Transferrin Receptor Type Research Articles

Viral Capsid, Antibody, and Receptor Interactions: Experimental Analysis of the Antibody Escape Evolution of Canine Parvovirus.

Canine parvovirus (CPV) is a small nonenveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late 1970s due to a host range switch of a virus similar to the feline panleukopenia virus that infected another host. The virus that emerged in dogs had altered capsid receptor and antibody binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we used in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bound two distinct epitopes, and one largely overlapped the host receptor binding site. We also generated mutated antibody variants with altered binding structures. Viruses were passaged with wild-type (WT) or mutated antibodies, and their genomes were deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the transferrin receptor type 1 binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we used an in vitro model system and deep genome sequencing to reveal the mutations that arose in the virus genome during selection by each of two monoclonal antibodies or their mutated variants. High-resolution structures of each of the Fab:capsid complexes revealed their binding interactions. The wild-type antibodies or their mutated variants allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and they likely have parallels for many other viruses.

A farewell to phlebotomy\u2014use of placenta-derived drugs Laennec and Porcine for improving hereditary hemochromatosis without phlebotomy: a case report

BackgroundHuman hepcidin, produced by hepatocytes, regulates intestinal iron absorption, iron recycling by macrophages, and iron release from hepatic storage. Recent studies indicate that hepcidin deficiency is the underlying cause of the most known form of hereditary hemochromatosis.Case presentationA 44-year-old Asian man who developed type 2 diabetes mellitus had elevated serum ferritin levels (10,191 ng/mL). Liver biopsy revealed remarkable iron deposition in the hepatocytes and relatively advanced fibrosis (F3). Chromosomal analysis confirmed the presence of transferrin receptor type 2 mutations (c.1100T>G, c.2008_9delAC, hereditary hemochromatosis type 3 analyzed by Kawabata). The patient received intravenous infusions of Laennec (672 mg/day, three times/week) or oral administration with Porcine (3.87 g/day) for 84 months as an alternative to repeated phlebotomy. At the end of the treatment period, serum ferritin level decreased to 428.4 ng/mL (below the baseline level of 536.8 ng/mL). Hemoglobin A1c levels also improved after treatment with the same or lower dose of insulin (8.8% before versus 6.8% after). Plural liver biopsies revealed remarkable improvements in the grade of iron deposition and fibrosis (F3 before versus F1 after) of the liver tissue.ConclusionThe discovery of hepcidin and its role in iron metabolism could lead to novel therapies for hereditary hemochromatosis. Laennec (parenteral) and Porcine (oral), which act as hepcidin inducers, actually improved iron overload in this hereditary hemochromatosis patient, without utilizing sequential phlebotomy. This suggests the possibility of not only improving the prognosis of hereditary hemochromatosis (types 1, 2, and 3) but also ameliorating complications, such as type 2 diabetes, liver fibrosis, and hypogonadism. Laennec and Porcine can completely replace continuous venesection in patients with venesection and may improve other iron-overloading disorders caused by hepcidin deficiency.

Evaluation of the Association of Transferrin Receptor Type 2 Gene Mutation (Y250X) with Iron Overload in Major β- Thalassemia.

Thalassemia is an inherited blood disorder in which the body produces defective hemoglobin. One of the important processes to reduce the complication of major β-thalassemia is blood transfusion that leads to elevated ferritin levels in the blood. Many patients who have major β-thalassemia may have hemochromatosis conditions resulting from iron metabolism disorders. In patients who have β-thalassemia, the mutation Y250X in the TFR2 gene may play a role in the incidence of hemochromatosis. This study aimed to determine the relationship between ferritin levels and Y250X mutation in major β-thalassemia patients. In the present study, 12 blood samples were divided into nine major β-thalassemia patients and three healthy controls. The DNA was isolated from blood samples and the amplification of the target region was performed based on the specific primers. Sanger sequencing was used to find genetic single nucleotide polymorphisms associated with iron overload. Blood parameters, such as hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and serum ferritin levels were analyzed and the recorded data showed the following results: 8.1±0.8 g/dL, 84.6±5.5 fL, 27±0.7 pg, respectively. The recorded data showed that the mean serum ferritin level in major β-thalassemia patients was 1921.7±848 ng/mL. The Y250X mutation was not found in major β-thalassemia patients and healthy controls.

Myotonic dystrophy kinase-related CDC42-binding kinase α, a new transferrin receptor type 2-binding partner, is a regulator of erythropoiesis.

