Low Transferrin Receptor Research Articles

Self-enhanced targeted nanomedicines based on iron starvation acclimation for tumor-specific therapy

Transferrin receptors (TfR)-targeted drug is a promising candidate for clinical tumor-targeted therapy due to the overexpressed TfR on the tumor-cell membrane. However, the variation of TfR expression levels in different tumor types seriously challenged the therapeutic effect of TfR-targeted drugs, hindering their development and clinical translation. In this work, we designed a novel TfR-targeted nanomedicine (DFO@lipo-Tf NPs) with self-enhanced targeting ability by up-regulating the expression of TfR on tumors through iron starvation acclimation, which significantly improves the drug accumulation in the tumor. The self-enhanced targeted nanoparticles (DFO@lipo-Tf NPs) were constructed by encapsulating deferoxamine (DFO) in transferrin (Tf) modified liposome. DFO has been proven to scavenge Fe3+ in the acid tumor environment, which can lead to iron starvation in tumor cells and further induce the increase of TfR expression. We then encapsulated the chemotherapeutic drug doxorubicin (DOX) into DFO@lipo-Tf NPs to form self-enhanced targeted chemotherapeutic nanomedicine (DFO/DOX@lipo-Tf NPs), which dramatically increase in vitro and in vivo therapeutic effect to HeLa cell-line tumor with inherently low TfR expression. This conceptual strategy provides an innovative design for advanced TfR-targeted nanomedicine by breaking through the bottleneck of targeting efficiency in tumor-targeted therapy, laying a foundation for future development of TfR-targeted nanomedicines.

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs.

DMT1‐mediated endosome‐mitochondria interactions regulates iron homeostasis and mitochondrial metabolism

Mitochondria‐early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE‐mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti‐import) and high ferroportin (FPN) (pro‐export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple‐negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome‐mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE‐mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re‐expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro‐Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE‐mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE‐mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1‐mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin‐dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re‐expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re‐expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE‐mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer

Immunochemical analysis of iron transporters and M2 macrophages in ovarian endometrioma and clear cell adenocarcinoma.

The association between iron ions and endometriosis-associated ovarian cancer (EAOC) has been previously investigated to elucidate EAOC carcinogenesis; however, the dynamics of iron deposition in the endometrial epithelium and endometrial stroma of ovarian endometrioma (OE) remains unknown. The present study aimed to determine the expression of iron transporters on the cell surface and the distribution of tumor-associated macrophages (TAMs) englobed with iron in the endometrial stroma. The current retrospective study investigated 20 OE and 18 ovarian clear cell adenocarcinoma (CCC) samples, using Perls Prussian blue staining and immunohistochemistry of iron transporters, including divalent metal transporter 1 (DMT1), transferrin receptor (TfR) and ferroportin (FPN). Additionally, samples were stained for CD68, CD11c, CD163 and CD206, and double-immunostained for iron and CD163 to define the distribution of macrophages. Iron transporters were identified on the endometrial epithelium of OE and CCC tumor cells, and TAMs were englobed with iron in the endometrial stroma of OE and CCC. Histological findings revealed DMT1 upregulation in OE and CCC, whereas lower TfR and FPN expression was observed in OE than in CCC. M2 macrophages were englobed with iron ions in the deep layers of the OE and CCC stroma. The endometrial epithelium located in the endometrial stroma of one patient with OE and the endometrial epithelium adjacent to CCC in two patients with CCC stained positive for the tumor proliferation marker Ki67. Epithelium infiltrating the stroma of OE may become the origin of cancer under the influence of M2 macrophages englobed with iron. These findings provide new perspectives on the malignant transformation of OE into EAOC and its possibility as a precancerous index.

Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine.

