Syngeneic bone marrow transplantation reduces the hearing loss associated with murine mucopolysaccharidosis type VII

MPS VII mice are deficient in beta-glucuronidase and share many clinical, biochemical, and pathologic characteristics with human mucopolysaccharidosis type VII (MPS VII). We have shown that syngeneic bone marrow transplantation (BMT) prolongs survival and reduces lysosomal storage in many organs of the MPS VII mouse. In this report, we quantify the hearing loss and determine the impact of syngeneic BMT on the development of deafness and the associated pathology in the MPS VII mouse. Eleven weeks after syngeneic BMT performed at birth, treated MPS VII mice had normal auditory-evoked brainstem responses (ABR), whereas untreated MPS VII mice had ABR thresholds 43 dB higher than normal. Treated MPS VII mice had beta-glucuronidase-positive cells in the temporal bone and in the subepithelial connective tissue of the external auditory canal. There was less thickening of the tympanic membrane and middle ear mucosa and decreased distortion of the ossicles and the cochlear bone. Although transplanted MPS VII mice had increased ABR thresholds by 33 weeks of age, four of the six had thresholds 12 to 32 dB lower than untreated mutants. These data indicate that syngeneic BMT in newborn MPS VII mice prevents early hearing loss and, in some animals, results in long-term improved auditory function.     

Disappearance of lysosomal storage in spleen and liver of mucopolysaccharidosis VII mice after transplantation of genetically modified bone marrow cells

Mice homozygous for the gusmps allele lack beta-glucuronidase activity and provide a useful model for human Mucopolysaccharidosis type VII (MPS VII), also known as Sly syndrome. Bone marrow (BM) transplantation was shown to correct the metabolic defect and to increase the life span of diseased animals. We have used this murine model in a preclinical study aimed at evaluating whether the techniques currently available for gene transfer into large mammalian and human BM cells will provide efficient enzyme replacement therapy in MPS patients. Autologous BM was transplanted into deficient mice after retrovirus-mediated transfer of the human beta-glucuronidase cDNA. Conditioning of recipients was performed by a single sublethal irradiation of 4.5 Gy, giving rise to low donor engraftment. In recipient mice analyzed until 145 days after gene transfer, the percentage of genetically modified hematopoietic cells was less than 5%. Nevertheless, beta-glucuronidase enzyme activity was detectable in various organs, including the brain, and disappearance of lysosomal storage was obvious in the liver and spleen. These results show that the autologous transplantation of genetically engineered BM cells could be beneficial in MPS patients.     

Nonablative neonatal marrow transplantation attenuates functional and physical defects of beta-glucuronidase deficiency

The toxicity of preparative regimens render neonatal bone marrow transplantation (BMT) for progressive childhood diseases a controversial treatment. Ablative BMT in neonatal mice with or without the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII) show high morbidity and developmental disruption of both brain and bone structure. In this investigation, BMT was performed with a high dose of congenic, normal bone marrow into nonablated newborn mice. Recipients had lifelong, multilineage, peripheral blood chimerism with the donor beta-glucuronidase-positive (GUS(+)) cells that was both well tolerated and therapeutic. Three daily injections of normal adult marrow increased the average life span by at least 6 months and corrected the functional breeding deficits typical of the MPS VII mice. Twelve months after injection, several structural features of femurs were more like that of normal mice than of untreated MPS VII mice. Periosteal circumference and bone cortical thickness were significantly improved in males and cortical density did not differ significantly from values in normal females. Significant reduction of lysosomal glycosaminoglycan storage corresponded directly with GUS enzyme activity and percentage of histochemically GUS(+) cells in visceral organs and hematopoietic tissues such as thymus, spleen, peripheral blood, and bone marrow. By all criteria tested, BMT into neonatal MPS VII mice in the absence of any preparative regimen is a successful therapy.     

