W Calvo

THE DEVELOPMENT OF RADIATION LATE EFFECTS TO THE BONE-MARROW AFTER SINGLE AND CHRONIC EXPOSURE

The marrow is a tissue distributed in numerous skeletal parts and works as an organ which is composed of a haemopoietic cell parenchyma and a supporting stroma. The pathophysiological mechanisms involved in the radiation-induced late effects depend mainly on the damage produced to each of these elements. Parenchymal cell damage ends with a failure of the stem cell pool to supply an adequate number of highly differentiated functional blood cells and is clinically manifested as aplastic anaemia or leukaemia. The effects of radiation on the haemopoietic stem cell can be measured by means of spleen colony forming units (CFU-S) in rodents. The self-maintaining capacity of the CFU-S was found to be lower than normal 16 weeks after a dose of 0.64 Gy. In larger animals it is only possible to measure the activity of some of the progenitor cells, estimating the number of granulocyte-macrophage colonies in culture (CFU-GM) as an indicator of stem cell changes. Their number in the blood is about 50 per cent of normal even 160 days after about 0.78 Gy. The stromal cells are also radiosensitive if measured with respect to their capacity to support long-term cell replication in vitro. Marrow fibrosis develops after single, repeated and chronic radiation exposure, and a dose of 40 Gy impairs the capacity of the marrow to support haemopoiesis.

Cryopreservation of blood mononuclear leukocytes and stem cells suspended in a large fluid volume - A preclinical model for a blood stem cell bank

SummaryIt was the purpose of this study to establish and evaluate a freezing and-thawing method for preservation of hemopoietic stem cells from the peripheral blood. Blood leukocytes collected by means of an IBM Blood-Cell-Separator were frozen in plastic bags using 10% DMSO and controlled cooling rates. Thawing was performed rapidly, and DMSO was diluted and removed prior to the in-vitro and in-vivo assays.The mean recovery of mononuclear cells collected from 82 leukaphereses was 86%. To assess the recovery of cryopreserved hemopoietic stem cells, the soft agar culture method adapted for the dog was used. There was no significant difference in the CFUc recovery per 1 × 106 mononuclear cells or in per leukapheresis after different Cryopreservation times (1–6 and 7–27 months).To evaluate the hemopoietic repopulation capability of cryopreserved blood stem cells, leukapheresis-derived leukocytes were transfused into 1200 R whole body x-irradiated dogs. The hemopoietic repopulation pattern at day 10 after transfusion of comparable numbers of fresh or frozen leukocytes was not significantly different, as measured in bone marrow smears and sections and by granulocyte concentration in the peripheral blood.ZusammenfassungEs war das Ziel der Untersuchungen, eine Einfriermethode für hämopoetische Blutstammzellen zu etablieren. Blutleukozyten wurden mittels eines IBM-Blood-Cell-Separator gewonnen und in Gegenwart von 10% DMSO in Plastik-Beuteln eingefroren, wobei der Kühlprozeß vorprogrammiert war. Der Auftauvorgang dauerte nur wenige Minuten; anschließend wurde das DMSO schrittweise verdünnt, bevor die aufgetaute Zellsuspension für in-vitro wie in-vivo-Versuche verwendet wurde.Die durchschnittliche Ausbeute an mononukleären Zellen von 82 Leukapheresen betrug 86%. Zur Beurteilung der Ausbeute von eingefrorenen hämopoetischen Blutstammzellen wurde die “soft agar culture” Methode in einer Modifizierung für den Hund verwendet. Die CFUc-Ausbeute pro l × 106 mononukleäre Zellen und pro Leukapherese war nach verschieden langer Einfrierdauer (1 bis 6 Monate und 7 bis 27 Monate) nicht signifikant unterschiedlich.Die hämopoetische Repopulationsfähigkeit eingefrorener Blutstammzellen wurde mittels Transfusion in ganzkörperbestrahlte Beagles untersucht. Dabei zeigte sich, daß das hämopoetische Repopulationsmuster am 10. Tag nach Transfusion vergleichbarer Zahlen an frischen oder eingefrorenen Leukozyten nicht signifikant unterschiedlich war, wie anhand von Knochenmarkausstrichen und histologischen Knochenmarkschnitten sowie anhand der Granulozytenkonzentration im peripheren Blut nachgewiesen werden konnte.

