Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1125-1129 ◽  
Author(s):  
Giuliana Montosi ◽  
Paola Paglia ◽  
Cinzia Garuti ◽  
Carlos A. Guzman ◽  
Judy M. Bastin ◽  
...  
Keyword(s):  
Single Gene ◽  
Hereditary Hemochromatosis ◽  
Phenotypic Expression ◽  
Hereditary Diseases ◽  
Transduction Efficiency ◽  
Hfe Gene ◽  
Wild Type ◽  
Iron Deficient ◽  
High Level

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.

Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1125-1129 ◽  
Author(s):  
Giuliana Montosi ◽  
Paola Paglia ◽  
Cinzia Garuti ◽  
Carlos A. Guzman ◽  
Judy M. Bastin ◽  
...  
Keyword(s):  
Single Gene ◽  
Hereditary Hemochromatosis ◽  
Phenotypic Expression ◽  
Hereditary Diseases ◽  
Transduction Efficiency ◽  
Hfe Gene ◽  
Wild Type ◽  
Iron Deficient ◽  
High Level

Abstract 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.

scholarly journals Loss of PTEN in Fallopian Tube Epithelium Results in Multicellular Tumor Spheroid Formation and Metastasis to the Ovary

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 884 ◽  
Author(s):  
Matthew Dean ◽  
Vivian Jin ◽  
Tova M. Bergsten ◽  
Julia R. Austin ◽  
Daniel D. Lantvit ◽  
...  
Keyword(s):  
Fallopian Tube ◽  
Single Gene ◽  
Similar Rate ◽  
Genetic Alterations ◽  
Wild Type ◽  
Spheroid Formation ◽  
Multicellular Tumor Spheroid ◽  
Ovarian Stroma

High-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube and then spread to the ovary. Our objective was to evaluate the role of multicellular tumor spheroids (MTS) in ovarian metastasis. By testing a panel of murine oviductal epithelial (MOE) cells with genetic alterations mimicking those seen in HGSOC, we found that loss of PTEN allowed MTS formation under ultra-low adhesion conditions. Confirming these results in vivo, MTS-like structures were observed in the oviducts of PAX8Cre/+ PTENflox/flox mice. MOE PTENshRNA cells could incorporate up to 25% wild type cells into MTS, while higher percentages of wild type cells resulted in a loss of MTS formation. MTS formation allowed MOE PTENshRNA cells to survive better under ultra-low adhesion conditions than control cells. MTS also attached to the ovarian stroma, as would be exposed during ovulation. Interestingly, MTS more robustly cleared monolayers of murine ovarian surface epithelia than murine ovarian fibroblasts. When xenografted into the ovarian bursa, OVCAR8 MTS were able to form tumors in the ovary at a similar rate as an equal number of OVCAR8 cells grown on traditional cell culture plastic. In conclusion, loss of a single gene (PTEN) allows the fallopian tube epithelia to form MTS, which survive better under ultra-low adhesion conditions, attach to the extracellular matrix exposed during ovulation, and colonize the ovary. These results suggest that MTS may contribute to seeding of the ovary in HGSOC patients.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

Hereditary Hemochromatosis Since Discovery of the HFE Gene

2001 ◽  
Vol 47 (7) ◽  
pp. 1147-1156 ◽  
Author(s):  
Elaine Lyon ◽  
Elizabeth L Frank
Keyword(s):  
Molecular Genetic ◽  
Hereditary Hemochromatosis ◽  
Phenotypic Expression ◽  
Protein Product ◽  
Population Screening ◽  
The Body ◽  
Hfe Gene ◽  
Molecular Testing ◽  
Disease Mechanism ◽  
Biochemical Tests

Abstract Background: Hereditary hemochromatosis is an inherited disorder of iron metabolism that is characterized by excessive iron deposition in major organs of the body. Chronic increased iron absorption leads to multiorgan dysfunction. Since the discovery of the gene responsible for the majority of cases, research has progressed rapidly to identify the gene product, the effects of mutations, and the implications for different populations. The protein product of the HFE gene is a transmembrane glycoprotein, termed HFE, that modulates iron uptake. Mutations in the HFE protein compromise its function and produce disease symptoms. Two mutations, C282Y and H63D, have been linked to the majority of disease cases. Approach: We reviewed the recent literature for the molecular basis of hereditary hemochromatosis. Genotypic information was combined with biochemical and clinical phenotypic information to achieve a better understanding of the disease mechanism. Content: This review provides a comprehensive discussion of known mutations in the HFE gene and their phenotypic expression. Diagnostic criteria using molecular genetic techniques in conjunction with traditional biochemical tests are provided. Current methods and limitations of molecular testing are examined in detail. A strategy for population screening and an algorithm for diagnosis that incorporates molecular testing are presented. Treatment by therapeutic phlebotomy and the use of blood obtained from hemochromatosis patients are discussed. Summary: Although the disease mechanism has not been completely elucidated, phenotypic and penetrance data are becoming available. Controversy still exists concerning the role of genetic testing in diagnosis and population screening.