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.

Transferrin Receptors in Erythropoiesis.

Erythropoiesis is a highly dynamic process giving rise to red blood cells from hematopoietic stem cells present in the bone marrow. Red blood cells transport oxygen to tissues thanks to the hemoglobin comprised of α- and β-globin chains and of iron-containing hemes. Erythropoiesis is the most iron-consuming process to support hemoglobin production. Iron delivery is mediated via transferrin internalization by the endocytosis of transferrin receptor type 1 (TFR1), one of the most abundant membrane proteins of erythroblasts. A second transferrin receptor—TFR2—associates with the erythropoietin receptor and has been implicated in the regulation of erythropoiesis. In erythroblasts, both transferrin receptors adopt peculiarities such as an erythroid-specific regulation of TFR1 and a trafficking pathway reliant on TFR2 for iron. This review reports both trafficking and signaling functions of these receptors and reassesses the debated role of TFR2 in erythropoiesis in the light of recent findings. Potential therapeutic uses targeting the transferrin-TFR1 axis or TFR2 in hematological disorders are also discussed.

Transferrin receptor binds virus capsid with dynamic motion

Canine parvovirus (CPV) is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Cross-species transmission of CPV occurs as a result of mutations on the viral capsid surface that alter the species-specific binding to the host receptor, transferrin receptor type-1 (TfR). The interaction between CPV and TfR has been extensively studied, and previous analyses have suggested that the CPV-TfR complex is asymmetric. To enhance the understanding of the underlying molecular mechanisms, we determined the CPV-TfR interaction using cryo-electron microscopy to solve the icosahedral (3.0-Å resolution) and asymmetric (5.0-Å resolution) complex structures. Structural analyses revealed conformational variations of the TfR molecules relative to the binding site, which translated into dynamic molecular interactions between CPV and TfR. The precise footprint of the receptor on the virus capsid was identified, along with the identity of the amino acid residues in the virus-receptor interface. Our "rock-and-roll" model provides an explanation for previous findings and gives insights into species jumping and the variation in host ranges associated with new pandemics in dogs.

Blood-Brain Barrier Transport, Plasma Pharmacokinetics, and Neuropathology Following Chronic Treatment of the Rhesus Monkey with a Brain Penetrating Humanized Monoclonal Antibody Against the Human Transferrin Receptor.

A monoclonal antibody (mAb) against the blood-brain barrier (BBB) transferrin receptor (TfR) is a potential agent for delivery of biologic drugs to the brain across the BBB. However, to date, no TfRMAb has been tested with chronic dosing in a primate model. A humanized TfRMAb against the human (h) TfR1, which cross reacts with the primate TfR, was genetically engineered with high affinity (ED50 = 0.18 ± 0.04 nM) for the human TfR type 1 (TfR1). For acute dosing, the hTfRMAb was tritiated and injected intravenously (IV) in the Rhesus monkey, which confirmed rapid delivery of the humanized hTfRMAb into both brain parenchyma, via transport across the BBB, and into cerebrospinal fluid (CSF), via transport across the choroid plexus. For chronic dosing, a total of 8 adult Rhesus monkeys (4 males, 4 females) were treated twice weekly for 4 weeks with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb via a 60 min IV infusion for a total of 8 doses prior to euthanasia and microscopic examination of brain and peripheral organs. A pharmacokinetics analysis showed the plasma clearance of the hTfRMAb in the primate was nonlinear, and plasma clearance was increased over 20-fold with chronic treatment of the low dose, 3 mg/kg, of the antibody. Chronic treatment of the primates with the 30 mg/kg dose caused anemia associated with suppressed blood reticulocytes. Immunohistochemistry of terminal brain tissue showed microglia activation, based on enhanced IBA1 immuno-staining, in conjunction with astrogliosis, based on increased GFAP immuno-staining. Moderate axonal/myelin degeneration was observed in the sciatic nerve. Further studies need to be conducted to determine if this neuropathology is induced by the antibody effector function, or is an intrinsic property of targeting the TfR in brain. The results indicate that chronic treatment of Rhesus monkeys with a humanized hTfRMAb may have a narrow therapeutic index, with associated toxicity related to microglial activation and astrogliosis of the brain.

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range.

