The low incidence of HIV-1 infection in patients with sickle cell disease (SCD) and inhibition of HIV-1 replication in vitro under the conditions of low intracellular iron or heme treatment suggests a potential restriction of HIV-1 infection in SCD. We investigated HIV-1 ex vivo infection of SCD peripheral blood mononuclear cells (PBMCs) and found that HIV-1 replication was inhibited at the level of reverse transcription (RT) and transcription. We observed increased expression of heme and iron-regulated genes, previously shown to inhibit HIV-1, including ferroportin, IKBα, HO-1, p21, and SAM domain and HD domain-containing protein 1 (SAMHD1). HIV-1 inhibition was less pronounced in hepcidin-treated SCD PBMCs and more pronounced in the iron or iron chelators treated, suggesting a key role of iron metabolism. In SCD PBMCs, labile iron levels were reduced and protein levels of ferroportin, HIF-1α, IKBα, and HO-1 were increased. Hemin treatment induced ferroportin expression and inhibited HIV-1 in THP-1 cells, mimicking the HIV-1 inhibition in SCD PBMCs, especially as hepcidin similarly prevented HIV-1 inhibition. In THP-1 cells with knocked down ferroportin, IKBα, or HO-1 genes but not HIF-1α or p21, HIV-1 was not inhibited by hemin. Activity of SAMHD1-regulatory CDK2 was decreased, and SAMHD1 phosphorylation was reduced in SCD PBMCs and hemin-treated THP-1 cells, suggesting SAMHD1-mediated HIV-1 restriction in SCD. Our findings point to ferroportin as a trigger of HIV-1 restriction in SCD settings, linking reduced intracellular iron levels to the inhibition of CDK2 activity, reduction of SAMHD1 phosphorylation, increased IKBα expression, and inhibition of HIV-1 RT and transcription.

ciency or ferroportin Q248H had lower circulating tumor necrosis factor-α concentrations than iron-replete children with WT ferroportin, 25 led us to hypothesize that ferroportin Q248H has reduced sensitivity to hepcidin but that this property would be observed at lower concentrations of hepcidin than those used in previous studies. 23 Here, we analyzed the sensitivity of ferroportin Q248H to hepcidin at various concentrations by determining the levels of ferroportin transiently expressed in cultured cells and in human primary monocytes derived from humans with different ferroportin genotypes. We also measured ferritin concentrations in these cells, levels that are indicative of cellular iron status. Finally, we examined the effect of Q248H on serum iron measures in patients with sickle cell anemia who have markedly increased macrophage iron export because of chronic hemolysis. Design and Methods Predictive analysis and worldwide allele frequenciesMinor allele frequencies for missense mutations in the single nucleotide polymorphism database (dbSNP) were determined using Kaviar and sequence data (http://db.systemsbiology.net/kaviar). 26Mutations were analyzed for effects on protein function using a 571-residue ferroportin protein (UniProtKB/Swiss-Prot Q9NP59) and the predictive analysis tools: SIFT (Sorting Intolerant From Tolerant; http://blocks.fhcrc.org/sift/SIFT.html) 27 and PolyPhen2 (Polymorphism Phenotyping version 2; using HumDiv model; http://genetics.bwh.harvard.edu/pph2/index.shtml). 28 Cells and media293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in the presence of 5% CO 2 . Ferroportin expression constructsHuman WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants cloned with c-Myc and histidine tags in a pcDNA3.1 expression vector were kindly provided by Dr. Hal Drakesmith. 23 To generate enhanced green fluorescent protein (EGFP)-tagged human ferroportin, the ferroportin coding sequences from WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants were amplified by polymerase chain reaction (PCR) with the following primers that included XhoI and Kpn1 restriction sites (shown in italics): forward primer, GCCTC-GAGATGACCAGGGCGGGAGATCAC and reverse primer, GCGGTACCGTAACAACAGATGTATTTGCTTGATTTTC. The PCR products were purified on agarose gel, digested with XhoI and Kpn1 (BioLabs, Ipswich, MA) and ligated into the pEGFP-N1 vector (Clontech, Mountain View, CA, USA) which was also digested with XhoI and Kpn1 and ligated. The ligation products were transformed into E. coli DH5α cells (Invitrogen) and kanamycin-resistant colonies were selected. WT ferroportin-EGFP, C326Y and Q248H ferroportin -GFP-expressing plasmids were purified using a Qiagen (Valencia, CA, USA) purification kit and sequenced using the Macrogen service (Rockville, MD, USA). Transfection and treatment293T cells were seeded in 6-well ...

