The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigenpresenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed doubletransgenic animals demonstrated that kidney mhc2dab:GFP hi ; cd45:DsRed hi cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish. (Blood. 2011;117(26):7126-7135) IntroductionIn recent years, the zebrafish (Danio rerio) has proven to be a unique vertebrate model for the study of hematopoiesis. 1 The use of the zebrafish to study the ontogeny of leukocyte subsets, 2 immune cell migration, 3,4 and host-pathogen interactions 5 has provided new insights into our understanding of innate immunity in the developing vertebrate embryo. A major focus of previous studies was on the neutrophil response, because several transgenic reporter lines have been generated that mark this granulocyte subset. Whereas neutrophils generally constitute the first line of defense against invading pathogens, the role of other immune cell subsets in the innate immune response has received less attention. In addition to neutrophils, macrophages are key in the response to pathogen challenge. In the zebrafish embryo, primitive macrophages have been demonstrated to be capable of clearing injected bacteria by phagocytosis. 6 However, the absence of markers specific to macrophages has limited the study of this myeloid cell subset in the zebrafish.The mononuclear phagocyte system (MPS) comprises monocytes, tissue macrophages, and dendritic cells (DCs), as well as their lineage-committed progenitors. 7 The primary function of mature MPS cells is the clearance of pathogens by phagocytosis. This activity is crucial during immune challenge to clear invasive pathogens. Mononuclear phagocytes also play an important role in the removal of apoptotic cell corpses, especially during embryonic development. In mice, embryonic macrop...

No abstract

al. Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A.

Background Serum markers currently used as indicators of iron status have clinical limitations. Hepcidin, a key regulator of iron homeostasis, is reduced in iron deficiency (ID) and increased in iron overload. We describe the first CLIA-validated immunoassay with excellent accuracy and precision to quantify human serum hepcidin. Its diagnostic utility for detecting ID in first-time blood donors was demonstrated. Methods A monoclonal competitive ELISA (C-ELISA) was developed for the quantitation of human hepcidin and validated according to CLIA guidelines. Sera from nonanemic first-time blood donors (n = 292) were analyzed for hepcidin, ferritin, transferrin, and serum iron. Logistic regression served to determine the utility of hepcidin as a predictor of ID. Results The C-ELISA was specific for human hepcidin and had a low limit of quantitation (4.0 ng/mL). The hepcidin concentration measured with the monoclonal C-ELISA was strongly correlated with a previously established, extensively tested polyclonal C-ELISA (Blood 2008;112:4292–7) (r = 0.95, P < 0.001). The area under the receiver operating characteristic curve for hepcidin as a predictor of ID, defined by 3 ferritin concentration thresholds, was >0.9. For predicting ID defined by ferritin <15 ng/mL, hepcidin <10 ng/mL yielded sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin <30 ng/mL yielded sensitivity of 67.6% and specificity of 91.7%. Conclusion The clinical measurement of serum hepcidin concentrations was shown to be a potentially useful tool for diagnosing ID.

Erythroferrone (ERFE) is a hormone produced by erythroblasts in the bone marrow in response to erythropoietin (EPO). Recent animal studies have shown that rather than being involved in regulation of baseline erythropoiesis, ERFE acts as a stress erythropoiesis-specific regulator of hepcidin expression. By suppressing hepcidin expression in the liver, EFRE contributes to increased dietary iron absorption and recycling of stored iron necessary for recovery of blood mass after hemorrhage. In addition, ERFE was found to be involved in hepcidin regulation in inherited iron loading anemias, such as b-Thalassemia. ERFE has potential as a clinical marker for assessing erythropoiesis in patients with blood disorders. To date, there have been no reports of a human ERFE assay development and validation. To study the biological function and potential clinical applications of ERFE in humans, we developed the first dual monoclonal sandwich ELISA for serum measurement. Purified recombinant ERFE was used as antigen to immunize mice and subsequently screen 3000 hybridomas for binding to human ERFE. The nine positive hybridomas were expanded and monoclonal antibody from each clone purified and isotyped. We biotinylated each antibody and queried all possible combinations of capture and detection antibodies for binding activity. We discovered at least two pairs of antibodies suitable for assay optimization. Two mAbs were selected, 4C1 and 2D2, as the capture and detection antibody, respectively. We used streptavidin-HRP to quantify binding and detection of ERFE. The ERFE ELISA standard curve ranges from 0-50 ng/mL. The assay's lower limit of detection (LLOD) is 0.15 ng/mL and lower limit of quantitation (LLOQ) is 0.17 ng/mL. We assessed the normal range of ERFE by analyzing serum from110 healthy first time blood donors with normal iron status determined by assessment of ferritin, plasma iron, and transferrin saturation. Serum ERFE in the first time blood donors ranged from 0.15 to 3.94 ng/mL with a mean of 0.83 ng/mL. To re-capitulate the animal data previously observed in mice (Kautz et al., Nat Genet. 2014; 46(7): 678-684), we examined the effect of blood donation on human serum ERFE concentrations (Figure 1). We analyzed sera from three donors which underwent platelet and plasma-apheresis at baseline and day 2, 3, 4, 5, 7, 9, 10, 14, and 120 (Li et al., J Clin Apher., 2016). It was estimated that 30ml of RBCs were lost in the procedure. Serum ERFE concentrations were elevated from baseline in each patient within 2 days and remained higher through 14 days. At 120 days serum ERFE returned to baseline levels. We went on to test the concept that serum ERFE concentrations would be elevated in blood disorders associated with ineffective erythropoiesis, we obtained serum samples from X-linked sideroblastic anemia probands and 15 of their family members. Nine of the probands had point mutation in the ALAS2 gene and two had a-globin duplications. We measured serum ERFE in the probands and family controls and discovered that ERFE was significantly increased in probands relative to familial controls (Figure 2). Family members had ERFE concentrations similar to healthy first time blood donors (<1 ng/ml). An additional study was conducted to examine ERFE in thalassemia patients whom are known to exhibit ineffective erythropoiesis due to mutations in the α- or β-globin genes that cause production of deformed red blood cells. We obtained sera from patients with both α- and β-thalassemia and discovered that both β+-thalassemia and β0-thalassemia patients had significantly higher serum ERFE concentrations than controls and a group of iron deficient (ID) controls (Figure 3). The β0-thalassemia patients had highly elevated serum ERFE which is likely due to the degree that erythropoiesis is affected. The data we present lends strong support to the quality of the dual monoclonal sandwich ELISA we have developed. The ERFE assay is very sensitive, has excellent reproducibility and spike recovery characteristics, and is easy to perform. The physiological and clinical data we present supports the assertion that the assay is specific for ERFE and will allow insight into a number of hematological diseases. Figure 1 Effect of plasma- or platelet-apheresis on ERFE. Figure 1. Effect of plasma- or platelet-apheresis on ERFE. Figure 2 ERFE in X-linked Sideroblastic Anemia. ****p<0.0001 Figure 2. ERFE in X-linked Sideroblastic Anemia. ****p<0.0001 Figure 3 ERFE in Iron Deficient and Thalassemic Patients. ****p<0.0001, **p<0.005 Figure 3. ERFE in Iron Deficient and Thalassemic Patients. ****p<0.0001, **p<0.005 Disclosures Han: Intrinsic Lifesciences.: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment. Ostland:Intrinsic LifeScienc s: Employment, Equity Ownership. Gutschow:Intrinsic LifeSciences: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership.

