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ABSTRACT: The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.
Drug metabolism and disposition: the biological fate of chemicals 05/2012; 40(8):1545-55. · 3.74 Impact Factor
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Thomas Sonnweber,
Claudia Ress,
Manfred Nairz,
Igor Theurl,
Andrea Schroll,
Anthony T Murphy,
Victor Wroblewski, Derrick R Witcher,
Patrizia Moser,
Christoph F Ebenbichler,
Susanne Kaser,
Günter Weiss
[show abstract]
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ABSTRACT: Obesity is often associated with disorders of iron homeostasis; however, the underlying mechanisms are not fully understood. Hepcidin is a key regulator of iron metabolism and may be responsible for obesity-driven iron deficiency. Herein, we used an animal model of diet-induced obesity to study high-fat-diet-induced changes in iron homeostasis. C57BL/6 mice were fed a standard (SD) or high-fat diet (HFD) for 8 weeks, and in addition, half of the mice received high dietary iron (Fe+) for the last 2 weeks. Surprisingly, HFD led to systemic iron deficiency which was traced back to reduced duodenal iron absorption. The mRNA and protein expressions of the duodenal iron transporters Dmt1 and Tfr1 were significantly higher in HFD- than in SD-fed mice, indicating enterocyte iron deficiency, whereas the mRNA levels of the duodenal iron oxidoreductases Dcytb and hephaestin were lower in HFD-fed mice. Neither hepatic and adipose tissue nor serum hepcidin concentrations differed significantly between SD- and HFD-fed mice, whereas dietary iron supplementation resulted in increased hepatic hepcidin mRNA expression and serum hepcidin levels in SD as compared to HFD mice. Our study suggests that HFD results in iron deficiency which is neither due to intake of energy-dense nutrient poor food nor due to increased sequestration in the reticulo-endothelial system but is the consequence of diminished intestinal iron uptake. We found that impaired iron absorption is independent of hepcidin but rather results from reduced metal uptake into the mucosa and discordant oxidoreductases expressions despite enterocyte iron deficiency.
The Journal of nutritional biochemistry 03/2012; · 4.29 Impact Factor
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ABSTRACT: Engineering monoclonal antibodies (mAbs) with improved binding to the neonatal Fc receptor (FcRn) is a strategy that can extend their in vivo half-life and slow their systemic clearance. Published reports have predominantly characterized the pharmacokinetics of mAbs after intravenous administration. Recently, studies in mice suggest FcRn may also play a role in affecting the subcutaneous bioavailability of mAbs. Herein, we examined whether five mAbs engineered with the T250Q/M428L Fc mutations that improved their FcRn interactions, and subsequently their in vivo pharmacokinetics after intravenous administration, had improved subcutaneous bioavailability compared with their wild-type counterparts in cynomolgus monkeys. Similar to the intravenous administration findings, the pharmacokinetic profiles of our variant mAbs after subcutaneous injection showed improved half-life or clearance. In contrast, a clear effect was not observed on the subcutaneous bioavailability. We expect that while FcRn may play a role in determining mAb subcutaneous bioavailability, multiple biopharmaceutical and physiological factors are likely to influence the success of engineering strategies aimed at targeting this pathway for improving bioavailability.
mAbs 03/2012; 4(2).
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Irene Pichler,
Cosetta Minelli,
Serena Sanna,
Toshiko Tanaka,
Christine Schwienbacher,
Silvia Naitza,
Eleonora Porcu,
Cristian Pattaro,
Fabio Busonero,
Alessandra Zanon, [......],
Carsten Gnewuch,
Eric Schadt,
Manfred Mitterer,
David Schlessinger,
Luigi Ferrucci, Derrick R Witcher,
Andrew A Hicks,
Günter Weiss,
Manuela Uda,
Peter P Pramstaller
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ABSTRACT: The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.
Human Molecular Genetics 01/2011; 20(6):1232-40. · 7.64 Impact Factor
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ABSTRACT: Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein.
We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis.
The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%-75% range 23.2-93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%-75% range 5.9-18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%-75% range 0.42-3.07 μg/L, n = 100).
The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.
Clinical Chemistry 11/2010; 56(11):1725-32. · 7.91 Impact Factor
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Igor Theurl,
Elmar Aigner,
Milan Theurl,
Manfred Nairz,
Markus Seifert,
Andrea Schroll,
Thomas Sonnweber,
Lukas Eberwein, Derrick R Witcher,
Anthony T Murphy,
Victor J Wroblewski,
Eva Wurz,
Christian Datz,
Guenter Weiss
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ABSTRACT: The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and the master regulator hepcidin. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. Many patients, however, present with both ACD and iron deficiency anemia (ACD/IDA), the latter resulting from chronic blood loss. We used a rat model of ACD resulting from chronic arthritis and mimicked ACD/IDA by additional phlebotomy to define differing iron-regulatory pathways. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In rats and humans with ACD, serum hepcidin concentrations are elevated, which is paralleled by reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects, and ACD/IDA persons, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Circulating hepcidin levels affect iron traffic in ACD and ACD/IDA and are more responsive to the erythropoietic demands for iron than to inflammation. Hepcidin determination may aid to differentiate between ACD and ACD/IDA and in selecting appropriate therapy for these patients.
