bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Iron Responsive Elements of the Hepcidin Promoter by In Vivo Imaging of Mice.
Authors: Flanagan Jonathan , Truksa Jaroslav , Peng Hongfan , Lee Pauline , Beutler Ernest 
Published: October 2019

Abstract
Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. It performs this function by modulating iron transfer by both the intestinal epithelial cells and macrophages. Low or high hepcidin levels result in increased or decreased iron absorption respectively. Hepcidin is mainly liver-derived and regulated at least, in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation have all been shown to influence hepcidin mRNA expression in intact animals. Regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating hepatocytes or mixtures of hepatocytes and macrophages or marrow cells with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains a mystery. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we took advantage of the fact that 2 hepcidin genes exist in mice. Hepcidin 1 is homologous to human hepcidin while hepcidin 2 is the result of a tandem duplication of murine hepcidin 1. In mice hepcidin 1 mRNA expression responds to iron and to inflammation, but hepcidin 2 responds only to iron. We established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals’ liver after transfecting hydrodynamically hepcidin promoter/luciferase constructs into mice. Murine hepcidin 1, murine hepcidin 2 and human hepcidin promoters were all shown to have basal luciferase activity. Luciferase expression was lower in HFE knockout mice than in wild type controls. When transfected mice were stimulated with iron or endotoxin, the murine hepcidin 1 and human hepcidin constructs were found to respond to both of these stimuli. The murine hepcidin 2 transfected mice showed a weak response to iron stimulation while there was no stimulation by endotoxin. These findings validate, in an in vivo system, what is known about these promoters from other studies. Subsequent experiments have focused on making shorter hepcidin promoter constructs to identify the key regions of the hepcidin promoter involved in iron induction of hepcidin expression. This strategy will allow us to map the iron response site of the hepcidin promoter and this should aid in identifying transcription factors and upstream stimuli involved in regulating expression of hepcidin by iron.
HAMP and HVJ as Candidate Modifier Genes in Type1 Hereditary Hemochromatosis with High Iron Burden and in Porphyria Cutanea Tarda (PCT).
Authors: Ajioka Richard S. , Phillips John D. , Weiss Robert B. , Dunn Diane M. , Kushner James P. 
Published: October 2019

Abstract
Homozygosity for the HFE C282Y mutation accounts for approximately 90 percent of HFE-associated (type 1) hereditary hemochromatosis. The clinical phenotype in C282Y homozygotes, however, ranges from simply an elevated percent saturation of transferrin to organ damage due to iron overload. Modifier genes have been proposed to explain this phenotypic variability. Hepatic siderosis is a nearly constant finding in patients with PCT. Approximately 20 percent of patients with PCT are homozygotes for the C282Y HFE mutation but the cause of hepatic iron loading in the remaining 80 percent is not known. Approximately one-third of patients with PCT are heterozygotes for mutations of the uroporphyrinogen decarboxylase (URO-D) gene (familial PCT) but pedigree studies indicate that clinical expression occurs only in those URO-D heterozygotes who develop hepatic siderosis. Most patients with PCT have no URO-D mutations (sporadic PCT) but virtually all sporadic cases have hepatic iron overload. Two genes known to affect iron homeostasis are hepcidin (HAMP) and hemojuvelin (HJV). Heterozygosity for HAMP and HJV mutations have been associated with marked iron overload in a small number of patients with type 1 hemochromatosis (Blood.2004; 103:2835–40; Blood Cells Mol Dis.2004; 33:338–43). We asked if mutations of HAMP or HJV could account for hepatic iron overload in highly penetrant C282Y homozygotes and in PCT patients with or without HFE mutations. We sequenced the HAMP and HJV genes in 96 hemochromatosis patients with grade 3–4 (scale 0–4) hepatic parenchymal cell stainable iron (HPCSI) and 96 PCT patients with variable degrees of hepatic siderosis. Ninety-four percent (90) of the hemochromatosis patients were C282Y homozygotes, 4.2 percent (4) were C282Y heterozygotes and 2.1 percent (2) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in either the HAMP or HJV genes. Eighty-three of the 96 PCT patients were genotyped at the HFE locus. Twenty-five percent (21) were C282Y homozygotes, 23 percent (19) were C282Y heterozygotes and 52 percent (43) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in the HJV gene of patients with PCT but two PCT patients were found to be heterozygotes for HAMP mutations. The first had the previously identified 212G→A transition leading to a G71D substitution. The second had a 248A→C transversion corresponding to K83R in the peptide. Both of these PCT patients were HFE 282 wild type homozygotes but both had grade 4 HPCSI. These data indicate that heterozygosity for mutations of HAMP or HJV rarely modifies the iron loading phenotype in either type 1 hemochromatosis or PCT. Other modifier loci must exist.
Role of HFE in Hepatic Iron Overload: Novel Findings Demonstrate Direct Effects of Mutant C282Y on Hepcidin and Calreticulin mRNA Expression.
Authors: Pinto Jorge P. , Ramos Pedro , de Almeida Sergio , Oliveira Susana , Breda Laura , Michalak Marek , Rivella Stefano , de Sousa Maria 
Published: October 2019

