Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice.

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy (60)Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.

Hematopoiesis-stimulating and anti-tumor effects of repeated administration of diclofenac in mice with transplanted fibrosarcoma cells.

Positive effects of repeated administration of diclofenac, an inhibitor of prostaglandin synthesis, in terms of prevention of tumor development and stimulation of hematopoiesis have been observed in C3H mice transplanted subcutaneously with G:5:113 fibrosarcoma cells. Fourteen-day treatment with diclofenac (3.75 microg/kg/day) started from day 5 after tumor cell transplantation. Measurements of tumors and hematological examinations were performed on day 30. The results strongly suggest the possibility that inhibitors of prostaglandin synthesis (non-steroidal anti-inflammatory drugs) may be used in oncological practice where the observed effects are highly desirable.

Radioprotection of haemopoietic stem cells by a single injection of bacterial lysate - IRS-19 administered to mice before or after irradiation.

Data in this report describes the effect of a single injection of bacterial lysate IRS-19 prior to irradiation of C57Bl/6 mice on recovery of colony-forming cells (CFC) after sublethal and lethal doses of radiation. The injection of IRS-19 promoted an earlier recovery of colony-forming cells in the bone marrow and spleen. For example, 5 and 9 days after 7.5 Gy irradiation, the number of CFU-S per femur was approximately 1.7-2.3-fold higher in IRS-19-injected mice than in saline-injected mice. Also, pretreatment of mice with IRS-19 induced an increase in the number of endogenous haemopoietic stem cells (endoCFU-S). In the postradiation period (5-21 days) significantly increased bone marrow and spleen cellularity and accelerated myelopoietic regeneration (committed progenitor granulocyte-macrophage-colony-forming cells, GM-CFC) in the bone marrow and spleen compared with saline-treated controls. At the time of presumed irradiation, (i.e. 24 h after administration of the drug to the non-irradiated mice), there was no significant difference between the control mice and mice treated with IRS-19 in numbers of femoral and spleen GM-CFC. In contrast, the number of nucleated femoral cells decreased significantly in the group treated with IRS-19. Moreover, treatment with IRS-19 caused a sustained increase in serum colony-stimulating activity which was followed by an enhanced repopulation of GM-CFC in the femoral marrow and spleen. Administration of the agent 24 h prior to irradiation rather than postirradiation appeared most effective with respect to radioprotection. Intravenous rather than i.p. and p.o. was the most effective route of administration in the mouse. Furthermore, single, high-dose injection appeared to be more effective than repeated, lower dose injections. Results suggest that the radioprotective properties associated with the administration of IRS-19 are largely a consequence of the induction of haemopoietic colony-stimulating activities and potentially the activation and/or enhancement of cytokine cascades in the recipient animals. These changes may ultimately impact the cell cycle profile of the haemopoietic cells and therefore their ability to withstand and/or recover from radiation insult.

Influence of age and K, Mg aspartate (Cardilan) on murine haemopoiesis.

We have studied the effect of age as well as the effect of short-term and long-term intake of K and Mg salts of aspartic acid (Cardilan) on haemopoiesis in ICR mice strain. The cellularity of the bone marrow does not change with aging, but the number of granulocyte-macrophage colony-forming cells (GM-CFC) and also the number of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) in two-year-old mice increased in the bone marrow. In two-year-old mice the number of leukocytes decreased in the peripheral blood with aging, mainly as a result of a decrease in mononuclear cells. Short-term drinking (STD) of Cardilan caused increased numbers of CFU-S and BFU-E in bone marrow and increased numbers of reticulocytes in the peripheral blood of one-year old animals (STD/12 months old). In the oldest mice (STD/24) increased weight and cellularity of the spleen and rapid increase of leukocytes and reticulocytes in the peripheral blood was recorded. After long-term drinking (LTD) of Cardilan the number of spleen GM-CFC rose markedly in one-year-old mice (LTD/12) and in two-year-old mice (LTD/24) the number of reticulocytes in the peripheral blood rose. Our results indicate that K and Mg salts of aspartic acid influence erythropoietic activity most widely.