Blood 04/2009; 113(21):5277-86. · 9.90 Impact Factor
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ABSTRACT: Iron absorption is inadequately increased in patients with chronic haemolytic anaemia, which is commonly complicated by iron overload. Growth differentiation factor 15 (GDF15) has been identified as a bone marrow-derived factor that abrogates hepcidin-mediated protection from iron overload under conditions of increased erythropoiesis. Increased concentrations of GDF15 have been reported in beta-thalassaemia patients and GDF15 has been found to suppress hepcidin expression in vitro. To further study the interdependencies of iron metabolism and erythropoiesis in vivo, the concentrations of hepcidin and GDF15 were determined in sera from 22 patients with pyruvate kinase deficiency (PKD) and 21 healthy control subjects. In PKD patients, serum hepcidin levels were 13-fold lower than in controls (2.0 ng/ml vs. 26.2 ng/ml) and GDF15 was significantly higher (859 pg/ml vs. 528 pg/ml). Serum hepcidin concentrations correlated positively with haemoglobin and negatively with serum GDF15. These results suggest that GDF15 contributes to low hepcidin expression and iron loading in PKD.
British Journal of Haematology 01/2009; 144(5):789-93. · 4.94 Impact Factor
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ABSTRACT: The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.
Molecular biology of the cell 11/2008; 19(12):5490-505. · 5.98 Impact Factor
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ABSTRACT: Individuals donating whole blood 13 times in a 2-year period without development of iron deficiency anemia (superdonors) are a self-selected population that is deferred for low hematocrit (Hct) level less frequently than other donors.
Iron metabolism was assessed in 138 superdonors through a questionnaire and measurement of Hct, serum ferritin, serum hepcidin, and serum growth differentiation factor 15 (GDF15). Genetic testing for HFE and JAK-2 mutations was also performed.
Iron deficiency (ferritin level, <30 microg/L) is present in more than 60 percent of superdonors. Behaviors altering iron status included casual use of iron supplements in males, but not in females, and cigarette smoking that produced increased Hct associated with decreased ferritin. The striking biochemical characteristic of superdonors is greatly decreased serum hepcidin, consistent with their need to absorb maximal amounts of dietary iron to replace that lost from blood donation. GDF15 is normal in most superdonors, indicating that GDF15 overexpression arising from the expanded erythroid pool necessary to replace donated red cells is not the biochemical mechanism for the decreased serum hepcidin. Mutations in JAK-2 were not found, indicating that undiagnosed polycythemia vera is not a common cause for successful repeated blood donation by superdonors. Mutations in HFE associated with hemochromatosis were present in superdonors at the same frequency as the normal population. However, superdonors heterozygous for the H63D mutation in HFE had significantly decreased hepcidin : ferritin ratios demonstrating for the first time that the heterozygous state for HFE mutations is associated with alterations in hepcidin expression.
Transfusion 10/2008; 48(10):2197-204. · 3.22 Impact Factor
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ABSTRACT: Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6(-/-) mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-gamma. The effects of DR6 are mostly amended when these immature DC are matured with IL-1beta/TNF-alpha, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.
Cancer Immunology and Immunotherapy 06/2008; 57(6):777-87. · 3.70 Impact Factor
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ABSTRACT: The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.
Proceedings of the National Academy of Sciences 05/2008; 105(17):6320-5. · 9.68 Impact Factor
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ABSTRACT: The hepatic peptide hormone hepcidin is considered the central regulator of iron metabolism. Characterizing the circulating levels of this peptide is critical to understanding its role in the development of clinically relevant syndromes, such as anemia of inflammation/chronic disease, and may provide insight into potential clinical interventions. While quantitative methods have been published for the determination of urinary hepcidin and serum prohepcidin, no definitive methods have been published for the determination of hepcidin in serum. In this report, we describe a quantitative method for the determination of both human and mouse hepcidin in serum and plasma. The method employs protein precipitation and solid-phase extraction followed by liquid chromatographic separation and tandem mass spectrometry detection. The method has a quantitative range of 0.25 ng/mL to 500 ng/mL serum for mouse hepcidin and 1 ng/mL to 500 ng/mL serum for human hepcidin. The method uses small sample volumes (50 microL for mice and 100 microL for humans) and 96-well formats for rapid sample processing. The method was used to establish baseline serum and plasma concentrations of hepcidin in normal C57Bl/6 mice and healthy human volunteers.