Antibody and receptor binding are key virus-host interactions that control host range and determine the success of infection. Canine and feline parvovirus capsids bind the transferrin receptor type 1 (TfR) to enter host cells, and specific structural interactions appear necessary to prepare the stable capsids for infection. Here, we define the details of binding, competition, and occupancy of wild-type and mutant parvovirus capsids with purified receptors and antibodies. TfR-capsid binding interactions depended on the TfR species and varied widely, with no direct relationship between binding affinity and infection. Capsids bound feline, raccoon, and black-backed jackal TfRs at high affinity but barely bound canine TfRs, which mediated infection efficiently. TfRs from different species also occupied capsids to different levels, with an estimated 1 to 2 feline TfRs but 12 black-backed jackal TfRs binding each capsid. Multiple alanine substitutions within loop 1 on the capsid surface reduced TfR binding but substitutions within loop 3 did not, suggesting that loop 1 directly engaged the TfR and loop 3 sterically affected that interaction. Binding and competition between different TfRs and/or antibodies showed complex relationships. Both antibodies 14 and E competed capsids off TfRs, but antibody E could also compete capsids off itself and antibody 14, likely by inducing capsid structural changes. In some cases, the initial TfR or antibody binding event affected subsequent TfR binding, suggesting that capsid structure changes occur after TfR or antibody binding and may impact infection. This shows that precise, host-specific TfR-capsid interactions, beyond simple attachment, are important for successful infection.IMPORTANCE Host receptor binding is a key step during viral infection and may control both infection and host range. In addition to binding, some viruses require specific interactions with host receptors in order to infect, and anti-capsid antibodies can potentially disrupt these interactions, leading to neutralization. Here, we examine the interactions between parvovirus capsids, the receptors from different hosts, and anti-capsid antibodies. We show that interactions between parvovirus capsids and host-specific TfRs vary in both affinity and in the numbers of receptors bound, with complex effects on infection. In addition, antibodies binding to two sites on the capsids had different effects on TfR-capsid binding. These experiments confirm that receptor and antibody binding to parvovirus capsids are complex processes, and the infection outcome is not determined simply by the affinity of attachment.

Both iron excess and iron depletion impair viability of rat H9C2 cardiomyocytes and L6G8C5 myocytes.

Iron is presumed to play an important role in the functioning of cardiomyocytes and skeletal myocytes. There is scarcity of direct data characterising the cells functioning when exposed to iron depletion or iron overload in a cellular environment. There is some clinical evidence demonstrating that iron deficiency has serious negative prognostic consequences in heart failure (HF) patients and its correction brought clinical benefit. The viability of the cells upon unfavourable iron concentration in the cell culture medium and the presence of the molecular system of proteins involved in intracellular iron metabolism in these cells have been studied. H9C2 rat adult cardiomyocytes and L6G8C5 rat adult skeletal myocytes were cultured for 24 h in optimal vs. reduced vs. increased iron concentrations. Intracellular iron content was measured by flame atomic absorption spectroscopy (FAAS). We analysed the mRNA expression of: ferritin heavy and light chains (FTH and FTL; iron storage proteins), myoglobin (MB, oxygen storage protein) ferroportin type 1 (FPN1; iron exporter), transferrin receptor type 1 (TfR1; iron importer), hepcidin (HAMP; iron metabolism regulator) using qPCR, the level of respective proteins using Western Blot (WB), and the viability of the cells using flow cytometry and cell viability tetrazolium reduction assay (MTS). Cardiomyocytes exposed to gradually reduced iron concentrations in the medium demonstrated a decrease in the mRNA expression of FTH, FTL, FPN1, MB, and HAMP (all R = -0.75, p < 0.05), indicating depleted iron status in the cells. As a consequence, the expression of TfR1 (R = 0.7, p < 0.05) was increased, reflecting a facilitated entrance of iron to the cells. The inverse changes occurred in H9C2 cells exposed to increased iron concentrations in the medium in comparison to control cells. The same pattern of changes in the mRNA expressions was observed in myocytes, and there was a strong correlation between analogous genes in both cell lines (all R > 0.9, p < 0.0001). WB analysis revealed the analogous pattern of changes in protein expression as an mRNA profile. Both iron depletion and iron excess impaired viability of cardiomyocytes and skeletal myocytes. Both rat cardiomyocytes and myocyte cells contain the set of genes involved in the intracellular iron metabolism, and both types of investigated cells respond to changing iron concentrations in the cultured environment. Both iron deficiency (ID) and iron overload is detrimental for the cells. This data may explain the beneficial effects of iron supplementation in patients with ID in HF.

