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Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies., Graphical abstract Image 1, Highlights • Disulfide bonds (DSBs) are critical to protein structure and function. • DSBs are rapidly oxidized by singlet oxygen and other oxidants to reactive intermediates. • These intermediates react with Cys-containing proteins to give new protein-protein cross-links. • This novel disulfide cross-linking pathway affects the functional activity of the proteins. • These cross-links can be diminished by reductants, but this does not repair the DSB damage.

Background Studies in chronic kidney disease populations suggest that the non–glomerular filtration rate (GFR) determinants of serum levels of the low-molecular-weight protein filtration markers cystatin C, β 2 -microglobulin (B2M), and beta-trace protein (BTP) are less affected by age, sex, and ethnicity than those of creatinine. Study Design Cross-sectional study. Setting & Participants Predominantly elderly participants selected from the Age, Gene/Environment Susceptibility Kidney Study (AGES-Kidney; N=683; mean [SD] age, 79 [4] years; GFR, 62 [17]mL/min/1.73 m 2 ) and from the Multi-Ethnic Study of Atherosclerosis Kidney Study (MESA-Kidney; N=273; mean [SD] age, 70.5 [9] years; GFR, 73 [19]mL/min/1.73 m 2 ). Predictors Demographic and clinical factors hypothesized to be associated with conditions affecting non-GFR determinants of the filtration markers. Outcomes Measured GFRs and estimated GFRs (eGFRs) based on creatinine, cystatin C, B2M, and BTP levels (eGFR cr, eGFR cys , eGFR B2M , and eGFR BTP , respectively). Residual associations of factors with eGFR after accounting for measured GFR as the parameter of interest. Results eGFR cys , eGFR B2M , and eGFR BTP had significantly less strong residual associations with age and sex than eGFR cr in both AGES-Kidney and MESA-Kidney and were not associated with ethnicity (black vs white) in MESA-Kidney. After adjusting for age, sex, and ethnicity, residual associations with most clinical factors were smaller than observed with age and sex. eGFR cys and eGFR B2M , but not eGFR BTP , had significant residual associations with C-reactive protein levels in both studies. Limitations Small sample size may limit power to detect associations. Participants may be healthier than the general population. Conclusions Similar to previous studies in chronic kidney disease, in community-dwelling elders, cystatin C, B2M, and BTP levels are less affected than creatinine level by age and sex and are not affected by ethnicity. Both cystatin C and B2M levels may be affected by inflammation. These findings are important for the development and use of GFR estimating equations based on low-molecular-weight serum proteins throughout the range in GFRs.

Objectives To determine CLLU1 gene levels and the relationship of that gene among other prognostic parameters in patients with chronic lymphocytic leukemia. Methods Bone-marrow infiltration pattern, β2-microglobulin (β 2-M), cluster of differentiation (CD)38, and ZAP-70 status were recorded. CLLU1 levels were assessed by real-time polymerase chain reaction (RT-PCR) and expressed as folds. The relationship between CLLU1 and other known prognostic parameters was evaluated. Results CLLU1 expression was positive in 81 patients and negative in 3 patients. The median (interquartile range [IQR]) CLLU1 level was 6.45 folds (3.75–16.57 folds) in patients with β 2-M normal values and 16.22 folds (3.91–62.00 folds) in patients with increased β 2-M (P = .15). Patients with a higher CD38 value than the median level had 3 times higher CLLU1 levels than the other group (P = .07). The median (IQR) CLLU1 level was 4.25 folds (2.75–13.71 folds) in patients with CLL who tested negative on ZAP-70, whereas it was 49.52 folds (15.06–446.36 folds) in those who tested positive via ZAP-70 (P = .005). Conclusions CLLU1 is a specific parameter to CLL, and its level corresponds well with the ZAP-70 level.

International audience; Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 min.

Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of beta 2-microglobulin (beta 2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of beta 2m (Delta N6 beta 2m). The crystal structure of Delta N6 beta 2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops-corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation-are unmasked and fold into a new two-stranded antiparallel beta-sheet. The beta-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular beta-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.

A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues.

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class II B loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African 'large' barb. This prompted us to study the number of MHC genes present in the genome of an African 'large' barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and II B genes. Our study revealed three beta2-microglobulin, five class Ia, four class II A, and four class II B genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.