Recovery of peripheral blood cells in irradiated mice pretreated with bacterial extract IRS-19.

The effect of antigenic bacterial lysate IRS-19 on the recovery of blood cells was studied in mice injured by a single dose of 7 Gy irradiation. The preirradiation administration of IRS-19 accelerated the recovery of leukocytes, reticulocytes and platelets in peripheral blood. The recovery of leukocytes 9-14 days after irradiation in protected animals was accompanied by a higher level of band forms of granulocytes as well as activated lymphoid and monocytoid cells.

Effects of immunomodulators on postirradiation recovery in the thymus.

The effect of immunomodulatory agents on reparation processes in the thymus was studied in mice injured by a single sublethal or lethal dose of ionizing radiation ranging between 6.5-9.5 Gy. Reparation of thymus weight was not influenced by pretreatment with immunomodulators. Furthermore, the morphological picture did not exhibit appreciable differences between non-protected and protected groups, except for greater proliferation of fibroblasts and macrophages in protected animals.

Combined modality radioprotection: enhancement of survival and hematopoietic recovery in gamma-irradiated mice by the joint use of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) and indomethacin.

We have reported previously [Fedorocko, P., Int. J. Radiat., Biol. 65:465, 1994] that liposomal muramyl tripeptidephosphatidyl ethanolamine (MTP-PE/MLV) given prior to irradiation results in augmented hemopoietic recovery and enhanced animal survival as evidenced by increased pluripotent stem cells (CFU-S) and progenitor cells committed to granulocyte and/or macrophage development (GM-CFC) or white blood cells, neutrophil counts, as well as by survival rates of lethally irradiated mice. In this report the effects of liposomal MTP-PE (radioprotective immunomodulator; 10 mg/kg i.p., 24 h before irradiation) and indomethacin (inhibitor of prostaglandin production; 2 mg/kg i.m., 24 h and 3 h before irradiation) were studied. Both of the agents were administered either alone or in combination. The results included the assessment of preirradiation hemopoietic effects of drugs and postirradiation hemopoietic recovery in terms of bone marrow cellularity, number of bone marrow GM-CFC, endogenous spleen colony formation (endoCFU-S), and the determination of the survival of lethally irradiated mice. Experimental evidence elevated by the increased preirradiation numbers of GM-CFC and hydroxyurea kill of GM-CFC as well as a simultaneous significant diminution in bone marrow cellularity indicated that the beneficial action of the combined treatment could be a consequence of increased cell proliferation in the hemopoietic tissue and mobilization with redistribution of stem cells from bone marrow into the circulation. In the postirradiation period (3-14 days), combined pretreatment of mice accelerated myelopoietic regeneration in the bone marrow compared to treatment with MTP-PE/MLV alone or indomethacin alone. Combined administration of MTP-PE/MLV (10 mg/kg, -24 h, i.p.) and indomethacin (2 mg/kg, -24 h and -3 h, i.m.) to mice, prior to lethal irradiation, exerted an additional radioprotective effect and protected 100% of the C57B1/6 mice.

Effects of cadmium on haemopoiesis in irradiated and non-irradiated mice: 1. Relationship to the number of myeloid progenitor cells.

The effects of single subcutaneous injection of cadmium chloride on haemopoiesis in normal (non-irradiated) or irradiated mice were investigated. Cadmium doses used ranged from 1-8 mg/kg body weight Twenty-four hours after treatment with cadmium (doses from 3 to 8 mg/kg) there were no significant changes in bone marrow cellularity and the granulocyte-macrophage progenitor cell (GM-CFC) number per femur in non-irradiated female ICR mice. Similarly, during the 30-day postinjection period bone marrow cellularity and marrow GM-CFC number in mice treated with a cadmium dose of 5 mg/kg were not significantly different from the control values. Cadmium significantly reduced the lethal effects of gamma rays. In addition, increasing the doses of cadmium administered 24 h prior to sublethal irradiation increased the number of endogenous haemopoietic stem cells (endoCFU-S) in a concentration-dependent manner. Pretreatment with cadmium also decreased the radiation damage to endoCFU-S and haemopoietic progenitor cells committed to granulocyte/macrophage development (GM-CFC). The survival of stem cells was higher and the regeneration of cellularity and GM-CFC of irradiated bone marrow was accelerated in mice pretreated with 5 mg Cd/kg body weight in comparison with saline-injected mice.