Trasferrin receptor 2 gene regulation by microRNA 221 in SH-SY5Y cells treated with MPP+ as Parkinson's disease cellular model

Parkinson's disease (PD) is one of the most frequent human neurodegenerations. The neurodegeneration in PD is related to cellular iron increase but the mechanisms involved in iron accumulation remain unclear. Transferrin receptor type 2 (TFR2) is a protein expressed on cell membrane and involved in the cellular iron uptake. We hypothesized that microRNA 221 could regulate the expression of TfR2 in an in vitro model of Parkinson's disease, SH-SY5Y cells treated with MPP+.The miRNA 221 was selected by in silico analysis of several miRNAs predicted to target the TFR2 gene in SHSY5Y cells treated with MPP+. Taqman miRNA assay was used to evaluate the expression of the selected miRNAs. Using a luciferase assay we demonstrated the inhibition of TFR2 by miRNA 221.We show that in PD cellular model, TFR2 expression is regulated by miRNA 221. TFR2 and miR 221 are inversely correlated in SHSY5Y cells during the treatment with MPP+. Moreover, overexpression of miRNA 221 decreases the expression of TFR2, respectively, at the mRNA and protein levels. The inhibition of endogenous miRNA 221 also is able to regulate TFR2.These data suggest that miRNA 221 regulate TFR2 in PD model.

Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid

Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells.

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.

Role of Multiple Hosts in the Cross-Species Transmission and Emergence of a Pandemic Parvovirus

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Substitutions in the Glycoprotein (GP) of the Candid#1 Vaccine Strain of Junin Virus Increase Dependence on Human Transferrin Receptor 1 for Entry and Destabilize the Metastable Conformation of GP

Candid#1 (Cd1) is an attenuated vaccine strain of Junin virus, the causative agent of Argentine hemorrhagic fever. Although several substitutions are present in Cd1, their importance for attenuation has not been established. We functionally characterized the substitutions present in the Cd1 glycoprotein (GP) and identified F427I in the transmembrane domain of the GP2 subunit as reducing infectivity in a reconstituted viral system. We further showed that this phenotype derives from the destabilization of the GP metastable conformation. Lastly, we identified an increased dependence of Cd1 GP on human transferrin receptor type 1 (hTfR-1) for entry, which may affect the tropism of the attenuated strain in vivo.

Hemojuvelin and Hepcidin Gene Mutations in Patients with Porphyria Cutanea Tarda from Southern France

Porphyria cutanea tarda (PCT), the most frequent subset of porphyria in western countries, is related to decreased activity of one of the key enzymes of haem synthesis, uroporphyrinogen decarboxylase (UROD) (1). Several well-known, often combined, factors may lead to overt PCT, including hepatitis C (HCV) infection, exposure to hepatotoxic drugs, alcohol abuse and liver iron overload. This last factor's pathological mechanisms in PCT may result partly from mutations affecting the various genes involved in the regulation of iron metabolism. Mutations in the HFE gene (C282Y and H63D) have been identified in PCT at an allele frequency up to 30% in several studies (1-4). Other studies have investigated additional genes involved in iron metabolism, namely the transferrin-receptor type 2 (TFR2) or the cytochrome P450 (CYP1A2), but results have remained inconclusive to date (5, 6). Mutations of other genes involved in iron metabolism should also be considered. Recently, new relevant biochemical actors have been identified in rare subsets of hereditary hemochromatosis (HH), i.e. the hemojuvelin (HJV) and hepcidin (HAMP) genes in juvenile haemochromatosis (JH) (7). Hemojuvelin protein has clearly been involved in the regulation of the expression of hepcidin, a key protein of iron metabolism also involved in JH (8). Hepcidin is an antimicrobial peptide that indirectly decreases intestinal iron absorption (9). To investigate the possible involvement in PCT of deleterious mutations of these two genes and their possible relationship with iron overload, a retrospective study was designed targeting 35 PCT patients from southern France.

