Mouse Model Of Thalassemia Intermedia Research Articles

Iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood.

BackgroundIron overload is one of common complications of β-thalassemia. Systemic iron homeostasis is regulated by iron-regulatory hormone, hepcidin, which inhibits intestinal iron absorption and iron recycling by reticuloendothelial system. In addition, body iron status and requirement can be altered with age. In adolescence, iron requirement is increased due to blood volume expansion and growth spurt. Heterozygous β-globin knockout mice (Hbbth3/+; BKO) is a mouse model of thalassemia widely used to study iron homeostasis under this pathological condition. However, effects of age on iron homeostasis, particularly the expression of genes involved in hemoglobin metabolism as well as erythroid regulators in the spleen, during adolescence have not been explored in this mouse model.MethodsIron parameters as well as the mRNA expression of hepcidin and genes involved in iron transport and metabolism in wildtype (WT) and BKO mice during adolescence (6–7 weeks old) and adulthood (16–20 weeks old) were analyzed and compared by 2-way ANOVA.ResultsThe transition of adolescence to adulthood was associated with reductions in duodenal iron transporter mRNA expression and serum iron levels of both WT and BKO mice. Erythrocyte parameters in BKO mice remained abnormal in both age groups despite persistent induction of genes involved in hemoglobin metabolism in the spleen and progressively increased extramedullary erythropiesis. In BKO mice, adulthood was associated with increased liver hepcidin and ferroportin mRNA expression along with splenic erythroferrone mRNA suppression compared to adolescence.ConclusionOur results demonstrate that iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood. The present study underscores the importance of the age of thalassemic mice in the study of molecular or pathophysiological changes under thalassemic condition.

Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia

β-thalassemia (β-Thal) is caused by defective β-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress.We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of β-thalassemia intermedia. In the absence of these transporters, the anemia of β-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of β-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved β-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of β-thalassemia.

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia.

Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myeloablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries, where the greatest demand for a β-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involves HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow, where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase-based integration machinery, a micro-LCR-driven γ-globin gene, and an MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose O6-benzylguanine plus bis-chloroethylnitrosourea. We showed in "healthy" human CD46-transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable γ-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near-complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and "portable" approach could permit a broader clinical application of thalassemia gene therapy.

Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of β-thalassemia

AbstractInherited anemias with ineffective erythropoiesis, such as β-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron, leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of HbbTh3/+ mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age, suggesting ERFE could be a factor suppressing hepcidin production in β-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age, and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis, as indicated by reduced spleen index, red cell distribution width, and mean corpuscular volume, but did not improve the anemia. Thus, ERFE mediates hepcidin suppression and contributes to iron overload in a mouse model of β-thalassemia.

Use of Minihepcidins As a “Medical Phlebotomy” in the Treatment of Polycythemia Vera

Polycythemia Vera (PV) is a myeloproliferative disorder in which the rate of erythropoiesis is massively increased. Most of the health concerns associated with PV are caused by increased red cell production, leading to increased risk of thrombosis due to red cell sludging in small vessels. PV is caused by JAK2 tyrosine kinase mutations that cause unregulated activation of the erythropoietin signaling pathway and massive overproduction of red cells. PV shows characteristic features of stress erythropoiesis, which is highly dependent on iron absorption and erythroid iron intake. The mainstay of therapy is therapeutic phlebotomy, the goal of which is to reduce the hematocrit (Hct) to 45% or less to minimize the risk of thrombosis. Since phlebotomy has no effect on bone marrow production of red cells the effect is transient and dependent on the development of an iron-deficient state for control of Hct. Poor compliance with phlebotomy and the intermittent nature of the intervention frequently leads to treatment failure. Iron deficiency or erythroid iron restriction may be effective in the treatment of PV by causing inhibition of erythropoietin signaling downstream of JAK2, mediated by inhibition of aconitase. We have previously shown that increased hepcidin level reduces transferrin saturation and erythroid iron intake and has a beneficial effect on the number of erythroid progenitors in a mouse model of Thalassemia Intermedia. Hepcidin mimetic peptides (minihepcidins (MH)) which replicate the biological effects of hepcidin may be beneficial in the treatment of PV by limiting iron availability to the developing erythrocyte, thereby overriding JAK2 stimulated erythropoiesis and reducing erythrocyte production.A mouse model of PV was generated by crossing a floxed heterozygous Jak2V617Ffl/fl (V617F is the most common mutation causing human PV) mouse with a mouse hemizygous for the Vav-iCre transgene expressed in hematopoietic cells. We generated a cohort of mice with the disease by engrafting Jak2V617Ffl/fl-Vav-iCre bone marrow into wild type (WT) mice. M009, which is a potent MH that decreases ferroportin expression in vitro and serum iron in vivo was administered subcutaneously at a supramaximal dose of 400 µg/mouse twice weekly. A high dose of M009 was used to ensure that a significant level of iron restriction was present throughout the study. In PK/PD studies in the rat equivalent doses of M009 had previously been shown to reduce serum iron by >80% for 48-72h post-dose. MH treatment for six weeks or vehicle control was initiated 2 months after bone marrow transplant, when the phenotype was fully established.Hct was 62.5 ± 4.3% (mean ± SD) in controls (n=5) at baseline, rising to 85.0 ± 4.0% at the end of study. In contrast in M009 treated animals Hct dropped from 69.3 ± 4.3% (n=6) to 38.8 ± 7.4%. Interim evaluation at 3 weeks indicated that the effect of M009 was evident early in the treatment period (Hct reduction of 36% in M009 treated animals versus 17% increase in vehicle controls). The effect of M009 on Hct was predominantly caused by a decrease in RBC (-34%) rather than MCV (-14%).These data indicate that MH-induced iron restriction can rapidly reduce the rate of erythropoiesis in this mouse model of human PV, leading to large reductions in Hct that would be highly clinically relevant if replicated in clinical trials. This effect was observed despite the fact that Hct continued to increase in control animals, indicative of even greater underlying disease activity during the treatment period. The dose of M009 that was used was high and probably supratherapeutic since circulating Hb was reduced below the level observed in WT mice. Nevertheless these results suggest that MH may be considered for development as a “medical phlebotomy” to provide continuous and improved control of accelerated erythropoiesis in the treatment of PV. DisclosuresCasu:Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.