The use of small interference RNA (siRNA) to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR) is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide) conjugated to cationic polymer branched polyethylenimine (bPEI), optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299) and low TfR expression (A549 and H460). The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP) at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

Protective effect of mitochondrial ferritin on cytosolic iron dysregulation induced by doxorubicin in HeLa cells

Doxorubicin (DOX) is an anticancer drug with cardiotoxic side effects mostly caused by iron homeostasis dysregulation. Mitochondria are involved in iron trafficking and mitochondrial ferritin (FtMt) was shown to provide protection against cellular iron imbalance. Therefore, we hypothesized that FtMt overexpression could limit DOX effects on iron homeostasis. Heart's homogenates of DOX-treated C57BL/6 mice were analyzed for cytosolic and mitochondrial iron-related proteins' expression and activity, revealing high cytosolic ferritin and ferritin-bound iron, low transferrin-receptor 1 and a strong hepcidin upregulation. Mitochondrial iron-related proteins (aconitase, succinate-dehydrogenase, frataxin) seemed, however, unaffected, although a partial inactivation of superoxide dismutase 2 was detected. Importantly, the ectopic expression of FtMt in human HeLa cells partially reverted DOX-induced iron imbalance. Our results, while confirming DOX effects on iron homeostasis, demonstrate that DOX affects more cytosolic than mitochondrial iron metabolism both in murine hearts and human HeLa cells and that FtMt overexpression is able to prevent most of these effects in HeLa cells.

Expression of curcin–transferrin receptor binding peptide fusion protein and its anti-tumor activity

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin–TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30–curcin–TfRBP9 was established. Then the recombinant vector pQE-30–curcin–TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin–TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin–TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin–TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin–TfRBP9 induced higher apoptosis rates in the HepG2 cells.

High Levels of Iron Status and Oxidative Stress in Patients with Metabolic Syndrome

Studies concerning oxidative stress (OxE) parameters have increased, mainly because of its important role in cardiovascular diseases and diabetes complications. The main objective of this study was to evaluate iron nutrition status and oxidative stress parameters in subjects that had developed metabolic syndrome (MetS). Subjects from the Research Program of Risk Factors for Cardiovascular Disease (n = 155) were studied (ages ranging from 45 to 65years old) and classified according to the Adult Treatment Panel III criterion. A blood sample was taken after a 12-h fasting period, and basal glucose, insulin, thiobarbituric acid reactive substances (TBARS), oxidized LDL (oxLDL), heme oxygenase (HO) activity, lipid profile, and iron nutrition status were determined. Eighty-five subjects were classified as MetS, and 70 non-MetS. Individuals with MetS showed higher Fe storage (high levels of ferritin, total body iron and low transferrin receptor), oxLDL, TBARS, and homeostatic model assessment for insulin resistance levels. The MetS group showed high levels of oxidative stress parameters (HO activity, oxLDL, and TBARS). The presence of MetS showed an association with LDL oxidation risk (multiple lineal regression according to sex and age, p < 0.001). High levels of triglycerides (p < 0.001) and waist circumference (p < 0.012) were associated with oxLDL levels, as well as an association between TBARS and oxLDL with ferritin levels. Through logistic regression analyses, the highest quartile of ferritin was associated with a threefold risk of developing MetS compared to the lowest quartile; also, TBARS showed a 21-fold risk for the development of MetS. Finally, elevated levels of oxidative stress parameters such us oxLDL, TBARS, HO, and Fe storage were associated to MetS.

Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams.

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.

Type 2 diabetic patients and their offspring show altered parameters of iron status, oxidative stress and genes related to mitochondrial activity

Type 2 diabetes (T2D) is directly related to alterations in iron status, oxidative stress and decreased mitochondrial activity, but the possible interaction of these parameters among T2D patients and their offspring is unclear. The whole study included 301 subjects: 77 T2D patients and one of their offspring and 51 control subjects with one of their offspring. The offspring were older than 20years old. We measured parameters of iron status (serum iron, ferritin and transferrin receptor), diabetes (pre and post-prandial glucose, insulin, lipids), oxidative stress (Heme oxygenase activity, TBARS, SOD, GSH, Vitamin E), as well as the expression of genes in blood leukocytes related to mitochondrial apopotosis (mitofusin and Bcl/Bax ratios). The offspring of T2D patients had increased levels of serum ferritin (P<0.01) and lower transferrin receptor (P<0.008); higher insulin (P<0.03) and total and LDL cholesterol; higher heme oxygenase and SOD activities increased TBARS and lower GSH; decreased mitofusin and Bcl/Bax expression ratios compared to offspring of normal subjects. These results suggest that the offspring of T2D patients could have an increased metabolic risk of develop a cardiovascular disease mediated by oxidative stress and iron status.