Blood 09/2007; 110(3):1048-54. · 9.90 Impact Factor
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ABSTRACT: The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-alpha (TNFalpha)). The T250Q/M428L anti-TNFalpha variant displayed an approximately 40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFalpha variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased approximately 500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFalpha T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFalpha variant displayed improved pharmacokinetics, characterized by an approximately 2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.
Journal of Biological Chemistry 02/2007; 282(3):1709-17. · 4.77 Impact Factor
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ABSTRACT: It is well established that the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. As such, modification of the interaction of IgG with FcRn has been the focus of protein-engineering strategies designed to generate therapeutic antibodies with improved pharmacokinetic properties. In the current work, we characterized differences in interaction of IgG between mouse and primate receptors using three humanized anti-tumor necrosis factor alpha antibodies with variant IgG(1) Fc regions. The wild-type and variant IgG showed a differential combination of improved affinity, modified dissociation kinetics, and altered pH-dependent complex dissociation when evaluated on the primate and murine receptors. The observed in vitro binding differences within and between species allowed us to more completely relate these parameters to their influence on the in vivo pharmacokinetics in mice and cynomolgus monkeys. The variant antibodies have different pharmacokinetic behavior in cynomolgus monkeys and mice, which appears to be related to the unique binding characteristics observed with the murine receptor. However, we did not observe a direct relationship between increased binding affinity to the receptor and improved pharmacokinetic properties for these molecules in either species. This work provides further insights into how the FcRn/IgG interaction may be modulated to develop monoclonal antibodies with improved therapeutic properties.
Drug Metabolism and Disposition 02/2007; 35(1):86-94. · 3.73 Impact Factor
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[show abstract]
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ABSTRACT: The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve
the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations,
which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in
both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-α (TNFα)).
The T250Q/M428L anti-TNFα variant displayed an ∼40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at
pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFα variant (P257I/Q311I) whose binding
kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for
murine FcRn was increased ∼500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction
was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFα T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these
molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When
administered intravenously to mice, the T250Q/M428L anti-TNFα variant displayed improved pharmacokinetics, characterized by
an ∼2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits
in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to
a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of
the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing
the disposition or elimination of monoclonal antibodies.
Journal of Biological Chemistry 01/2007; 282(3):1709-1717. · 4.77 Impact Factor
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ABSTRACT: Ghrelin was discovered for its ability to bind the growth hormone secretagogue receptor (GHSR1a) and stimulate growth hormone release. However, much research conducted with this novel stomach hormone is focused on proposed roles for it to participate in regulating energy balance. Exogenous administration of ghrelin stimulates food consumption in experimental animals and humans, presenting the hormone as the first to stimulate appetite after peripheral administration and implicates it for an etiology of obesity. The hormone also presents other exceptional characteristics that solicit need for future study. The peptide is modified by acylation with a mediumchain fatty acid on its third residue, and it is that ghrelin peptide that binds GHS-R1a. Enzymes or transfer proteins responsible for such acylation and de-acylation remain unknown. Specific assays for both acyl- and des-acyl ghrelin are not available nor are methods to prevent de-acylation in blood samples. Such knowledge is important because des-acyl ghrelin is reported to bestow biology distinct from that of ghrelin and that signal may actually oppose those prescribed for its acylated parent. This review of ghrelin data relating to obesity recognizes the complexity of ghrelin endocrinology and attempts to be cautious when discussing studies that measured ghrelin during different physiological states. Although much more exploration is needed, we placed more emphasis on reviewing studies during different physiological states when conclusions are less dependent on measurement of ghrelin. Despite these shortcomings, we conclude that there is ample evidence indicating ghrelin participates in regulating energy balance.
Metabolic syndrome and related disorders 02/2006; 4(1):37-42.
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ABSTRACT: Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus, and rabbit IL-1beta but only weakly binds to mouse and rat IL-1beta. Biacore experiments demonstrated that Hu007 and the type-I IL-1 receptor competed for binding to IL-1beta. Increasing salt concentrations decrease the association rate with only moderate effects on the dissociation rate, suggesting that long-range electrostatics are critical for formation of the initial complex. To understand the ligand-binding specificity of Hu007, we have mapped the critical residues involved in the recognition of IL-1beta. Selected residues in cynomolgus IL-1beta were mutated to the corresponding residues in mouse IL-1beta, and the effects of the changes on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically, substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobic interactions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N- and C-terminal regions of cIL-1beta with those found in mouse IL-1beta (V3I/S5Q/F150S) decreased the binding affinity of Hu007 to IL-1beta by about 1000-fold. Conversely, mutating the corresponding residues in mouse IL-1beta to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1beta were protected from exchange because of antibody binding. The results from this study demonstrate that Hu007 binds to a region located in the open end of the beta-barrel structure of IL-1beta and blocks binding of IL-1beta to its receptor.