Effects of cadmium on haemopoiesis in irradiated and non-irradiated mice: 2. Relationship to the number of circulating blood cells and haemopoiesis.

The effect of administration of cadmium alone in non-irradiated mice as well as the effect of pre-irradiation administration of cadmium on the reparation processes of haemopoiesis were investigated in mice irradiated by a dose of 7.5 Gy. The pre-irradiation administration of cadmium accelerated the reparation processes in the bone marrow and spleen as well as the number of leukocytes and thrombocytes in the peripheral blood. The administration of cadmium alone caused a temporary weight decrease of the thymus and reduced number of erythrocytes, reticulocytes and haemoglobin values in the peripheral blood. The temporary rapid increase in the number of leukocytes on the 21st day after cadmium administration was investigated.

Combined radioprotective effect of Broncho-Vaxom and WR-2721 on hemopoiesis and circulating blood cells.

Possibilities of combined radioprotection, using preirradiation WR-2721 administration and post- or preirradiation Broncho-Vaxom administration in lethally whole-body gamma-irradiated mice were investigated. Combined modality treatments were more effective than individual treatments alone. The ameliorative influence of the combined radioprotection could be explained by the hemopoietic stem cell protection with WR-2721 and the stimulation by Broncho-Vaxom.

Administration of the bacterial extract Broncho-Vaxom enhances radiation recovery and myelopoietic regeneration.

In the present study, we show that the bacterial extract Broncho-Vaxom (BV, 500 micrograms/mouse; free of endotoxin) has radiation recovery activity when administered i.p. 24 h before sublethal irradiation. In the postirradiation period (5-12 days), pretreatment of mice with BV induced significantly increased bone marrow cellularity and accelerated myelopoietic regeneration (committed progenitor granulocyte-macrophage colony-forming cells; GM-CFC) in the bone marrow compared with saline-treated controls. The earlier hemopoietic recovery in BV-injected mice was not associated with an increase in the number of bone marrow GM-CFC and CFU-S (colony-forming units-spleen) within 24 h after injection. Simultaneously, a significant diminution in bone marrow cellularity occurred. In addition, the percentage of both GM-CFC and CFU-S in the S-phase of the cell cycle was significantly increased 24 h after a single treatment. In our experiments colony stimulating activity (CSA) in the serum of treated mice was not observed within 24 h after injection. Administration of BV 24 h prior to lethal irradiation, resulted in an increase in the number of surviving mice. Combined administration of BV (24 h) and indomethacin (24 h and 3 h) to mice, prior to irradiation, caused an additional radioprotective effect. These results demonstrate that BV stimulates myelopoietic regeneration and suggest a mechanism by which this treatment protects mice from otherwise lethal irradiation.

Radioprotective effects of WR-2721, Broncho-Vaxom and their combinations: survival, myelopoietic restoration and induction of colony-stimulating activity in mice.

The possibilities of combined radioprotection, using preirradiation WR-2721 administration and post- or preirradiation Broncho-Vaxom administration in lethally whole-body gamma-irradiated mice were investigated. LD50/30 dose reduction factors (DRFs) for mice treated with WR-2721 (200 mg/kg i.p. 30 min before irradiation), Broncho-Vaxom (25 mg/kg i.p. 24 h before irradiation), or both agents were 1.92, 1.17 and 2.07, respectively. These results demonstrated at least additive radioprotective effects of both agents, manifested in increased survival of irradiated mice. Radioprotection from 17 Gy was optimal when WR-2721 in combination with Broncho-Vaxom was given 30 min before irradiation and Broncho-Vaxom 24 h before or 4-8 h after irradiation. Combined modality treatments were also more effective than individual treatments alone in accelerating the bone marrow GM-CFC restoration. During the first days after irradiation enhanced colony-stimulating activity (CSA) of the lungs was observed in mice with postirradiation injection of Broncho-Vaxom alone or in mice injected with WR-2721 and Broncho-Vaxom (8 h after irradiation), as well as in mice only irradiated.