Immunologic characterization suggests reduced alloimmunization in a murine model of thalassemia intermedia.

Transfusion therapy remains a mainstay of treatment for patients with thalassemia major and to a lesser extent for the less anemic patients with thalassemia intermedia. We have previously reported a role for regulatory T cells (Tregs) in the control of antibody responses in wild-type C57BL/6 (WT) mice exposed to allogeneic red blood cell transfusions. As an initial step to study and characterize immune regulation in thalassemias, we performed an immunologic cell-type characterization of C57BL/6 Hbb(th-1)/Hbb(th-1) mouse model of thalassemia intermedia (Thal) in steady state as well as after transfusions with allogeneic blood. The myeloid and lymphocyte compartments including Tregs and T helper (Th) responses were analyzed in transfusion naive Thal and WT mouse spleens. The effect of allogeneic transfusions on Treg and global T helper responses was also measured. We found elevated levels and activity of splenic Tregs in Thal mice with lower Th type 1/Th type 2 ratios before as well as after transfusion. Furthermore, pretransfused Thal mice had altered ratios of the splenic myeloid compartment with increased proportion of macrophages but lower frequency of conventional dendritic cells. Surprisingly, transfusions resulted in lower alloimmunization levels in Thal compared to WT mice. These data suggest that this experimental model of thalassemia intermedia has an intrinsic alteration in splenic immunoregulation with an increased resistance to alloimmunization, raising the possibility that studying this animal model may help to identify potential immunoregulatory networks to inhibit alloimmunization.

Aln-TMP: A Subcutaneously Administered RNAi Therapeutic Targeting Tmprss6 For The Treatment Of β-Thalassemia

The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6 expression significantly ameliorated the disease phenotype in the Hbbth3/+ mouse model of Thalassemia Intermedia (Blood. 2013; 14;121(7):1200-8). The Tmprss6 gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6 removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6 in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+ mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia.To this end, we developed ALN-TMP, a subcutaneous RNAi therapeutic targeting hepatic Tmprss6 for the treatment of β-Thalassemia. ALN-TMP employs the GalNAc conjugate siRNA delivery platform that safely and effectively delivers siRNA to the liver for hepatic gene silencing. Preclinical animal data demonstrate ALN-TMP exhibits robust and durable dose-dependent gene suppression as single dose administration of ALN-TMP leads to > 80% Tmprss6 gene suppression for up to 3 weeks post-dose. This leads to concomitant increases in Hepcidin gene expression (>2x baseline levels) and subsequent decreases in total serum iron and TfSat (>50% decrease from baseline). The degree to which ALN-TMP modulates Hepcidin and serum iron mobilization is nearly identical to that observed in the previous Hbbth3/+ mouse studies and suggests ALN-TMP is a potent RNAi therapeutic with the potential of producing disease modifying effects in β-Thalassemia. Disclosures:Butler:Alnylam: Employment. Fishman:Alnylam: Employment. Racie:Alnylam Pharmaceutical, Inc: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Fitzgerald:Alnylam: Employment.