Background β 2 ‐Microglobulin and cystatin C may have advantages over creatinine in assessing risk associated with kidney function. We therefore investigated whether emerging filtration markers, β 2 ‐microglobulin and cystatin C, are prospectively associated with risk of the development of peripheral artery disease ( PAD ). Methods and Results We conducted nested case‐control studies among women within the Nurses’ Health Study (1990–2010) and among men within the Health Professionals Follow‐up Study (1994–2008) with the use of archived blood samples collected before PAD diagnosis. During follow‐up, symptomatic PAD was confirmed in 144 women and 143 men. Controls were matched 3:1 based on age, race, smoking status, fasting status, and date of blood sampling. Conditional logistic regression models were used to estimate relative risks ( RRs ) and were adjusted for plasma creatinine and cardiovascular risk factors. In women, the RR s (95% CI) per 1‐SD) increment were 1.16 (0.85 to 1.58) for β 2 ‐microglobulin and 0.94 (0.69 to 1.28) for cystatin C. Corresponding RR s in men were 1.50 (1.08 to 2.09) for β 2 ‐microglobulin and 1.54 (1.07 to 2.22) for cystatin C. There was no association between creatinine and PAD risk in women, whereas the association in men ( RR 1.41, 95% CI 1.10 to 1.81) disappeared after adjustment for either β 2 ‐microglobulin or cystatin C. In pooled analyses of men and women, only β 2 ‐microglobulin was associated with PAD risk ( RR 1.31, 95% CI 1.04 to 1.64). Conclusions In pooled analyses, β 2 ‐microglobulin was associated with an increased risk of symptomatic PAD ; a similar association with cystatin C was observed only in men. The findings suggest that β 2 ‐microglobulin may capture the atherosclerosis‐promoting or atherosclerosis‐related elements of kidney dysfunction better than creatinine.

Hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor-alpha (r-TNF alpha) and melphalan has been shown to result in a sepsis-like syndrome due to leakage of r-TNF alpha from the perfusion system to the systemic circulation. We have studied renal function parameters in 11 cancer patients, who underwent 12 perfusions. Three patients, perfused with melphalan only, served as controls, All patients treated with r-TNF alpha developed a sepsis syndrome and needed volume replacement ansi inotropes to remain normotensive; controls had an uneventful postoperative course, Creatinine clearance decreased transiently on the day of perfusion in both groups (mean preperfusion clearance 118 ml/min, mean post-perfusion clearance 68 ml/min, p

Beta-2 microglobulin (β2m) is an essential component of the major histocompatibility complex (MHC) class I proteins and in the nervous system β2m is predominantly expressed in motor neurons. As β2m can promote nerve regeneration, we investigated its potential role in amyotrophic lateral sclerosis (ALS) by investigating its expression level as well as the effect of genetically removing β2m on the disease process in mutant superoxide dismutase 1 (SOD1 (G93A) ) mice, a model of ALS. We observed a strong upregulation of β2m in motor neurons during the disease process and ubiquitous removal of β2m dramatically shortens the disease duration indicating that β2m plays an essential and positive role during the disease process. We hypothesize that β2m contributes to plasticity that is essential for muscle reinnervation. Absence of this plasticity will lead to faster muscle denervation and counteracting this process could be a relevant therapeutic target. ispartof: Frontiers in Cellular Neuroscience vol:7 pages:249-249 ispartof: location:Switzerland status: published

International audience; The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.

A nanomechanical biosensor based on microcantilevers was implemented to test low molecular weight (small) compounds for their ability to stabilize beta(2)-microglobulin (beta(2)-m) in its native conformation. beta(2)-m was immobilized on the top face of silicon microcantilevers and it was demonstrated that pH induced unfolding of the immobilized beta(2)-m drives a specific microcantilever bending. This beta(2)-m microcantilever assay was then implemented to probe the effect of a pilot set small ligands on beta(2)-m conformational stability. Among the tested ligands, congo red was the only one able to protect beta(2)-m from unfolding, that is known to be the primary trigger of its self-polymerization into fibrils and in turn of the onset of amyloidosis. These findings disclose the high potentiality of nanomechanical sensors in the field of protein conformation related diseases, as they bring the unique advantage of directly screening compounds for their specific pharmacological activity rather than for generic binding preferences, effectively shortcutting the identification of the active ones. (C) 2012 Elsevier B. V. All rights reserved.