Acute and long-term alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after partial-body irradiation: irradiation of the upper body with a single myeloablative dose

The acute and long-term effects of a single dose of partial-body irradiation on the granulocyte/macrophage progenitor cell compartment were studied in dogs. A myeloablative dose of 11.7 Gy (dose rate 6.5 cGy/min) was given to the upper body which contains approximately 70% of the total bone marrow mass. The lower part of the body (pelvis, lower extremities and tail) was shielded by a lead box. In the non-irradiated bone marrow, the concentration of the GM-CFC/10(5) mononuclear cells was slightly decreased within the first 7 days and showed some fluctuations around the normal value for several weeks thereafter. In the irradiated bone marrow, virtually no GM-CFC could be detected on day 1 after exposure. Beginning on day 7, a continuous increase took place up to day 21 when the GM-CFC concentration reached between 25% (sternum) and 43% (humerus) of the initial value. No further increase took place up to day 80. Between day 120 and 380 a secondary increase was observed which reached near-normal bone marrow GM-CFC concentrations. The blood GM-CFC concentration first showed a strong depression followed by a transient increase between day 10 and 30. This coincided with GM-CFC normalization in the protected bone marrow as well as with the initial phase of regeneration in the irradiated sites. A prolonged secondary long-lasting depression between day 33 and 120 amounted to 20 to 50% of normal values. This depression was closely related to the stagnation in the GM-CFC recovery in the irradiated bone marrow sites. The GM-CFC concentration in the blood was found to be supranormal at day 380 when the bone marrow GM-CFC had recovered. The colony stimulating activity in the serum showed an increase within the first 20 days after exposure, that is, within the same interval the bone marrow GM-CFC concentration experienced the strongest alterations, and was inversely related to the changes in the blood granulocyte values.

A NEW MODEL OF RADIATION-INDUCED MYELOPATHY: A COMPARISON OF THE RESPONSE OF MATURE AND IMMATURE PIGS

PURPOSE The development of an experimental model of radiation-induced myelopathy in the pig which would facilitate the study of the effects of clinically relevant treatment volumes. METHODS AND MATERIALS The effects of local spinal cord irradiation, to a standard 10 x 5 cm field, have been evaluated in mature (37-42.5 weeks) and immature (15.5-23 weeks) pigs. Irradiation was with single doses of 60Co gamma-rays at a dose-rate of 0.21-0.65 Gy/min. The incidence of paralysis was used as an endpoint. RESULTS Irradiation of mature animals resulted in the development of frank paralysis with animals showing combined parenchymal and vascular pathologic changes in their white matter. These lesions, in common with those seen in patients, had a clear evidence of an inflammatory component. The latency for paralysis was short, 7.5-16.5 weeks, but within the wide range reported for patients. However, it was shorter than that reported in other large animal models. The ED50 value (+/- SE) for paralysis was 27.02 +/- 0.36 Gy, similar to that in rats taking into account dose-rate factors. The irradiation of immature pigs only resulted in transient neurological changes after doses comparable to those used in the mature animals, ED50 value (+/- SE) 26.09 +/- 0.37 Gy. The reasons for these transient neurological symptoms are uncertain. CONCLUSION A reliable experimental model of radiation-induced myelopathy has been developed for mature pigs. This model is suitable for the study of clinically relevant volume effects.

Cellular Composition of Human Fetal Bone Marrow

The cellular composition of the human marrow was studied in thin sections of undecalcified bones of 24 fetuses aged from 11 to 22 weeks of gestation, embedded in methacrylate and stained by the Giemsa

The Effect of Tritiated Water on the Development of the Rat Oocyte after Maternal Infusion during Pregnancy

SummaryThe effect of continuous exposure in utero to tritiated water (HTO) was studied in the post-natal oocyte development of Wistar rats. Pregnant rats were given daily doses of 290 μCi to 5800 μCi HTO intravenously from the ninth day of pregnancy until term. During the first 21 days of life, a dose-dependent reduction of oocyte number was observed. A dose of 1450 μCi decreased the total oocyte number to 50 per cent of the controls. Accelerated maturation of oocytes was not demonstrable. The highest dose of 5800 μCi produced total aplasia in the ovaries. The number of oocytes is apparently one of the most sensitive parameters of β-irradiation damage. A comparison with data after continuous 3H-thymidine (3H-TdR) application showed a ten times higher effect of 3H-TdR compared with HTO.The mechanisms for this phenomenon are discussed, and it is concluded that the final position of the tritium compound in the cells is the most important factor in the radiobiological effect.

Bone Marrow Structure and Its Possible Significance for Hematopoietic Cell Renewal

The authors review the progress made during the last quarter of a century in the fields of hematopoietic cellular proliferation and differentiation in relation to the bone marrow structure and the microenvironment provided by the marrow stroma in which unlimited self-renewal occurs. The marrow is conceived of as an organ in which the stroma originates from local mesenchymal elements which form a vascularized and innervated matrix, seeded later by blood-borne stem cells. Transplantation studies using total-body-irradiated dogs show that stem cells derived from the marrow, as well as those from the blood and from the fetal liver, are able to repopulate a marrow rendered aplastic by irradiation. By grafting equal numbers of GM-CFU from peripheral blood and bone marrow, a faster hemopoietic reconstitution is provided by blood-derived stem cells. The most efficient stem cells in the long range are those derived from fetal liver. Bone marrow and peripheral blood GM-CFU differ in some in vitro characteristics such as radiation sensitivity. These peripheral blood cells are more radiosensitive than those derived from the marrow. Autografting of bone marrow mononuclear cell fractions obtained by velocity sedimentation techniques demonstrates that the fraction of small mononuclear cells holds a repopulating potential similar to that of circulating blood stem cells. The cells collected in fraction 2 of a discontinuous albumin gradient contain most of the blood stem cells and repopulate the marrow without causing GVHD, while cells collected in fractions 3 and 4 contain a minimal amount of stem cells and cause severe GVHD.