Abstract
Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.
Novel Rearrangements and Mutations in the Alpha-Globin Gene Cluster Detected by a New Alpha-Globin Dosage Assay, Cation Exchange HPLC and Comprehensive DNA Sequencing.
Authors: Hantash Feras M. , Gallivan Monica V. , Mario Mikula , Kelsey Starn , Wang Sheng-Biao , Sun Weimin , Strom Charles M. 
Published: October 2019

Abstract
The alpha globin gene cluster contains two highly homologous alpha globin genes, HBA1 and HBA2, that code for identical proteins. Mutations in the alpha globin gene cluster are predominantly large deletions causing the loss of either one copy of the alpha-globin gene (e.g. -α3.7 and -α4.2 deletions) or both copies (e.g. --THAI, --FIL or --MED). A few large deletions encompassing the regulatory HS-40 region have also been described in alpha-thalassaemia patients. Point mutations and small base pair insertions or deletions have also been detected in HBA1 and HBA2 genes. Seven common large deletions in the alpha globin gene cluster are detected by a gapped-PCR assay. These common mutations and some other types of rearrangements can be detected by Southern blot, a laborious and time consuming method. However, these methods may not accurately identify the total number of copies of alpha globin like genes. We designed a single-tube alpha globin gene dosage assay (αGDA) using semi-quantitative fluorescent PCR (SQF PCR) for detecting the total number of alpha globin genes. Primers that amplify specific fragments from HBA1 and HBA2 genes, a fragment between the alpha globin pseudogenes, and three fragments flanking and including the HS-40 regulatory region were included in a single PCR reaction together with primers that amplify fragments from 3 different normalization genes. Using the αGDA, we were able to detect in patient samples varying copy numbers of alpha globin genes and to identify the nature of DNA rearrangements between HBA1 and HBA2. We also identified novel alpha globin conversion events that were verified by DNA sequencing. We also designed a complimentary comprehensive DNA sequencing assay to detect point mutations and small base pair insertions or deletions in the HBA1 and HBA2 genes. Using this method, and in combination with cation exchange HPLC and agarose gel electrophoresis, novel mutations in alpha globins were identified and submitted to the globin gene server, including Hb Linwood (α2 40 Lys>Gln), Hb Creve Coeur (α2 24 Tyr>Asp), and Hb Westborough (α-3.7 130 Ala>Val). The simplicity of αGDA will allow the replacement of the laborious Southern blot analysis to detect large deletions in the alpha globin gene cluster and to provide accurate information of total a-globin gene dosage, while the DNA sequencing assay will allow the detection of known and novel variants.
Towards a New Therapy for Hemophilia A: Evaluation of Multiple Approaches To Prolong the In Vivo Efficacy of Recombinant Factor VIII.
Authors: Murphy John E. , Pan Clark , Mei Baisong , Strauss Jonathan , Tjandra Hendri , Tang Liang , Esmon Pamela , Newgren James , Jesmok Gary , Landskroner Kyle , Parmathi Mohan , Radtke Klaus-Peter , Severs Joanne , Teare John , Jiang Haiyan , Fournel Michael 
Published: October 2019

Abstract
Prophylactic therapy with rFVIII has been shown to have a significant positive impact on the treatment of Hemophilia A. One of the impediments to effective prophylaxis is the requirement for frequent injections necessitated by the 12–14 hour circulating half-life of FVIII. We have evaluated a number of approaches to modify FVIII to reduce the need for frequent injections. In one approach, the active form of FVIII was stabilized by addition of a disulfide linkage between the A2 and A3 domains. As previously reported1, these molecules exhibited prolonged FVIII activity in a number of in vitro assays following activation. In vivo characterization of these molecules is in progress. Other approaches focused on increasing the circulating half-life of FVIII. In an attempt to reduce FVIII clearance by the liver receptor LRP, site-directed mutations in the reported LRP binding region of the FVIII A2 domain were generated. Twenty mutants, with single amino acid changes, were analyzed in mouse and rabbit recovery studies and no significant increase in recovery was observed. Since the point mutants may not have covered a large enough surface of the LRP binding domains, polyethlylene glygol (PEG) modification was used to disrupt a larger surface area. Previously, attachment of PEG moieties to FVIII had been shown to lead to an increase in plasma half-life in animal models; however, standard pegylation chemistries result in significant product heterogeneity and thus may not be suitable for commercial production. We have developed a novel method, based on protein engineering, to introduce PEG to specific cysteine residues on the surface of FVIII. Using this method, different molecular weight PEGs have been conjugated to sites in the A1, A2 or A3 domains of FVIII. Analysis of the pegylated proteins confirms that the attachment of PEG is highly specific to the engineered site. The pegylated products retain activity based on the two stage chromogenic assay, but exhibit reduced activity when analyzed by the one stage coagulation assay. Pharmacokinetic studies, performed in mouse and rabbit, show that pegylation increases half-life in a manner that is proportional to PEG molecular weight. Using a number of injury models in hemophilic mice, the pegylated molecules have been shown to be efficacious in stopping bleeds. The prospects for using site-specific pegylation of FVIII to produce a therapeutic for treatment of hemophilia A will be discussed.
Identifying Novel Blood Proteins and Protein Isoforms Using Tandem Mass Spectra.
Authors: Fermin Damian , Allen Baxter B. , Blackwell Thomas W. , Menon Rajasree , Adamski Marcin , States David J. 
Published: October 2019