Inflammation is strongly associated with Plasmodium falciparum malaria and predicts erythropoietin, soluble transferrin receptor, and zinc protoporphyrin concentrations in severely anemic Zanzibari preschool children

Plasmodium falciparum malaria (Pf) contributes to severe anemia (SA) in African children, and inflammation is likely to be an important mechanism. We investigated whether Pf and inflammation predicted zinc protoporphyrin (ZPP), transferrin receptor (TfR) and erythropoietin (EPO) concentrations in a community‐based sample of 225 SA [hemoglobin (Hb) ≤ 7 g/dL] Zanzibari children (median age 13 m). Mean Hb (6.3 g/dL), ZPP (286 μmol/mol heme), serum ferritin (48 μg/L) and C‐reactive protein (CRP, 23 mg/L) suggested prevalent iron deficiency and severe inflammation. EPO was high (261 mIU/mL, normal 8–18) and appropriate for the degree of anemia. TfR was high (19 mg/L, &lt; 8) but low for the degree of anemia, suggesting dyserythropoiesis. 76% of children had Pf‐positive blood film, which was strongly correlated with CRP (p&lt;0.01). 17% had fever, which was related to Pf (p&lt;0.01) and less strongly to CRP (p=0.03). Increasing CRP quartile was associated with higher EPO (p for trend = 0.02) and lower TfR (p&lt;0.01) and ZPP (p=0.03). Increasing quartile of Pf parasitemia showed trends with EPO, TfR and ZPP that were similar in direction but less significant. In multivariate analysis with ZPP, EPO or TfR as dependent variables, Pf was not significant once CRP was added. In conclusion, inflammation strongly predicted ZPP, EPO and TfR concentrations in SA children in a manner consistent with iron sequestration and blunted bone marrow.

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

Randomized comparison of 3 types of micronutrient supplements in Ghanaian infants

Assuring adequate micronutrient status of infants is a challenge. We compared the efficacy and acceptability of “Sprinkles” (SP), “Nutritabs” (NT, a crushable tablet) and “Nutributter” (NB, a peanut based spread), when added to home-prepared complementary foods in Ghana. All products contained iron (SP 12.5 mg Fe as ferrous fumarate; NT & NB 9 mg Fe as ferrous sulfate), zinc, vitamins A and C and folic acid but differed in the content of energy and other nutrients. Infants (n = 313) were randomly assigned to receive SP, NT or NB daily from 6 to 12 mo of age. At 6 and 12 mo, infants were measured and blood samples were collected. Non-Intervention (NI) infants, who were eligible but not randomly selected for the intervention (n=96), were assessed at 12 mo only. All of the supplements were well accepted, and compliancewas similar among intervention groups. The 3 intervention groups did not differ in iron status or hemoglobin at 12 mo, but all 3 had significantly higher ferritin and lower transferrin receptor than the NI group. Mean (± SD) hemoglobin (g/L) was significantly higher in NT (112 ± 14) and NB (114 ± 14) but not in SP (110 ± 14) infants, compared to NI infants (106 ± 14). Results for other indices of micronutrient status are pending. Controlling for initial size, at 12 mo the NB group had significantly higher (effect size ~0.3) weight-for-age and length-for-age than the SP and NT groups, which were similar to the NI group. We conclude that all 3 supplements were well accepted and had a similar effect on iron status, but only NB increased infant growth. Supported by Nestle Foundation and USAID.

Markers of Iron Overload Are Poor Prognostic Factors in Stem Cell Transplantation.

