokines, and then injected i.v. into rats previously submitted to total body irradiation, caused significant increase in the spleen weight, as well as formation of spleen colonies quite larger and with higher erythroid differentiation - though less numerous than in the controls (i.e., in the rats injected just with untreated lymphocytes). This behaviour may be due to the tendency to merge together of two or more colonies in a single giant colony, and supports the interpretation that immune-competent lymphocytes activated either with PHA or with lymphokines may stimulate spleen colony growth and erythroid differentiation, though at different extents.