Haptoglobin modifies the hemochromatosis phenotype in mice

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3353-3355 ◽  
Author(s):  
Emanuela Tolosano ◽  
Sharmila Fagoonee ◽  
Cinzia Garuti ◽  
Linda Valli ◽  
Nancy C. Andrews ◽  
...  
Keyword(s):  
Genetic Disorder ◽  
Hereditary Hemochromatosis ◽  
Reticuloendothelial System ◽  
Heme Iron ◽  
Hfe Gene ◽  
Plasma Hemoglobin ◽  
Sufficient Cause ◽  
Common Genetic Disorder ◽  
Free Plasma

Abstract Classic hereditary hemochromatosis (HH) is a common genetic disorder of iron metabolism caused by a mutation in the HFE gene. Whereas the prevalence of the mutation is very high, the clinical penetrance of the disease is low, suggesting that the HFE mutation is a necessary but not sufficient cause of clinical HH. Several candidate modifier genes have been proposed in mice and humans, including haptoglobin. Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. It delivers free plasma hemoglobin to the reticuloendothelial system, thus reducing loss of hemoglobin through the glomeruli and allowing heme-iron recycling. To gain insight into the role of haptoglobin as a modifier gene in HH, we used Hfe and haptoglobin double-null mice. Here, we show that Hfe and haptoglobin compound mutant mice accumulate significantly less hepatic iron than Hfe-null mice, thus demonstrating that haptoglobin-mediated heme-iron recovery may contribute significantly to iron loading in HH. (Blood. 2005;105:3353-3355)

scholarly journals Role of β-Lactamases and Porins in Resistance to Ertapenem and Other β-Lactams in Klebsiella pneumoniae

2004 ◽  
Vol 48 (8) ◽  
pp. 3203-3206 ◽  
Author(s):  
George A. Jacoby ◽  
Debra M. Mills ◽  
Nancy Chow
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACT High-level resistance to ertapenem was produced by β-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 β-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 μg/ml and expression of outer membrane proteins was diminished could be selected.

scholarly journals Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

2001 ◽  
Vol 75 (23) ◽  
pp. 11284-11291 ◽  
Author(s):  
David A. Einfeld ◽  
Rosanna Schroeder ◽  
Peter W. Roelvink ◽  
Alena Lizonova ◽  
C. Richter King ◽  
...  
Keyword(s):  
Adenovirus Type ◽  
Wild Type ◽  
Base Modification ◽  
Model Following ◽  
Vector Distribution ◽  
High Level ◽  
Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

scholarly journals HFE Gene Mutation Status Predicts Response to Gemtuzumab Ozogamicin in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1307-1307 ◽  
Author(s):  
Etienne Paubelle ◽  
Alice Marceau ◽  
Florence Zylbersztejn ◽  
Mickael Dussiot ◽  
Ivan Cruz Moura ◽  
...  
Keyword(s):  
Total Dose ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Gemtuzumab Ozogamicin ◽  
Hfe Gene ◽  
Study Cohort ◽  
Dependent Manner ◽  
Wild Type ◽  
Significant Difference ◽  
Hfe Mutations