Reduction of lysosomal storage in murine mucopolysaccharidosis type VII by transplantation of normal and genetically modified macrophages

This study examined the ability of macrophages to serve as target cells of gene therapy for mucopolysaccharidosis (MPS) type VII using a murine model. Bone marrow cells were harvested from syngeneic normal mice and differentiated to macrophages. These cells were given to nonmyeloablated MPS VII mice. After transplantation, donor cells populated the liver and spleen. The pathologic improvement at day 38 after transplantation was significant and glycosaminoglycan storage was reduced. To develop gene therapy using this system, a retroviral vector expressing human beta-glucuronidase (HBG) was used to infect macrophages cultivated from MPS VII mice and given to nonmyeloablated MPS VII mice. At 38 days after transplantation, HBG-positive cells were still observed histochemically and pathologic improvement was significant. These observations suggest that macrophage transplantation is a promising method for treatment of murine MPS VII without myeloablation, and macrophages may be good target cells for ex vivo gene therapy for MPS VII.     

Behavior and therapeutic efficacy of beta-glucuronidase-positive mononuclear phagocytes in a murine model of mucopolysaccharidosis type VII.

Bone marrow transplantation (BMT) is relatively effective for the treatment of lysosomal storage diseases. To better understand the contribution of specific hematopoietic lineages to the efficacy of BMT, we transplanted beta-glucuronidase-positive mononuclear phagocytes derived from either the peritoneum or from bone marrow in vitro into syngeneic recipients with mucopolysaccharidosis type VII (MPS VII). Cell surface marking studies indicate that the bone marrow-derived cells are less mature than the peritoneal macrophages. However, both cell types retain the ability to home to tissues rich in cells of the reticuloendothelial system after intravenous injection into MPS VII mice. The half-life of both types of donor macrophages is approximately 7 days, and some cells persist for at least 30 days. In several tissues, therapeutic levels of beta-glucuronidase are present, and histopathologic analysis demonstrates that lysosomal storage is dramatically reduced in the liver and spleen. Macrophages intravenously injected into newborn MPS VII mice localize to the same tissues as adult mice but are also observed in the meninges and parenchyma of the brain. These data suggest that macrophages play a significant role in the therapeutic efficacy of BMT for lysosomal storage diseases and may have implications for treatments such as gene therapy.     

Correction of murine mucopolysaccharidosis VII by a human beta-glucuronidase transgene.

We recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31). Affected mice, of genotype gusmps/gusmps, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarge vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report we describe the consequences of introducing the human beta-glucuronidase gene, GUSB, into gusmps/gusmps mice that produce virtually no murine beta-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human beta-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gusmps/gusmps mice and that human beta-glucuronidase corrects the murine mucopolysaccharidosis storage disease.     

Effects of bone marrow transplantation on the cardiovascular abnormalities in canine mucopolysaccharidosis VII

The genetic mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting from defective catabolism of glycosaminoglycans (GAGs). Echocardiographic abnormalities in dogs with MPS type VII (Sly syndrome, beta-glucuronidase deficiency) included mitral valve thickening and insufficiency, large aortic dimensions in both the long and short axes, and thickened aortic valves. Grossly, at post mortem examination, there was nodular thickening of the mitral valve, a prominent ductus diverticulum, and a dilated aorta with thickened walls. Histologically, cytoplasmic vacuolation was seen in cells of the mitral valves, coronary arteries, and aorta. By electron microscopy, the cells of the mitral valve were packed with electron-lucent cytoplasmic vacuoles. The mean residual activity of beta-glucuronidase in the aorta and myocardium was <1% of normal, the mean hexosaminidase A activity >2. 5 times normal, and the mean GAG concentrations more than twice normal. In three MPS VII dogs that received heterologous BMT at 6 weeks of age, the echocardiographic abnormalities were improved, and the histopathologic and ultrastructural pathology was reduced. In the aorta and myocardium, the mean beta-glucuronidase activity of the BMT group was 4.5% and 11% of normal, respectively, and the hexosaminidase A activity and GAG concentrations were normalized. Bone Marrow Transplantation (2000) 25, 1289-1297.     