These results do not support recommending dietary heme or nonheme iron restrictions for HFEC282Y homozygotes diagnosed through screening in North America.

Pulmonary hypertension is a complication of sickle cell disease that is associated with increased mortality. Whether this complication is associated with hemolysis has been questioned. Systolic pulmonary artery blood pressure can be estimated from echocardiography-determined tricuspid regurgitation velocity (TRV). A velocity of 2.5 m/s or higher suggests possible pulmonary hypertension. A retrospective review of hospital records from adult patients with sickle cell disease undergoing echocardiography in 2006 and 2007 was performed at a tertiary level hospital. Echocardiographic, demographic, and clinical laboratory data were collected. Echocardiographic results were available for 105 adult sickle cell patients. Of these, 62 (59%) had a TRV ≥2.5 m/s and 24 (22.8%) had a TRV ≥3.0 m/s. Mitral valve regurgitation was observed in 44% and left ventricular abnormalities (defined by either hypertrophy or dilation) in 28% of cases. Elevated TRV had independent and significant associations with greater age, higher serum lactate dehydrogenase (LDH) concentration, and lower hemoglobin concentration. We confirmed that elevated TRV is common among hospital-based adults with sickle cell disease. Significant, independent associations were found with both elevated LDH concentration and degree of anemia, suggesting that hemolytic and other mechanisms contribute to pulmonary hypertension in patients with sickle cell disease.

; for the Hemochromatosis and Iron Overload Study Research InvestigatorsPurpose: We assessed the effectiveness of educational interventions for conveying clinical findings and information about hereditary hemochromatosis (HH) and iron overload (IO) to individuals evaluated clinically after initial screening for HH/IO with serum ferritin (SF) concentration, transferrin saturation (TS), and HFE genotyping. Methods: A questionnaire mailed to 2300 cases and controls 1 month after a letter summarizing clinical findings measured understanding of results and recommendations, knowledge of HH/IO, and satisfaction with information received. Results: Of 1622 (70.5%) participants completing relevant items, 83.6% were satisfied with receiving initial screening results by mail, 93.4% found information clear and easy to understand, 89.2% generally felt they got enough information, but 47.5% still had questions. C282Y/C282Y homozygosity with normal TS/SF predicted the best understanding of genetic results.Many with no mutations thought relatives were at risk. Iron levels created most confusion, and a third incorrectly recalled treatment recommendations. Having any abnormal result, lower education, older age, and being non-white, and/or non-English speaking predicted lower understanding. Conclusions: Combining genotypic and phenotypic screening for HH/IO creates additional difficulties in communicating results-particularly to those with low health literacy. Explaining aberrant iron TS and SF levels and low-risk genotypes, follow-up recommendations, and risk to relatives will need creative, culturally appropriate strategies. Genet Med 2007:9(11):778 -791. Key Words: genetic counseling, genetics education, health literacy, hemochromatosis screening, HFE geneHereditary hemochromatosis (HH) is a common disorder that causes some affected individuals to develop iron overload (IO) because of increased intestinal absorption of iron. This may lead to serious complications including hepatic cirrhosis, liver cancer, diabetes mellitus, cardiomyopathy, arthropathy, endocrinopathy, and a shortened lifespan. 1,2 Early detection can prevent development of IO in susceptible individuals, and therapeutic phlebotomy can reverse both the IO and some of its complications in those already symptomatic. 3 Because of these effective interventions and the fact that HH occurs in 0.3-0.5% of whites of Western European descent, it is a candidate for population-based screening. 4 The success of such a screening program depends not only on correctly identifying those who are at risk, but also on ensuring that those who screen positive receive additional diagnostic assessment, understand the potential manifestations of the condition, learn how symptoms can be prevented or treated, and notify others in their family who could be at risk. Because misunderstandings may diminish the benefits of screening, it is important to determine the efficacy of patient education and to see if it is affected by factors such as ethnicity, preferred language, educational level, ag...