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P < .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P < .001), high β2-microglobulin (P < .001), unmutated variable region of immunoglobulin heavy chain gene status (P < .001), high CD38 and ζ-chain-associated protein kinase 70 expression (P < .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

Even in the era of newer and sophisticated prognostic markers, beta2- microglobulin (B2M) remains a simple but very powerful predictor of treatment-free survival (TFS) and overall survival (OS) in patients with chronic lymphocytic leukaemia (CLL). However, B2M levels are heavily influenced by the patient’s glomerular filtration rate (GFR) and this study aimed to evaluate whether GFR-adjusted B2M (GFR-B2M) had improved prognostic value compared to unadjusted B2M in a cohort of over 450 consecutive CLL patients from two separate institutions. Multivariate analysis identified a significantly shorter TFS in patients who were ZAP- 70 + (P < 0AE001), with increased GFR-B2M (P < 0AE001), and del(11q) or del(17p) as detected by fluorescence in situ hybridization (FISH; P < 0AE001). When OS was evaluated by multivariate analysis, age 65 years or older (P < 0AE001) and poor risk FISH abnormalities (P < 0AE001) had a confirmed adverse prognostic impact, but the predictive value of GFR-B2M was lost in the validation analysis. In all survival models, B2M did not attain independent significance unless GFR-B2M was eliminated from the analysis. In conclusion, GFR-B2M is a better predictor of TFS than unadjusted B2M in CLL patients.

Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37 degrees C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular beta-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (DeltaG(U-F)(H2O)). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation.

Background: Fetal serum beta(2)-microglobulin (beta M-2) has been reported as a reliable indicator of fetal infectious diseases. Objectives: To evaluate serum beta M-2 as a marker of congenital toxoplasmosis or cytomegalovirus (CMV) infection in neonates. Methods: beta M-2 was retrospectively measured in 72 neonatal serum samples from preterm neonates. Of these, 32 originated from neonates with serological evidence of congenital toxoplasmosis (n = 12) and CMV infection (n = 20), while 40 samples from neonates in which both infections were serologically excluded served as controls. beta M-2 levels were compared between the infection and control groups. Results: Mean (+/-SEM) beta M-2 levels were significantly higher in the groups of neonates infected with Toxoplosma (5.64 +/- 0.61 mg/l) (p = 0.014) and CMV (6.06 +/- 0.66 mg/l) (p lt 0.0001) than in the control group (3.80 +/- 0.2). Against the cut-off level of 5 Mg/l, beta M-2 was normal in 36 of the 40 uninfected neonates examined, indicating a specificity of 90%. In contrast, it was elevated in 66.7% (8/12) and 65% (13/20) of the Toxoplasma and CMV-infected neonates, respectively, indicating an overall sensitivity of 66%. Conclusions: In the absence of urogenital disorders, an increase in beta M-2 in neonates is likely to be infection-induced. We showed that serum beta M-2 is increased in congenital toxoplasmosis and CMV infection in the first weeks of life. Copyright (C) 2008 S. Karger AG, Basel.

beta 2-Microglobulin (beta 2m) is the light chain component of class I major histocompatibility complex (MHC-1). beta 2m is an intrinsically amyloidogenic protein that call assemble into amyloid fibrils ill vitro and ill vivo. Several recent reports Suggested that the polypeptide loop comprised between beta-strands D and E of beta 2m is important for protein stability and for the protein propensity to aggregate as amyloid fibrils. In particular. the roles of Trp60 for MHC-I assembly and beta 2m stability have been highlighted by showing that the beta 2m Trp60 -> Gly mutant is more stable and less prone to aggregation than the wild type protein. To further analyse such properties, the Trp60 -> Cys and Asp59 -> Pro beta 2m mutants have been expressed, purified, and their crystal structures determined. The stability to thermal denaturation and propensity to fibrillar aggregation have also been analysed. The experimental evidences gathered on the two mutants reinforce the hypothesis that conformational strain in the DE loop call affect beta 2m stability and amyloid aggregation properties. (C) 2008 Elsevier Inc. All rights reserved.