In utero transplantation of fetal liver cells in the mucopolysaccharidosis type VII mouse results in low-level chimerism, but overexpression of beta-glucuronidase can delay onset of clinical signs

Mice with the lysosomal storage disease mucopolysaccharidosis (MPS) VII, caused by a deficiency of beta-glucuronidase (GUSB), have signs of disease present at birth. Bone marrow transplantation (BMT) or retroviral vector-mediated gene transfer into hematopoietic stem cells can partially correct the disease in adult mice, and BMT performed at birth results in a better clinical outcome. Thus, treatment in utero may result in further improvement. However, this must be done without cyto-ablation, and the donor cells do not have a competitive repopulating advantage over host cells. Transplantation in utero of either syngeneic fetal liver hematopoietic stem cells marked with a retroviral vector, or allogeneic donor cells that constitutively express high levels of human GUSB from a transgene, resulted in only about 0.1% engraftment in the adult. Immuno-affinity enrichment of stem and progenitor cells of 5- to 10-fold resulted in significantly higher GUSB activities at 2 months of age, but by 6 months engraftment was about 0.1%. Attempts to further increase the number of stem and progenitor cells were deleterious to the recipients. Nevertheless, GUSB expressed during the first 2 months of life in MPS VII fetuses could delay the onset of overt signs of disease. This suggests that the expression of some normal enzyme activity beginning in fetal life may offer the possibility of slowing the progression of the disease until more definitive postnatal transplantation or gene transfer to stem cells could be accomplished.     

Active site mutant transgene confers tolerance to human beta-glucuronidase without affecting the phenotype of MPS VII mice

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of beta-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gus(mps/mps)) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human beta-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gus(mps/mps)) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gus(mps/mps)) mouse. However, they are now tolerant to immune challenge with human beta-glucuronidase. This "tolerant MPS VII mouse model" should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.     

A genetically myeloablated MPS VII model detects the expansion and curative properties of as few as 100 enriched murine stem cells

Causes of transplantation failures are often difficult to assess due to our inability to monitor hematopoietic stem cell (HSC) homing, distribution, and amplification in situ. We have developed a mouse model that permits histochemical localization of 1000-fold enriched HSC and quantification of their long-term expanded progeny in situ. The mice are genetically myeloablated (c-kit receptor mutated, W41/W41) and are beta-glucuronidase null (GUSB ; gus(mps)/gus(mps)). The GUSB- mice with mucopolysaccharidosis type VII (MPS VII), like a large number of human patients with similar diseases, have systemic lysosomal storage disease that leads to premature death. Congenic GUSB+, Lineage(lo), Sca-1(hi), c-Kit(hi), Hoechst(lo) HSC, at doses of 30, 100, 250, and 425 cells, implanted and amplified in adult W41/W41, gus(mps)/gus(mps) recipients in a dose-dependent manner. At autopsy, primary recipients of 100 and 425 donor cells had histologically identifiable donor GUSB+ cells in multiple sites and showed both myeloid and lymphoid expansion in bone marrow. Donor cells were rare in the liver and spleen of 100-cell recipients, but lysosomal storage was significantly reduced. The life span was significantly extended in engrafted recipients of 250 (36.7 +/- 3.84 weeks,p = 0.0316) and 425 (40.7 +/-1.53 weeks,p = 0.0033) cells compared to untreated mice (26.4 +/- 1.53 weeks). Secondary hosts of marrow from the recipients of 425 cells demonstrated continued expansion of the GUSB+ cells. Results indicate the genetically myeloablated MPS VII mice can be used to trace and enumerate donor cells long-term and to follow early engraftment events in situ.     

Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2-3 days of age with a retroviral vector (RV) expressing canine beta-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5-60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6-17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal.     

Missense models [Gustm(E536A)Sly,Gustm(E536Q)Sly,and Gustm(L175F)Sly] of murine mucopolysaccharidosis type VII produced by targeted mutagenesis

Human mucopolysaccharidosis VII (MPS VII, Sly syndrome) results from a deficiency of beta-glucuronidase (GUS) and has been associated with a wide range in severity of clinical manifestations. To study missense mutant models of murine MPS VII with phenotypes of varying severity, we used targeted mutagenesis to produce E536A and E536Q, corresponding to active-site nucleophile replacements E540A and E540Q in human GUS, and L175F, corresponding to the most common human mutation, L176F. The E536A mouse had no GUS activity in any tissue and displayed a severe phenotype like that of the originally described MPS VII mice carrying a deletion mutation (gus(mps/mps)). E536Q and L175F mice had low levels of residual activity and milder phenotypes. All three mutant MPS models showed progressive lysosomal storage in many tissues but had different rates of accumulation. The amount of urinary glycosaminoglycan excretion paralleled the clinical severity, with urinary glycosaminoglycans remarkably higher in E536A mice than in E536Q or L175F mice. Molecular analysis showed that the Gus mRNA levels were quantitatively similar in the three mutant mouse strains and normal mice. These mouse models, which mimic different clinical phenotypes of human MPS VII, should be useful in studying pathogenesis and also provide useful models for studying enzyme replacement therapy and targeted correction of missense mutations.     

Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors

Gene transfer vectors based on lentiviruses can transduce terminally differentiated cells in the brain; however, their ability to reverse established behavioral deficits in animal models of neurodegeneration has not previously been tested. When recombinant feline immunodeficiency virus (FIV)-based vectors expressing beta-glucuronidase were unilaterally injected into the striatum of adult beta-glucuronidase deficient [mucopolysaccharidosis type VII (MPS VII)] mice, an animal model of lysosomal storage disease, there was bihemispheric correction of the characteristic cellular pathology. Moreover, after the injection of FIV-based vectors expressing beta-glucuronidase into brains of beta-glucuronidase-deficient mice with established impairments in spatial learning and memory, there was dramatic recovery of behavioral function. Cognitive improvement resulting from expression of beta-glucuronidase was associated with alteration in expression of genes associated with neuronal plasticity. These data suggest that enzyme replacement to the MPS VII central nervous system goes beyond restoration of beta-glucuronidase activity in the lysosome, and imparts improvements in plasticity and spatial learning.     

Treatment of lysosomal storage disorders: Cell therapy and gene therapy

Most lysosomal storage diseases have central nervous system (CNS) involvement. No effective treatment is available at present. We investigated the usefulness of brain-directed gene therapy and cell therapy using mouse models of lysosomal storage diseases. For gene therapy to the CNS, a recombinant adenovirus encoding beta-galactocerebrosidase gene was injected into the cerebral ventricle of neonatal twitcher mice, a murine model of Krabbe disease. Improvements in neurological symptoms and a prolonged lifespan were observed. Brain activity of beta-galactocerebrosidase was increased significantly and the concentration of a cytotoxic metabolite, psychosine, was decreased. Pathological observations of the brain were also improved in treated twitcher mice. For cell therapy to the CNS, a neural stem cell line derived from human fetal brain was genetically engineered to overexpress beta-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mice, a model of beta-glucuronidase deficiency. Transplanted human neural stem cells were found to integrate and migrate in the host brain and to produce large amounts of beta-glucuronidase. Brain contents of the substrate of beta-glucuronidase were reduced and widespread clearing of lysosomal storage was observed in treated MPS VII mice. These data suggest that brain-directed gene/cell therapy may be useful in the treatment of neurological alterations in lysosomal storage diseases.     

In utero fetal liver cell transplantation without toxic irradiation alleviates lysosomal storage in mice with mucopolysaccharidosis type VII.

Lysosomal storage diseases, such as Mucopolysaccharidosis type VII (MPS VII), cause progressive loss of mobility and intellect and result in early death. Treatment of progressive diseases must occur before the blood-brain barrier closes. In MPS VII mice, normal donor hematopoietic cells secrete the missing enzyme beta-glucuronidase (GUSB) that reverses disease manifestations. Correction of lysosomal storage is limited to the visceral organs unless transplantation is preceded by high-dose irradiation. We hypothesize that irradiation opens the blood-brain barrier allowing passage of corrective cells. Here we transplanted genetically myeloablated MPS VII fetuses to determine whether earlier treatment without toxic irradiation is systemically corrective. Cells with a selective advantage in utero were identified. Donor fetal liver cells (FLC), a substitute for difficult to obtain murine cord blood cells, were increased 10-fold in the host peripheral blood over equivalent numbers of adult marrow cells injected simultaneously and were stable long term in both primary and secondary hosts. GUSB- MPS VII fetuses injected with GUSB+ FLC were assessed longitudinally after birth. Donor FLC replaced host stem cell descendants, prolonged life dramatically, and reduced bone dysplasia and lysosomal storage in all tissues long term. GUSB, donor leptomeningeal cells, and microglia were present in the brain at 11 months postinjection. Lysosomal storage in cortical neurons and glia, although not completely corrected, was reduced. We conclude that in utero intervention without toxic pretreatment in this model reduces the storage disease long term and improves the length and quality of life despite exerting only minor effects on the brain.     

Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells

OBJECTIVE: Ionizing radiation-induced myeloablation can be rescued via bone marrow transplantation (BMT) or administration of cytokines if given within 2 hours after radiation exposure. There is no evidence for the existence of soluble factors that can rescue an animal after a lethal dose of radiation when administered several hours postradiation. We established a system that could test the possibility for the existence of soluble factors that could be used more than 2 hours postirradiation to rescue animals. MATERIALS AND METHODS: Animals with an implanted TheraCyte immunoisolation device (TID) received lethal-dose radiation and then normal bone marrow Lin- cells were loaded into the device (thereby preventing direct interaction between donor and recipient cells). Animal survival was evaluated and stem cell activity was tested with secondary bone marrow transplantation and flow cytometry analysis. Donor cell gene expression of five antiapoptotic cytokines was examined. RESULTS: Bone marrow Lin- cells rescued lethally irradiated animals via soluble factor(s). Bone marrow cells from the rescued animals can rescue and repopulate secondary lethally irradiated animals. Within the first 6 hours post-lethal-dose radiation, there is no significant change of gene expression of the known radioprotective factors TPO, SCF, IL-3, Flt-3 ligand, and SDF-1. CONCLUSION: Hematopoietic stem cells can be protected in lethally irradiated animals by soluble factors produced by bone marrow Lin- cells.     

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors

Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR-/- MPS VII mice. The availability of both MR+/+ and MR-/- mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human beta-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.     

Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.     

Growth retardation and depigmentation of hair after high-dose busulfan and congenic hematopoietic cell transplantation in mice.

Busulfan, a myeloablative but non-immunosuppressive alkylating agent, is used extensively in clinical bone marrow transplantation (BMT), but the effects of high-dose administration have not been previously evaluated in preclinical BMT settings with young murine recipients. We compared the survival and growth of C57BL/6 mice given graded single doses of busulfan (10-100 mg/kg) or total body irradiation (TBI; 900 cGy) at age 9 days and hematopoietic cell transplantation (HCT; transplantation of congenic bone marrow and spleen cells) 24 h later. The 30-day survival was 87-100% in mice transplanted after 10-40 mg/kg busulfan and 79% after TBI, but fell to 54% and 33%, respectively, after 80 mg/kg and 100 mg/kg busulfan, suggesting that this latter dosage range represents the LD50 for single-dose busulfan in young C57BL/6 mice given stem cell rescue. The weights of 10-week-old mice given HCT after lower doses of busulfan ranged from 87% of control at 10 mg/kg to 64-69% of control in mice conditioned with 35-65 mg/kg busulfan or TBI. Impairment of weight gain was most striking (approximately 50% of control) in mice transplanted after 80-100 mg/kg busulfan. Despite retardation of somatic growth, the brain weights of busulfan-conditioned mice remained at least 90% of control, and there were no obvious neuropathological alterations in the brains of these animals. All mice treated with at least 20 mg/kg busulfan or TBI lost hair by 3-4 weeks after transplant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioprotection of hemopoiesis conferred by aqueous extract from chlorococcal algae (Ivastimul) administered to mice before irradiation

In experiments on mice, single i.p. injections of aqueous extract from chlorococcal freshwater algae (Ivastimul) were found to increase their radioresistance. During the same period the number of spleen colony-forming units (CFUs) in the bone marrow and spleen and their proliferation activity increased. The amount of granulocyte-macrophage colony-forming cells (GM-CFC) in the bone marrow grows and the colony-stimulating activity (CSA) of the blood serum of mice is elevated at an early period after injection of the substance. The recovery of the CFUs and GM-CFC pools in femoral bone marrow after irradiation proceeds at a faster rate in Ivastimul-treated animals than in control groups. The activation of the pool of hemopoietic stem cells and stimulatory effects of Ivastimul on granulocytopoiesis act favorably on the repair of radiation damage and on increased percentage of animals surviving lethal doses of gamma radiation.