Transfusional iron overload is common in survivors of acute leukemia and hematopoietic stem cell transplantation and might cause long-term liver dysfunction. Routine evaluation for iron overload in such patients is recommended because excess iron can be readily removed from the body via phlebotomy or chelation. Iron overload might be associated with worse survival after stem cell transplantation in these diseases. We were interested in determining whether levels of the iron binding protein ferritin or the serum transferrin receptor (TfR) were predictive for survival. In a prospective study, we examined the correlation between iron parameters at the time of transplantation and overall survival. Serum ferritin, transferrin saturation, and TfR were measured before preparative regimen on patients who underwent hematopoietic stem cell transplantation between 1999 and 2004 for the diagnosis of aplastic anemia, MDS or acute leukemia (n=79). We used the number of transfusions before transplantation as a measure of iron load. Among these iron markers, serum ferritin correlated the most with the number of transfusions, regardless of remission status. High ferritin (>1,500 ng/ml), low TfR (≤4 μg/ml) and low TfR/log ferritin ratio (≤2) were associated with shorter survival (p=0.005, 0.04, and 0.001 respectively)(Figure 1). Among acute leukemia patients in remission, there was no difference in overall survival between patients with high or low iron markers. Markers of iron excess (serum ferritin >1,500 ng/ml, TfR/log ferritin ratio ≤2) at the time of stem cell transplantation is associated with shorter survival in MDS, aplastic anemia and acute leukemia with active disease. These results demonstrate that knowledge of patient ferritin and TfR levels can aid in risk stratification. The results also suggest that patients with high levels of ferritin may benefit from iron chelation before treatment. [Display omitted]

Altered iron homeostasis involvement in arsenite-mediated cell transformation

Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenite-transformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation.

Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2

AbstractIron-regulatory proteins (IRP14144) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2-/- mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. Here, we report that ablation of IRP2 results in iron-limited erythropoiesis. TfR expression in erythroid precursors of IRP2-/- mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRP-dependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2-/- erythroid cells. IRP2-/- mice represent a new paradigm of genetic microcytic anemia. We postulate that IRP2 mutations or deletions may be a cause of refractory microcytic anemia and bone marrow iron depletion in patients with normal transferrin saturations, elevated serum ferritins, elevated red cell protoporphyrin IX levels, and adult-onset neurodegeneration.

High serum transferrin receptor level in anemia of chronic disorders indicates coexistent iron deficiency.

Blood transferrin receptor (TR) level is largely determined by the quantum of erythropoiesis and by intracellular iron content of the cells of the erythroid lineage. Hence, a high serum TR level has been found to be useful in distinguishing iron deficiency anemia (IDA) from anemia of chronic disorders (ACD). In order to examine its potential role in the diagnosis of concomitant iron deficiency in ACD, we determined serum TR levels in 130 cases of ACD, in 25 cases of IDA, and in 40 normal adults. As expected, all patients of IDA had significantly higher serum TR levels compared to the normal subjects (4.2-19.2 microg/dL vs. 1.3-3.0 microg/dL) (P < 0.002). In 11/25 cases of IDA, the total iron-binding capacity (TIBC) was in the normal range although bone marrow iron store was absent and serum TR levels were high, thereby highlighting the superiority of TR level in the diagnosis of iron deficiency compared to TIBC. Although 54% (70/130) patients of ACD had normal or low serum TR levels (0.9-3.0 microg/dL) as expected, in 46% (60/130) of ACD patients, serum TR levels were high (3.2-11.0 microg/dL). Mean corpuscular volume, red cell distribution width, and transferrin saturation were significantly lower (P < 0.001) in the latter group of patients compared to the former, and these parameters resembled those in IDA patients. Also, serum iron was lower and TIBC was higher in this group of ACD patients compared to those with normal or low serum TR. All these features point to an "IDA-like" profile of ACD patients with high TR and support the possibility of co-existent iron deficiency in this subgroup of ACD patients. In light of these observations it would be prudent to treat ACD patients with high serum TR levels with iron replacement therapy.

Aberrantly low transferrin receptor expression on human monocytes is associated with nonpermissiveness for Legionella pneumophila growth.

