Plasma iron circulates bound to transferrin (Trf), which solubilizes the ferric ion and attenuates its reactivity. Diferric Trf interacts with cell-surface Trf receptor (Trfr) to undergo receptor-mediated endocytosis into specialized endosomes. Endosomal acidification leads to iron release, and iron is transported out of the endosome through the activity of divalent metal transporter 1 (DMT1, formerly Nramp2), a transmembrane iron transporter that functions only at low pH. Trf and Trfr then return to the cell surface for reuse, completing a highly efficient cycle. Although the Trf cycle is assumed to be the general mechanism for cellular iron uptake, this has not been validated experimentally. Mice with hypotransferrinaemia (hpx) have little or no plasma Trf. They have severe anaemia, indicating that the Trf cycle is essential for iron uptake by erythroid cells. Other hpx tissues, however, are generally normal, and there is a paradoxical increase in intestinal iron absorption and iron storage. To test the hypothesis that the Trf cycle has unique importance for erythropoiesis, we disrupted the Trfr gene in mice. This results in elimination of the Trf cycle, but leaves other Trf functions intact. Mice lacking Trfr have a more severe phenotype than hpx mice, affecting both erythropoiesis and neurologic development. Furthermore, haploinsufficiency for Trfr results in impaired erythroid development and abnormal iron homeostasis.

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Objective-Intraplaque hemorrhage increases the risk of plaque rupture and thrombosis. The release of hemoglobin (Hb) from extravasated erythrocytes at the site of hemorrhage leads to iron deposition, which may increase oxidation and inflammation in the atherosclerotic plaque. The haptoglobin (Hp) protein is critical for protection against Hb-induced injury. Two common alleles exist at the Hp locus and the Hp 2 allele has been associated with increased risk of myocardial infarction. We have demonstrated decreased anti-oxidative and anti-inflammatory activity for the Hp 2 protein. We tested the hypothesis that the Hp 2-2 genotype is associated with increased oxidative and macrophage accumulation in atherosclerotic plaques. Methods and Results-The murine Hp gene is a type 1 Hp allele. We created a murine type 2 Hp allele and targeted its insertion to the Hp locus by homologous recombination. Atherosclerotic plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 2-2 mice were associated with increased iron (Pϭ0.008), lipid peroxidation (4-hydroxynonenal and ceroid) and macrophage accumulation (Pϭ0.03) as compared with plaques from C57Bl/6 ApoE Ϫ/Ϫ Hp 1-1 mice. Conclusions-Increased iron, lipid peroxidation and macrophage accumulation in ApoEϪ/Ϫ Hp 2-2 plaques suggests that the Hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque hemorrhage. Key Words: atherosclerotic plaque Ⅲ hemoglobin Ⅲ inflammation Ⅲ iron Ⅲ macrophages T he major cause of acute coronary thrombosis is atherosclerotic plaque rupture and the precursor lesion has been termed the high-risk plaque. [1][2][3][4][5][6] Pathological features of highrisk plaques include a large lipid necrotic core, thin fibrous cap, inflammatory infiltrate, and intraplaque hemorrhage. [1][2][3][4][5][6] Extracorpuscular hemoglobin (Hb) released from red blood cells after intra-plaque hemorrhage represents a potent stimulus for inflammation within the plaque. It is becoming apparent that the frequency of microvascular hemorrhages has been severely underestimated and may occur in up to 40% of all advanced atherosclerotic plaques. 7 An important defense mechanism to counteract the effects of intra-plaque hemorrhage is mediated by haptoglobin (Hp), an abundant serum protein whose primary function is to bind to extracorpuscular Hb, thereby attenuating its oxidative and inflammatory potential. 8 Hp also promotes the clearance of extracorpuscular Hb via the CD163 scavenger receptor present on macrophages. 9 This scavenging pathway is the only mechanism that exists for removing free Hb released at extravascular sites, ie, at sites of hemorrhage within the atherosclerotic plaque.In humans there exist 2 classes of alleles for Hp, designated 1 and 2. The Hp polymorphism is a common polymorphism. In the western world, 16% of the population is Hp 1-1 (homozygous for the Hp 1 allele), 36% is Hp 2-2 (homozygous for the Hp 2 allele), and 48% is Hp 2-1 (heterozygote). 8 The Hp 2 allele is found only in humans. All other mammals, including higher primates have only th...

Chronic granulomatous disease (CGD) is an innate immunodeficiency with a genetic defect of the nicotinamide adenosine dinucleotide phosphate, reduced, oxidase components. This leads to decreased reactive oxygen species (ROS) production, which renders patients susceptible to life‐threatening infections. Over the course of 30 years, we diagnosed CGD in 84 patients from 61 families using functional, molecular, and genetic studies. The incidence of CGD in Israel is 1.05 per 100,000 live‐births in the Jewish population and 1.49 in the Israeli Arab population. We diagnosed 52 patients (62%) with autosomal recessive inheritance (AR‐CGD) and 32 (38%) with X‐linked recessive inheritance (XLR‐CGD). Consanguinity was detected in 64% of AR‐CGD families (14% in Jews and 50% in Israeli Arabs). We found 36 different mutations (23 in XLR‐CGD and 13 in AR‐CGD patients), 15 of which were new. The clinical spectrum of CGD varied from mild to severe disease in both XLR and AR forms, although the AR subtype is generally milder. Further, residual ROS production correlated with milder clinical expression, better prognosis and improved overall survival. Patients with recurrent pyogenic infections developed fibrosis and hyperinflammatory states with granuloma formation. The management of CGD has progressed substantially in recent years, evolving from a fatal disease of early childhood to one of long‐term survival. Our present cohort displays an encouraging 81% overall long term survival. Early hematopoietic stem cell transplantation is advisable before tissue damage is irreversible. Successful transplantation was performed in 18/21 patients. Therapeutic gene modification could become an alternative cure for CGD. Am. J. Hematol. 92:28–36, 2017. © 2016 Wiley Periodicals, Inc.

Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2.

Targeted mutagenesis was used to produce two mutations in the murine hemochromatosis gene (Hfe) locus. The first mutation deletes a large portion of the coding sequence, generating a null allele. The second mutation introduces a missense mutation (C282Y) into theHfe locus, but otherwise leaves the gene intact. This mutation is identical to the disease-causing mutation in patients with hereditary hemochromatosis. Mice carrying each of the two mutations were bred and analyzed. Homozygosity for either mutation results in postnatal iron loading. The effects of the null mutation are more severe than the effects of the C282Y mutation. Mice heterozygous for either mutation accumulate more iron than normal controls. Interestingly, although liver iron stores are greatly increased, splenic iron is decreased. We conclude that the C282Y mutation does not result in a null allele.

Hereditary hemochromatosis is most commonly caused by homozygosity for a point mutation (C282Y) in the human hemochromatosis gene (HFE). The mechanism by which HFE regulates iron absorption is not known, but the C282Y mutation results in loss of cell surface expression of the human hemachromatosis protein (HFE) and hyperabsorption of iron by the duodenal enterocyte. Mice homozygous for a deletion in the mouse hemochromatosis gene (Hfe) or a mutation equivalent to that seen in human hereditary hemochromatosis IntroductionHereditary hemochromatosis is one of the most common inherited disorders of whites, affecting nearly 5 per 1000 people of Northern European extraction. 1 The gene mutated in hereditary hemochromatosis, the human hemochromatosis gene (HFE), is a major histocompatibility complex (MHC) class I-like gene. 2 The HFE mutation most frequently found in hereditary hemochromatosis results in a cysteine-to-tyrosine conversion at amino acid 282 in a region of the human hemachromatosis protein (HFE) corresponding to the ␣3 domain in class I proteins. This mutation results in the loss of interaction with ␤ 2 -microglobulin and decreased cell surface expression of HFE. 3 The HFE-␤ 2 microglobulin heterodimer interacts with the transferrin receptor (TfR), resulting in a conformational change causing a reduction in the affinity of the receptor for diferric transferrin (Tf (Fe) 2 ). 4-6 Despite increasing information regarding HFE structure and expression, 7,8 the mechanism by which HFE regulates iron absorption remains undefined.Iron absorption is mediated by the duodenal enterocyte. Absorption is affected by the rate at which iron is imported from the gut lumen into the enterocyte and the rate at which it is exported to the plasma. An importer (natural resistance-associated macrophage protein 2 (Nramp2), divalent cation transporter 1 (DCT1), divalent metal transporter 1 (DMT1)) and an exporter (ferroportin1, ironregulated transporter 1 (Ireg1), metal transporter protein 1 (MTP1)) in enterocytes have been identified, but whether or not HFE directly affects the activity of these iron transporters is not known. 9-13 The murine hemochromatosis gene (Hfe) has been altered by recombination to produce both knockout mice and mice homozygous for the C282Y mutation (knockin). 14,15 Both mutant strains develop iron overload with aging and serve as useful models for hemochromatosis. We used Hfe knockout and knockin mice to determine the contribution of mouse hemachromatosis protein (Hfe) to the regulation of intestinal iron absorption in iron-replete, iron-loaded, and iron-deficient states and in mice with stimulated erythropoiesis. Materials and methods Mice and dietsAll mice used in these experiments were of the 129/SvEvTac strain. 15 Mice were maintained in the Animal Resource Center at the University of Utah following approved guidelines. For dietary studies, mice were maintained on a standard rodent diet (Harlan Teklad TD 8640) containing approximately 0.33 g of iron per kilogram, a diet supplemented with 20 g carbony...

Abstract-Two common alleles exist at the haptoglobin (Hp) locus, and the Hp2 allele is associated with an increased incidence of cardiovascular disease, specifically in diabetes mellitus (DM). Oxidative stress is increased in Hp2 mice and humans with DM. Oxidative modification of the apolipoprotein A-I inhibits reverse cholesterol transport. We sought to test the hypothesis that reverse cholesterol transport is impaired in Hp2 DM mice and humans. In vitro, using serum from non-DM and DM individuals, we measured cholesterol efflux from 3 H-cholesterol-labeled macrophages. In vivo, we injected 3 H-cholesterol-loaded macrophages intraperitoneally into non-DM and DM mice with the Hp1-1 or Hp2-2 genotype and monitored 3 H-tracer levels in plasma, liver, and feces. In vitro, in DM individuals only, we observed significantly decreased cholesterol efflux from macrophages incubated with serum from Hp2-1 or Hp2-2 as compared with Hp1-1 individuals (PϽ0.01). The interaction between Hp type and DM was recapitulated using purified Hp and glycated Hb. In vivo, DM mice loaded with 3 H-cholesterol-labeled macrophages had a 40% reduction in 3 H-cholesterol in plasma, liver, and feces as compared with non-DM mice (PϽ0.01). The reduction in reverse cholesterol transport associated with DM was significantly greater in Hp2-2 mice as compared with Hp1-1 mice (54% versus 25% in plasma; 52% versus 27% in liver; 57% versus 32% in feces; PϽ0.03). reverse cholesterol transport is decreased in Hp2-2 DM. This may explain in part the increased atherosclerotic burden found in Hp2-2 DM individuals.