Development and histogenesis of the thymus in dog. A light and electron microscopical study.

The development of the thymus was studied with histological, transmission electron microscopic (TEM) and histochemical methods in 100 dog fetuses (beagle), between day 19 of gestation and day 21 after birth. Thymus development could be divided in three stages: 1/Formation of epithelial palisades; 2/Initiation of lymphopoiesis; 3/Differentiation of the medulla and Hassalls bodies (HB). The epithelial anlagen were seen at day 23 of gestation showing the characteristic palisade structure of the endodermally derived epithelium. Ten days later the beginning of lymphoiesis and the reticularization of the epithelial cells could be seen. The first HB could be found at day 38 when cortical-medullary differentiation is recognized. The histochemical observation demonstrated a rich content of PAS positive coarse granules in the cytoplasm of reticulo-epithelial (RE) cells. On the other hand, the HB showed a diffuse PAS positive reaction. The ultrastructural investigations demonstrated the presence of desmosomes connecting RE cells to one another. Desmosomes were not found between RE and lymphocytes. The growth of the developing thymus into the mesenchymal matrix resulted in the lobulation of the organ by connective tissue cells and fibers. The first mast cells were seen at day 35 of gestation, most abundantly in the interlobular connective tissue (ICT) although a few were present in the cortex and somewhat more in the medulla, near the HB. At the end of development small groups of neutrophil cell precursors appeared in the ICT and the cortex. Cysts were not present up to day 21 after birth.

Fetal liver transplantation in the dog. I. Restoration of hemopoiesis with cryopreserved fetal liver cells from DLA-identical siblings.

Fetal liver cells (FLC) were obtained from beagle fetuses 52 days postconception, and were cryopreserved prior to transplantation into ten sibling recipients that had previously been exposed to total-body irradiation delivered in 3 fractions of 6 Gy each at 4 days, 2 days, and 2 hr before grafting. Donors and hosts were genotypically identical for dog leukocyte antigens (DLA)-A, B, and D. A rapid and lasting engraftment was achieved in all animals following the transfer of 0.2x108 to 1.6x108 mononuclear FLC/kg body weight, which were equivalent to 0.9x104 to 19.8x104 granulocyte/macrophage progenitor cells (CFU-GM)/kg. Between days 14 and 20 posttransplant pretreatment levels were detected for blood granulocytes, between days 23 and 28 for circulating platelets, and between days 35 and 40 for the erythrocyte count and hemoglobin concentration. Increasing the number of CFU-GM transfused resulted in an accelerated granulocyte and platelet recovery. Bone marrow cells were of donor origin throughout the observation interval, but declining proportions of host lymphocytes circulated in the peripheral blood during the initial recovery phase. In two dogs, skin alterations that might indicate slight graft-versus-host disease (GVHD) were noted following days 20 and 70, respectively. Six recipients had to be sacrificed due to inanition, probably secondary to radiation-induced pancreatic insufficiency two to three months after grafting. The results of this study indicate that cryopreserved FLC are highly effective in restoring hemopoiesis in DLA-compatible sibling dogs. Transplantation of canine FLC may prove valuable in analyzing mechanisms pathogenetically related to graft rejection or to the development of GVHD following the transfer of T-cell-depleted hemopoietic grafts at a preclinical stage.

Fetal liver transplantation in the dog. II. Repopulation of the granulocyte-macrophage progenitor cell compartment by fetal liver cells from DLA-identical siblings.

The restoration of the granulocyte-macrophage progenitor cell (CFU-GM) compartments in blood and bone marrow, and the recovery of blood monocytes were followed for up to one year in ten beagles that had been exposed to fractionated (3x6 Gy) total-body irradiation before being transfused with cryopreserved fetal liver cells (FLC) from sibling donors that were genotypically matched for dog leukocyte antigens. Grafts contained 0.2–1.6x108 mononuclear cells and 0.9–19.8x104 CFU-GM/kg body weight. Numbers of circulating monocytes rose parallel to granulocyte numbers after day 6 and became normal by day 18 posttransplant. In bone marrow aspirates, low numbers of CFU-GM were detected on day 3 and their incidence per 105 mononuclear cells was normal after day 14. Circulating CFU-GM were present in significant numbers by day 7 and their elevated concentration per milliliter of blood after day 14 continued for one year. Dextran sulfate injection mobilized normal numbers of CFU-GM into the blood early after transplantation, and spontaneously circulating CFU-GM in a later phase did not differ from blood progenitors of normal animals with respect to radiation sensitivity and sedimentation velocity. Thus, FLC transplantation effected a rapid restoration of granulopoiesis and monocytopoiesis, which was reflected at both the level of mature blood cells and the compartments of CFU-GM in blood and bone marrow, underlining the high repopulating capacity of fetal liver stem cells.