Abstract BACKROUND: The protein defective in type 1 hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins, associates with beta2-microglobulin and is implicated in membranous protein recycling. Gemtuzumab ozogamicin (GO), a monoclonal antibody directed against CD33 linked to a cytotoxic agent, has been used with controversial effects in acute myeloid leukemia (AML). Internalization of GO is required for its anti-leukemic effect. Therefore, we hypothesized that H63D or C282Y HFE gene mutations may impair GO activity by preventing its internalization. METHODS: Wild type, C282Y and H63D HFE leukemic cells and primary cell were used to assess effects of GO alone or in combination with cytarabine on cell proliferation and apoptosis. Flow cytometry, confocal and AMNIS stream analysis were used to evaluate GO internalization. HFE mutations were analyzed by PCR analysis on DNA from patients included in clinical studies. Post-hoc subgroup analysis was performed to assess in vivo the role of HFE status on GO efficacy and toxicity among the 280 patients of the ALFA-0701 study as a study cohort (patients aged 50-70 years; GO 3mg/m² on days 1, 4, and 7 of chemotherapy and on day 1 of the first and second induction ; total dose 15 mg/m² ) and then on the GOELAMS-LAM 2006 IR study (patients aged 18-60 years; GO 6 mg/m² on day 4 of chemotherapy during the induction and the first consolidation; total dose 12 mg/m²) and UK NCRI AML17 study (patients aged 18-81 years; GO 3 vs 6 mg/m² on day 1 of chemotherapy but not during consolidation) as validating cohorts. RESULTS: GO induced cell death by apoptosis in AML cell lines and primary cells in a dose-dependent manner and synergistically in combination with cytarabine. However, the IC5O of GO was significantly higher in HFE mutated cells (125 vs 10 ng/mL p<0.001). To further understand this phenomenon, the CD33 internalization was analyzed upon GO or anti-CD33 antibody exposure. In line with the cytotoxic effect, CD33 was significantly less internalized in HFE mutant cells (17.4% vs 65.1% after 1 hour, p<0.01). HFE mutations were screened in 242 of the 280 ALFA-0701 patients with DNA available. There were 155 non-mutated patients (64%), 68 (28%) heterozygous for H63D, 11 (5%) heterozygous for C282Y, and 8 (3%) homozygous for H63D, which is consistent with the prevalence of the various mutations in the French population. Median age was 62 years (50-71) and the M/F ratio was 0.5, equally distributed among the different groups. No significant difference was observed with respect to the diagnosis of various hematological parameters, including white blood cell count, blasts number, cytogenetic subgroups, molecular mutation incidences (FLT3, NPM1, CEBPA, IDH, DNMT3A). In the ALFA-0701 study cohort, HFE wild-type (WT) patients had a higher overall survival (OS) when treated in the GO arm (median, not reached vs 19.5 months, p=0.0193). In contrast, OS was similar among patients with heterozygotes HFE mutations treated in the GO and the control arm (median, 19.9 vs 21.9, p=0.9675). In confirmatory cohorts, GO treatment led to a trend to increased OS in the GOELAMS-LAM 2006 IR cohort only in HFE WT patients (4-year OS with GO 62% vs 48%, p=0.08) but not in mutated patients (4-year OS with GO 73% vs 64%, p=0.45). Furthermore, in this cohort in the FLT3 WT patients subgroup, GO further improve OS in WT patients (4-year OS with GO 72% vs 50%, p=0.017), but not in patients with heterozygous HFE mutation (4-year OS with GO 80% vs 65%, p=0.23) In the UK NCRI AML17 cohort, which used GO only during induction, 245 patients were randomized between GO at 3mg/m2 and 6mg/m2 and evaluated for HFE status. Overall there was no effect of GO dose on outcomes, and no evidence of either any heterogeneity by HFE, nor any subgroup, which showed a differential effect of GO dose. CONCLUSIONS: Future studies should focus on optimising the fractionated schedule for GO at the 3mg/m2. Fractionated high doses (3x3mg/m2) during induction and single dose during consolidation seems to be the best schedule. Most importantly, the effect of GO treatment differed between HFE WT and heterozygote mutated AML patients. GO only increased OS among HFE WT patients. This is likely related to impaired internalization of the CD33 target. Our data suggest that HFE status should be used as a companion test to predict outcome of AML treated with GO. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of the Cold-Box Region in the 5′ Untranslated Region of the cspA mRNA in Its Transient Expression at Low Temperature in Escherichia coli

1998 ◽  
Vol 180 (1) ◽  
pp. 90-95 ◽  
Author(s):  
Li Fang ◽  
Yan Hou ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Untranslated Region ◽  
Cold Shock ◽  
Wild Type ◽  
Mrna Stabilization ◽  
High Level ◽  
Cold Box ◽  
In Cells

ABSTRACT Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced inEscherichia coli. However, when the 5′ untranslated region (5′ UTR) of cspA mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the “cold box” existing in the 5′ UTRs ofcspA, cspB, and csdA. Here, we demonstrate that the overproduction of the 5′ UTR of not onlycspA but also cspB and csdA mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the cspA 5′ UTR its derepression function was abolished. The amount of mRNA from the chromosomal cspA gene was much higher in cells overproducing the wild-type 5′ UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5′ UTR. The stability of the chromosomal cspA mRNA in cells overproducing the wild-type 5′ UTR was almost identical to that in cells overproducing the cold-box-deleted 5′ UTR. Therefore, the derepression ofcspA caused by overproduction of 5′ UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in cspA transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with acspA mutant strain containing a cold-box-deletedcspA gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.

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