Human leukocyte antigen (HLA) class I molecules are expressed on the surface of all nucleated cells and present antigenic peptides to cytotoxic T cells, thereby playing an important role in initiating the cellular anti-tumor immune response. We previously reported that loss of HLA class I expression in diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS) and the testis is a common event. Loss of expression and mutations of the light chain of the HLA class I molecule, beta(2)-microglobulin (beta(2)m) have been described in a variety of human tumors and cell lines. In our study, we screened 15 DLBCL cases with a combined loss of HLA class I and beta(2)m expression for mutations in the latter gene by direct sequencing. Frame shift mutations in repetitive sequences within the beta(2)m gene leading to loss of functional beta(2)m were detected in 2 cases. Loss of heterozygosity (LOH) and fluorescent in situ hybridization (FISH) analysis for chromosome 15 exhibited loss of the remaining copy of the beta(2)m gene in both cases but also hemizygous deletions and monosomies in 6 additional cases. Since similar mutations in the beta(2)m gene have been associated with microsatellite instability (MSI), we used 8 markers to study MSI involvement in DLBCL. Low MSI was more frequent (33%) as compared to nodal DLBCL (n=15) but did not correlate with the beta(2)m mutations. Our data indicate that multiple mechanisms lead to downregulation of beta(2)m and concomitant loss of HLA class I expression in DLBCL. (C) 2002 Wiley-Liss, Inc.

Recent investigations have shown that malignant transformation may down-regulate the expression of class I HLA molecules, β2-microglobulin (β2m) and members of the antigen-processing machinery. In the present study, we HLA-genotyped and identified at a biochemical level the three (HLA-A25, -B8, -Cw7) class I alleles expressed by the previously described [D'Urso CM et al (1992) J Clin Invest 87: 284–292] β2m-defective human melanoma FO-1 cell line and tested their ability to interact with calnexin, calreticulin and the TAP (transporter associated with antigen processing) complex. All these alleles were found to bind calnexin, but not calreticulin or the poorly expressed TAP complex, both in parental and β2m-transfected FO-1 cells, demonstrating a complex defect of class I expression in FO-1 cells. In these conditions, Cw7 heavy chains interacted with calnexin more strongly than A25 and B8, and preferentially accumulated in the endoplasmic reticulum, in both a calnexin-associated and a calnexin-free form. In addition, they could be transported to the cell surface at low levels even in the absence of β2m, without undergoing terminal glycosylation. These results establish a parallel between HLA-C and the murine Db and Ld molecules which have been found to be surface expressed and functional in β2m-defective cells. They also demonstrate distinctive features of HLA-C molecules. We propose that the accumulation of several assembly intermediates of HLA-C might favour the binding of peptide antigens not readily bound by HLA-A and -B molecules in neoplastic cells with suboptimal class I expression. © 1999 Cancer Research Campaign

Hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor-alpha (r-TNF alpha) and melphalan has been shown to result in a sepsis-like syndrome due to leakage of r-TNF alpha from the perfusion system to the systemic circulation. We have studied renal function parameters in 11 cancer patients, who underwent 12 perfusions. Three patients, perfused with melphalan only, served as controls, All patients treated with r-TNF alpha developed a sepsis syndrome and needed volume replacement ansi inotropes to remain normotensive; controls had an uneventful postoperative course, Creatinine clearance decreased transiently on the day of perfusion in both groups (mean preperfusion clearance 118 ml/min, mean post-perfusion clearance 68 ml/min, p

Hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor-alpha (r-TNF alpha) and melphalan has been shown to result in a sepsis-like syndrome due to leakage of r-TNF alpha from the perfusion system to the systemic circulation. We have studied renal function parameters in 11 cancer patients, who underwent 12 perfusions. Three patients, perfused with melphalan only, served as controls, All patients treated with r-TNF alpha developed a sepsis syndrome and needed volume replacement ansi inotropes to remain normotensive; controls had an uneventful postoperative course, Creatinine clearance decreased transiently on the day of perfusion in both groups (mean preperfusion clearance 118 ml/min, mean post-perfusion clearance 68 ml/min, p

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes–macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes–macrophages with high transduction efficiency. The accumulation of 55Fe delivered by55Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of 55Fe delivered by 55Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the55Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.