Abstract
Blood is a complex fluid that samples all tissues in the human body. Despite complete sequence determination of the human genome, defining genes and gene products remains a challenge. Here, we apply tandem mass spectroscopy as new source of unbiased data to interrogate genomic sequence and identify novel protein coding sequences. A six-frame translation of the Human genome was used as the query database to search for novel blood proteins in the data from the HUPO PPP. Significance is assessed using a Poisson statistical model incorporating the length of the matching sequence and the frequency of spectrum matches observed in searching the database [Nat Biotech 2006 24(3):333–8]. Matches are binned by X!Tandem hyperscore, and statistics for each score class are considered independently. The overall probability that the matches to an ORF occurred at random is calculated as the product of the probability that the matches in each score category occurred at random. The expected number of random matches, E, is calculated as the product of the probability that an ORF match occurred at random multiplied by the number of ORFs searched. The confidence in an ORF identification is 1/(1+E). An open reading frame is considered significant if confidence is greater than 95%. Expanding recently published work [Genome Biol2006; 7(4):R35], we have identified 837 significant open reading frames coding for 18852 peptides falling within 914 exons of 413 genes. Out of 8856 candidate ORFs outside the boundaries of known genes, 3246 of them achieved a confidence >= 0.95. We also required the XG ORFs to be supported by at least 3 distinct ESTs. Twenty four of the XG ORFs were found to have a significant alignment to the mouse genome. Of these, 13 of the alignments encompassed a coding region for one of the diagnostic peptides associated with the ORF. Gene models for the XG ORFS were derived from the GENSCAN prediction made for their coding regions. This analysis suggests that alternative splicing of blood protein genes is common and that much remains to be learned about the protein constituents of blood.
Anti-Inflammatory Effect of Blocking the Interaction between Platelet Glycoprotein Ibα and the Leukocyte Integrin αMβ2.
Authors: Han Qiang , Li Zhijie , Peng Yuandong , Wayne C. , Lopez Jose A. 
Published: October 2019

Abstract
The interaction between the leukocyte integrin Mac-1 (α M β 2) and platelet glycoprotein (GP) Ibα allows leukocytes to firmly adhere to platelet thrombi, enabling their subsequent transmigration into the vessel wall. This interaction is blocked by the GP Ibα monoclonal antibody AP1, which we have previously mapped to the GP Ibα sequence Arg218 to Tyr228. In the current investigation we examined the effectiveness of a GP Ibα-based peptide containing the AP1 epitope in blocking platelet-leukocyte interactions under flow and in an inflammation model based on corneal wound healing. The peptide bound the α M I-domain specifically and saturably and was able to inhibit several GP Ibα-dependent functions under flow:
the adhesion of GP Ib-IX complex-expressing CHO cells to immobilized α M I-domain, the adhesion of THP-1 cells - a monocytic cell line expressing α Mβ 2 - to immobilized glycocalicin (soluble extracellular region of GP Ibα ), adhesion of activated THP-1 cells to surface-adherent GP Ib-IX complex-expressing CHO cells, and the binding of neutrophils to immobilized glycocalicin or a platelet monolayer.
We therefore tested whether the peptide could inhibit inflammatory processes that may be dependent on the interactions between platelets and neutrophils. Neutrophils play an important role in corneal wound healing, but because the cornea is avascular, they must migrate to the wound from the limbal vessels at the periphery of the cornea. Therefore we chose this model of mouse corneal wounding as we have previously demonstrated that antibody-induced platelet depletion markedly reduces neutrophil localization and emigration from the limbal vessels and therefore postulated that platelets may directly facilitate neutrophil emigration. In our model, a 2 mm wound was made at the center of the cornea by removing the epithelium without injuring the stroma. Neutrophil infiltration across the cornea, from periphery to center, was assessed at 12 and 30 hr after the injury. Mice were injected with either AP1 peptide or control peptide (scrambled AP1 peptide) three times at 4 hour intervals beginning immediately after corneal wounding. Mice treated with AP1 peptide showed markedly fewer neutrophils infiltrating all regions of the cornea. These results indicate that the interaction between GP Ibα and Mac-1 is required for efficient leukocyte extravasation. They also suggest that drugs targeting the GP Ibα-α Mβ 2 axis could have anti-inflammatory properties without compromising hemostasis, as the peptide had minimal effects on the interaction of GP Ibα with von Willebrand factor.
Sbds Deficient Neutrophils Exhibit Normal Numbers, Chemotaxis and Phagocytic Functions, but Impaired NADPH Oxidase Activity.
Authors: Zhang Siyi , Sun Chunxian , Zhong Jian , Glogauer Michael , Rommens Johanna M. 
Published: October 2019