Growth of Legionella pneumophila within human monocytes is iron dependent. A person with monocytes uniquely nonpermissive to L. pneumophila growth was identified whose monocytes expressed an abnormally low number of transferrin receptors in the nonactivated state, similar to the typically low level expressed in the interferon-gamma-activated state. The monocytes failed to up-regulate transferrin receptor expression appropriately in response to iron-transferrin. After treatment for chronic periodontal disease, the subject's monocytes converted to a permissive state. In contrast to the nonpermissive state, the permissive monocytes had normal transferrin receptor expression and up-regulated transferrin receptor expression appropriately in response to iron-transferrin. Thus, a nonpermissive state for L. pneumophila intracellular multiplication is associated with low levels of transferrin receptor expression in nonactivated monocytes and with an inability to up-regulate transferrin receptor expression in response to iron-transferrin. This nonpermissive state may be related to chronic inflammatory conditions such as periodontal disease.

Anemia associated with advanced prostatic adenocarcinoma: effects of recombinant human erythropoietin.

Nine patients with hormone-refractory metastatic prostatic adenocarcinoma and anemia were treated with recombinant human erythropoietin (rHuEpo) at a median dose of 150 U/kg BW 3 times a week subcutaneously. Baseline hemoglobin (Hb) ranged from 70 to 116 g/L, and the study duration was 12 weeks (median patient participation period was 8 weeks). Four patients demonstrated a median Hb increase of 20 g/L and were considered responders. Three patients showed a median increase of 17 g/L but required blood transfusion once, and were therefore considered as partial responders. Baseline erythropoietic status showed a significant correlation between serum Epo and Hb. Inadequate Epo production, evaluated by the observed/predicted log Epo ratio, was found in two patients. Defective bone marrow activity, demonstrated by low transferrin receptor (TfR), and hypoferremia in spite of abundant iron stores were also shown. Hemorheological investigations showed elevated plasma viscosity. Our results indicate that suppression of erythropoiesis can be mainly explained by the depressed marrow activity. The altered hemorheology might contribute to the anemia. This anemia could possibly be corrected with rHuEpo.

Early Prediction of Response to Recombinant Human Erythropoietin in Patients With the Anemia of Renal Failure by Serum Transferrin Receptor and Fibrinogen

Recombinant human erythropoietin (rHuEpo) has been shown to be effective in correcting the anemia of chronic renal failure, but the dose needed may be variable. The reason for this variation is not known, but several factors could be involved, such as iron deficiency, inflammation, aluminum intoxication, hyperparathyroidism, blood losses, or marrow dysfunction. Treatment with rHuEpo was given intravenously thrice weekly after hemodialysis to 64 consecutive unselected patients with the anemia of chronic renal failure. The starting dose was 50 U/kg/dose, which was increased to 75 and 100 U/kg/dose if no response was observed after 1 and 2 months of treatment. After a minimum follow-up of 6 months, response was evaluated as early (hematocrit [Hct] > or = 30% before 3 months) or late (Hct > or = 30% after 3 months) response, or failure (target Hct not attained). We examined the value of various laboratory parameters (baseline values and early changes) as predictors of response to rHuEpo. The best prediction by pretreatment parameters only was obtained with baseline serum transferrin receptor (TfR) (< or > or = 3,500 ng/mL) and fibrinogen (< or > or = 4 g/L): 100% response rate when both parameters were low, versus only 29% when they were both high, and versus 67% when one was low and the other high. When the 2-week TfR increment was greater than 20%, the response rate was 96%. When TfR increment was less than 20%, the response rate was 100% when baseline TfR and fibrinogen were low, 12% when fibrinogen was elevated, and 62% when fibrinogen was low but baseline TfR high. The predictive value of baseline TfR and fibrinogen and of the 2-week increment of TfR was confirmed by life table analysis and stepwise discriminant analysis. Major reasons for failure or late response were identified and included subclinical inflammation, iron deficiency, functional iron deficiency, marrow disorders, hemolysis, bleeding, and low Epo dose. We conclude that response to rHuEpo can be predicted early by pretreatment fibrinogen and TfR, together with early changes of TfR levels. These prognostic factors illustrate the importance of the early erythropoietic response, subclinical inflammation, and functional iron deficiency. Early recognition of a low probability of response in a given patient could help identify and correct specific causes of treatment failure to hasten clinical improvement and avoid prolonged ineffective use of an expensive medication.