Treatment With Minihepcidin Peptide Improves Anemia and Iron Overload In a Mouse Model Of Thalassemia Intermedia

IntroductionBeta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). MethodsWe used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. ResultsIn animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells.At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation.Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). ConclusionMinihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures:Casu:Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Abstract 481Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia.Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to <150ug/dl in treatment group), as well as liver iron accumulation. Histopathological evaluation and Prussian’s Perl Blue staining indicated that iron was sequestered by macrophages, which led to an increase in spleen iron concentration.The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes.In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures:Guo:Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

The Effect of Dietary Iron on Tissue Iron Levels in Intact and Splenectomized Mice Affected by β-Thalassemia.

Patients with β-thalassemia hyper absorb dietary iron, most of which is stored in the liver. They also suffer from ineffective erythropoiesis (IE) which leads to hepatosplenomegaly, often requiring a splenectomy. We have been conducting a series of studies utilizing the th3/+ mouse model of thalassemia intermedia to investigate the absorption, distribution and erythroid utilization of iron. Here we focus on changes in the iron content of liver and spleen resulting from diets containing low (2.5 ppm), sufficient (35 ppm) and high (200 ppm) levels of iron, and assess the impact of splenectomy on its distribution. The high iron diet was standard rodent chow while the others were defined diets. Th3/+ mice were either bred or generated by transplantation of th3/+ hematopoietic stem cells from E14.5 fetal livers into lethally irradiated wild type (+/+) recipients. Wild type controls were similarly obtained. Splenectomy of bred and recipient mice was performed at 5 weeks of age and bone marrow transplantation (BMT) at 8 weeks. Non-transplanted mice were placed on the test diets at 8 weeks of age, and transplanted mice at 11 weeks. All animals were sacrificed after 4 weeks on the test diets, and livers and spleens harvested for determination of their iron content by atomic absorption. Group sizes ranged from 3 to 10 mice (median 7). In general, the mean organ iron content of mice fed the high iron diet was not significantly different from that of the animals fed the iron sufficient diet, while those fed the low iron diet had reduced levels of tissue iron. Over the course of the 4-week feeding study, the iron content of the livers and spleens of +/+ mice fed the 35-ppm diet increased 39% and 202%, respectively, while the corresponding values of those fed the 2.5-ppm diet were −21% and 30%. The changes in the liver and spleen of th3/+ mice were 79% and 32% (35-ppm diet) and 14% and 12% (2.5-ppm diet) compared to the values at baseline. The latter values, those at 8 weeks of age, were 1.8- and 30-fold higher in the th3/+ mice, the massive accumulation of iron in the spleen undoubtedly resulting from IE. Where iron intake (liver plus spleen) was low, it went preferentially to the spleen, undoubtedly to sustain erythropoiesis. Groups of splenectomized +/+ mice were also fed the three diets for 4 weeks. The mean iron content of their livers was similar to that of non-splenectomized animals. Similar studies of th3/+ mice are now in progress. A second set of studies is being conducted in transplanted +/+ and th3/+ mice, the goal being to determine whether or not the absorption and distribution of iron is the same as in bred animals. Again, the organ iron content of those mice fed the high iron diet was similar to that of the animals fed the iron sufficient diet. In the case of the transplanted +/+ animals fed iron sufficient diets, the mean iron contents of the livers and spleens were 64% and 186% increased after 4 weeks of feeding, values not markedly different from those of bred animals. The corresponding values on the 2.5-ppm diet were 27% and 72%, again the pattern being similar. The transplanted th3/+ animals accumulated significantly less iron in these organs than those that were bred. However, the rate at which they accumulated this iron was 10 to 20 times higher than that of the other groups studied, including the transplanted +/+ mice, perhaps reflecting a synergistic effect of BMT and IE on iron absorption. Mice fed the 35-ppm diet had only 75% and 46% as much iron in their livers and spleens, the animals fed the 2.5-ppm diet having even less (35% and 23%) while again showing preferential diversion of iron to the spleen. Splenectomizing the animals resulted in further increasing the liver iron, more that 2.5-fold in those fed the low iron diet. The hemoglobin levels of all the mice evaluated were unchanged as a result of the dietary studies, except for a 20% decrease seen in bred +/+ mice fed the low iron diet. We are currently studying splenectomized transplanted th3/+ mice as well as doing feeding studies of 5-months duration. In summary, a low iron diet has a marked effect on the iron levels of liver and spleen, which are accentuated under conditions of IE. Secondly, more iron is absorbed under conditions of IE than is needed for erythropoiesis, the excess being shuttled to the liver for storage.