Pre- and postirradiation hemopoietic effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) administered to C57Bl/6 mice before irradiation.

The effect of muramyl tripeptide (MTP-PE) encapsulated in liposomes (multilamellar vesicles, MLV) on hemopoiesis and hemopoietic recovery after sublethal single irradiation with a dose of 6 Gy was investigated. MTP-PE/MLV during 24 h after administration stimulated the monocyte/macrophage system and accelerated recovery from radiation-induced damage of hemopoiesis. The above reparative processes were accompanied by hyperplasia of granulocytes in the bone marrow on days 6-8 after irradiation, acceleration of erythropoiesis in the spleen and recovery of granulocyte count in the peripheral blood. The repair of thymic and splenic lymphoid tissues as well as recovery of the number of mononuclear cells in peripheral blood was not influenced by MTP-PE/MLV application.

Pre-irradiation haematological effects of the bacterial extract Broncho-Vaxom and postirradiation acceleration recovery from radiation-induced haematopoietic depression.

Effects of the bacterial extract Broncho-Vaxom (BV), free of endotoxin, on haematopoietic parameters of peripheral blood, bone marrow, spleen and thymus were investigated in normal and irradiation-myelosuppressed C57BL/6 mice. BV in normal (nonirradiated) mice induced transient lymphocytopenia and increased granulocytes, particularly neutrophils, six to nine hours after administration. Elevated numbers of peripheral blood macroreticulocytes were observed during the third hour. In the splenic red pulp, enhancing haemopoietic activity, foci of fibroblasts began to appear more frequently from the first hour to 24 h. In lymphatic follicles of the spleen and thymus, the incidence of macrophages or monocytes began to increase three hours after BV application. Twenty-four hours later thymus weight decreased by 27%. In the bone marrow, enhanced proliferation of eosinophils was observed from hour nine, with maximum proliferation at hour 24. BV treatment 24 h before irradiation had a notable radio-protective effect, manifested by accelerated haemopoietic recovery during the postirradiation period. Accelerated myelopoiesis, erythropoiesis and megakaryocytopoiesis resulted in accelerated peripheral blood recovery. However, BV had no effect on thymus reparation as suggested by similar effects in BV protected and BV nonprotected animals during the entire period which followed a 6.5 Gy dose.

Radioprotection of mice by the bacterial extract Broncho-Vaxom: haemopoietic stem cells and survival enhancement.

Pretreatment of mice with 50-1000 micrograms of the bacterial extract Broncho-Vaxom (BV, free of endotoxin) before sublethal irradiation induced an increase in the number of endogenous haemopoietic stem cells (E-CFU). The degree of radioprotection was dependent on both the time of administration and the dose of BV. An optimal E-CFU survival was observed when 500 micrograms of BV was administered i.p. 24 h before irradiation. BV did not affect the day 9 CFU-S survival in the bone marrow directly after irradiation. However, 5, 9 and 12 days after irradiation, the number of day 9 CFU-S was almost 2-fold higher in the bone marrow of BV injected mice. Pretreatment with BV protected C57B1/6 mice in a dose-dependent manner from the lethal effect of ionizing radiation. A single dose (50, 100, 250, or 500 micrograms) of bacterial lysate injected i.p. 24 h before 9.5 Gy gamma-rays (LD100/21) protected 16%, 25%, 80%, and 94% of C57B1/6 mice, respectively. The dose reduction factor in the case when the BV (500 micrograms per mouse) was administered at that time was 1.18 (95% CL 1.12, 1.25).