Biochemistry 09/2005; 44(33):11106-14. · 3.42 Impact Factor
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Gustavo Matute-Bello,
W Conrad Liles,
Charles W Frevert,
Shireesha Dhanireddy,
Kimberly Ballman,
Venus Wong,
Richard R Green,
Ho Yeong Song, Derrick R Witcher,
Joseph A Jakubowski,
Thomas R Martin
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ABSTRACT: The Fas/FasL system is both proapoptotic and proinflammatory. FasL is inhibited by decoy receptor-3 (DcR3), a naturally occurring decoy receptor. We determined the effects of systemic blockade of the Fas/FasL system by a DcR3 analog (DcR3-a) in mice with pneumococcal pneumonia.
Streptococcus pneumoniae (7.2 x 105 or 1.9 x 107 cfu/mL) was instilled intratracheally into untreated C57Bl/6 mice, C57Bl/6 mice treated with DcR3-a, or Fas-deficient lpr mice, and the mice were studied 48 h later.
After instillation of the lower bacterial dose, disruption of the Fas/FasL system by either DcR3-a or the lpr mutation resulted in improved clearance of bacteria in the lungs (mean +/- SE, 4.6+/-2.1 x 10(6) and 3.5 +/- 1.6 x 10(6) cfu/lung, respectively, vs. 21.9+/-9.3 x 10(6) cfu/lung in untreated C57Bl/6 mice; P<.05) and decreased percentage of polymorphonuclear neutrophils in bronchoalveolar lavage fluid (mean +/- SE, 19.3%+/-9.5% and 20.2%+/-7.8%, respectively, vs. 55.0%+/-12.2% in untreated C57Bl/6 mice; P<.05). These changes were associated with decreased lung concentrations of the proinflammatory cytokines tumor necrosis factor- alpha and macrophage inflammatory protein-2 and with a decrease in apoptotic cells in the alveolar walls.
Blockade of the Fas/FasL system by DcR3-a in the lungs improves clearance of bacteria in mice with pneumococcal pneumonia.
The Journal of Infectious Diseases 03/2005; 191(4):596-606. · 6.41 Impact Factor
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Ling Liu,
Chunjin Ding,
Wei Zeng,
Josef G Heuer,
Jonathan W Tetreault,
Timothy W Noblitt,
Giao Hangoc,
Scott Cooper,
Kellie A Brune,
Ganesh Sharma,
Niles Fox,
Scott W Rowlinson,
Danise P Rogers, Derrick R Witcher,
Peter K Lambooy,
Victor J Wroblewski,
James R Miller,
Hal E Broxmeyer
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ABSTRACT: In a search for novel growth factors, we discovered that human interleukin-20 (IL-20) enhanced colony formation by CD34+ multipotential progenitors. IL-20 had no effect on erythroid, granulocyte-macrophage, or megakaryocyte progenitors. IL-20 transgenic mice increased the numbers and cell cycling of multipotential but not other progenitors. IL-20 administration to normal mice significantly increased only multipotential progenitor cells, demonstrating that IL-20 significantly influences hematopoiesis, with specificity toward multipotential progenitors. This is the first cytokine with such specificity identified.
Blood 12/2003; 102(9):3206-9. · 9.90 Impact Factor
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ABSTRACT: Fas ligand (FasL)-induced lung inflammation has recently been suggested to play an important role in the pathogenesis of acute respiratory disease syndrome (ARDS). In order to further explore this connection, we established a FasL-induced murine model of pulmonary inflammation. Instillation of recombinant FasL (rFasL) into the lung induced neutrophil infiltration and increased pulmonary permeability, as evidenced by increased total protein in the airspace; both occur in patients with ARDS. These effects were accompanied with a rapid induction of proinflammatory mediators: cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Pretreatment with a FasL antagonist, a decoy receptor 3 analogue (DcR3 analogue), reduced neutrophil infiltration into the airspace and resulted in a highly significant reduction in the levels of GM-CSF, MIP-2 and KC in bronchoalveolar lavage (BAL) fluid. We postulate that rFasL may be responsible for induction of proinflammatory chemokines and cytokines in the lung, which in turn attract neutrophil infiltration into the airspace. This proinflammatory process and the associated pulmonary permeability may, in part, explain the association of FasL with severe pulmonary inflammation, such as ARDS, and shed new light on FasL and its role in lung injury.
Immunology 11/2003; 110(2):225-33. · 3.32 Impact Factor