Abstract
Shwachman-Diamond syndrome (SDS) is a recessive disease with multi-system involvement, and is characterized by hematological dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. Recurrent infections are a major cause of morbidity for SDS patients, due to neutropenia and proposed defects in neutrophil functions. SDS is caused by mutations in SBDS, a highly conserved gene that has been suggested to be involved in RNA metabolism and/or ribosome biogenesis. A conditional Cre-Lox Sbds allele was generated in the mouse using gene targeting technology, as complete ablation of Sbds has been shown to lead to early embryonic lethality. To investigate neutrophil functions and their specific sensitivity to loss of Sbds, we elected to delete the gene by breeding mice with the conditional allele to mice with Cre recombinase under the control of the lysozyme M promoter. While Sbds was confirmed to be ablated in mature neutrophils in adult offspring mice, their numbers and morphology were found to be normal in peripheral blood. Sbds-ablated neutrophils were also found to have normal chemotatic and chemokinetic abilities, and they demonstrated phagocytic function comparable to neutrophils of control mice. In contrast, NADPH oxidase activity was observed to be greatly reduced in response to phorbol-12-myristate 13-acetate stimulation, while the expression levels and phosphorylation of the NADPH oxidase complex components remained intact. To evaluate the net effect of the loss of Sbds in vivo, clearance of Pseudomonas aeruginosa from infected lungs was investigated, and found to be significantly reduced in mice with Sbds-abated neutrophils compared their wild type littermates. In summary, loss of Sbds does not to affect total peripheral neutrophil numbers directly, or chemotaxis and phagocytic abilities, but was essential for a robust NADPH oxidase response. These findings suggest that neutrophils are compromised with loss of Sbds, but that at least some of the neutrophil deficiencies of SDS patients are likely due to microenvironment deficiencies, or early hematopoiesis abnormalities.
The Neutrophil Specific CD177 Is a Novel Counter-Receptor for Endothelial PECAM-1.
Authors: Sachs Ulrich , Andrei-Selmer Cornelia , Maniar Amudhan , Paddock Cathy , Newman Peter , Chavakis Triantafyllos , Santoso Sentot 
Published: October 2019

Abstract
CD177 is a glycosyl-phosphatidylinositol-linked member of the Ly-6 superfamily that is expressed on approximately 50% of human neutrophils, though 3–5% of Caucasians and African Americans are CD177 null. CD177 carries the clinically-important neutrophil antigen, HNA-2a - alloantibodies to which can result in profound neutropenia. Although the role of CD177 in neutrophil function is not known, it has been found to be aberrantly expressed on the neutrophils of most patients suffering from polycythemia vera, and in approximately half of patients with essential thrombocythemia. To further elucidate the function of CD177, we produced a CD177/IgG Fc fusion protein, and used it identify its binding partner on endothelial cells. Flow cytometric analysis revealed that CD177/Fc bound tightly and specifically to human umbilical vein endothelial cells, and an antigen-capture ELISA screen employing a panel of monoclonal antibodies (mAbs) to endothelial cell surface proteins further revealed the target antigen to be the endothelial cell junctional protein, PECAM-1. Surface plasmon resonance analysis showed tight binding between PECAM-1 and CD177, having a Kd of 8.17 x 10−7 M. U937 cells stably transfected with CD177 bound immobilized PECAM-1, and this heterophilic interaction could be blocked by mAbs specific for Ig domains 5 or 6 of the PECAM-1 extracellular domain, or by mAbs specific for CD177. In contrast, no inhibition was observed when mAbs against Ig domains 1 and 2 were applied. The same mAbs were also found to be effective inhibitors of neutrophil transendothelial migration. Taken together, these data establish PECAM-1/CD177 as a previously unrecognized heterophilic receptor/counter-receptor pair that functions in the multi-step adhesion and signaling cascade of events that take place during the inflammatory response.