Binding Site on the Transferrin Receptor for the Parvovirus Capsid and Effects of Altered Affinity on Cell Uptake and Infection

Canine parvovirus (CPV) and its relative feline panleukopenia virus (FPV) bind the transferrin receptor type 1 (TfR) to infect their host cells but show differences in the interactions with the feline and canine TfRs that determine viral host range and tissue tropism. We changed apical and protease-like domain residues by introducing point mutations and adding or removing glycosylation signals, and we then examined the interactions of those mutant TfRs with the capsids. Most substitutions had little effect on virus binding and uptake. However, mutations of several sites in the apical domain of the receptor either prevented binding to the capsids or reduced the affinity of receptor binding to various degrees. Glycans within the virus binding face of the apical domain also controlled capsid binding. CPV, but not the related feline parvovirus, could use receptors containing a canine TfR-specific glycosylation to mediate efficient infection, while addition of other N-linked glycosylation sites into the virus binding face of the feline apical domain reduced or eliminated both binding and infection. Replacement of critical feline TfR residue 221 with every amino acid had effects on binding and infection which were significantly associated with the biochemical properties of the residue replaced. Receptors with reduced affinities mostly showed proportional changes in their ability to mediate infection. Testing feline TfR variants for their binding and uptake patterns in cells showed that low-affinity versions bound fewer capsids and also differed in attachment to the cell surface and filopodia, but transport to the perinuclear endosome was similar.

Commentary [Research Highlights

Drug delivery to the central nervous system remains a vexing problem because of the presence of the blood-brain barrier (BBB), whose endothelial cell tight junctions limit the paracellular flux of hydrophilic molecules. One solution may be vector-mediated delivery to the brain. This employs chimeric peptide technology, wherein the drug is conjugated to a molecular carrier of protein nature (e.g. cationized albumin or a transferrin receptor antibody). Transferrin receptor antibodies selectively target BBB endothelium due to the high levels of receptor expressed by these cells, thereby triggering receptor-mediated transport across the BBB via transcytosis. Chitosan, a naturally occurring, abundant, and biocompatible polysaccharide has found widespread pharmaceutical application. In particular, the cationic nature of chitosan facilitates its interaction with negative charges on the brain endothelium, Building on their previous work on brain-deliverable chitosan-polyethylene glycol nanoparticles functionalized with monoclonal anti-transferrin antibody, Karatas and colleagues have now designed caspase-3 inhibitor-loaded chitosan nanospheres conjugated with an anti-mouse transferrin receptor monoclonal antibody that selectively recognizes the transferrin receptor type 1 on the cerebral vasculature. Caspase-3 has been implicated in neuronal cell death in cerebral ischemia, as well as in the pathophysiology of other neurological disorders. When given intravenously, the caspase-3-loaded nanospheres were rapidly transported across the BBB and dose-dependently decreased infarct volume, neurological deficit, and ischemiainduced caspase-3 activity in mice subjected to focal cerebral ischemia/reperfusion. Because significant opening of the BBB starts only 6 h after ischemia/reperfusion, the rapid transfer of nanospheres to the brain was not likely caused by an increased permeability of the BBB. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17, after closure of the BBB. These observations convincingly demonstrate that transferrin monoclonal antibody-conjugated chitosan nanospheres are an efficient delivery system for bringing neuroprotective, small peptide drugs into the brain. This opens intriguing new avenues for the treatment of central nervous system disorders in which the administration of therapeutic agents is handicapped by insufficient diffusion across the BBB. Further studies are eagerly awaited to see if this technology will be applicable to polypeptide factors, for example, brain-derived neurotrophic factor and ciliary neurotrophic factor.

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.

Suppressive Effects of Retinoids on Iron-Induced Oxidative Stress in the Liver

We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all-trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor alpha gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.

Transferrin fails to provide protection against Fas-induced hepatic injury in mice with deletion of functional transferrin-receptor type 2

We reported previously that Fas-induced hepatic failure in normal mice was attenuated or prevented by exogenous transferrin (Tf), particularly apoTf. Here we show in C57BL6J/129 mice with genetic inactivation of transferrin receptor 2 (TfR2(Y245X)), that Fas-induced hepatotoxicity (apoptosis; rise in plasma aspartate aminotransferase (AST) levels) was comparable to that in wild-type mice, but was not modified by pretreatment with Tf. Rises in plasma AST were preceded by a decline in serum iron levels. AST elevations and iron declines were more profound in female than in male mice. Female mice also showed higher baseline levels of Bcl-xL in hepatocytes, which declined significantly upon treatment with agonistic anti-Fas antibody. These data confirm the cytoprotective function of Tf, and show a novel property of TfR2. Both apoptotic Fas responses and cytoprotective effects of Tf were associated with significant shifts in plasma iron levels, which quantitatively differed between male and female mice.