Erythroferrone (ERFE) is a hormone produced by erythroblasts in response to erythropoietin. ERFE acts as a regulator of hepcidin expression during stress erythropoiesis. By suppressing hepcidin expression, ERFE contributes to the mobilization of dietary and stored iron necessary for recovery from blood loss after hemorrhage. Furthermore, overproduction of ERFE plays a pathogenic role in β-thalassemia and other anemias with ineffective erythropoiesis, where it contributes to hepcidin suppression and consequent iron overload. Development of a method to quantify serum ERFE in mice would improve our ability to study the pathobiology of this erythroid regulator. A dual polyclonal sandwich ELISA was developed to quantify ERFE in mouse serum. Purified recombinant mouse ERFE was used to immunize rabbits and goats, and high titer antibodies were purified from serum via protein A. Western blotting of reduced ERFE protein demonstrated that both the capture and detection antibody specifically recognized mouse ERFE, weakly recognized recombinant human ERFE, but neither antibody recognized mouse, rabbit or human TNF-alpha. Antibody was biotinylated and screened to determine the optimal antibody pairs. ELISA optimization established the standard curve range from 0 to 4 ng/ml. With a 10% sample dilution the lower limit of quantitation (LLOQ) was 0.1 ng/ml. Average spike recovery of ERFE in 3 different mouse sera (0.75 - 24 ng/ml) ranged from 93-105% (mean 99%). Dilutional linearity of the same spiked samples ranged from 93-104% (mean 99%). Intra- and inter-assay precision was 4.9% and 3.9%, respectively, over a concentration range of 0.57 - 16.3ng/ml. The effect of phlebotomy on serum ERFE in 6- and 8-week-old male C57BL/6 mice (n=3 each) was examined 24 h after removal of 0.5 ml of blood (Figure 1). At time 0, all the mice had serum ERFE levels below the limit of detection, but serum ERFE had increased to a mean of 1.9 ng/ml at 24 hours (P=0.03). This increase in ERFE is associated with a decrease in hepcidin from 209 ng/ml to 104 ng/ml (p<0.0001). In a mouse model of β-Thalassemia (th3/+) there was significantly greater ERFE (1.2 ng/ml, n=5) compared to wild type (wt, <0.1 ng/ml, n=3, p < 0.04). Crossing the th3/+ mice with transferrin receptor 1 (TfR1) heterozygous mice (th3/+ TfR1+/-) produced a partial rescue of ERFE (0.6 ng/ml, n=5). Erfe knockout mice (th3/+ erfe-/- and th3/+ TfR1+/- erfe-/-) produced significantly less ERFE (p<0.02) than their respective single and double mutants (<0.1 ng/ml [n=7] and <0.1 ng/ml [n=4], respectively) (Figure 2). Collectively, this validated ELISA can quantify mouse serum ERFE in both healthy and diseased mouse models and can be used to study the pathobiology of this erythroid regulator in diseases associated with ineffective erythropoiesis. Disclosures Gutschow: Intrinsic LifeSciences: Employment, Equity Ownership. Han:Intrinsic LifeSciences: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees. Nemeth:Silarus Therapeutics: Consultancy, Equity Ownership; Ionis Pharmaceuticals: Consultancy; Protagonist: Consultancy; La Jolla Pharma: Consultancy; Intrinsic LifeSciences: Consultancy, Equity Ownership; Keryx: Consultancy. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership. Ostland:Intrinsic LifeSciences: Employment, Equity Ownership.

Introduction: Hepcidin is the principal regulator of iron absorption and iron recycling, and is required to provide iron for erythropoiesis. Hepcidin is a peptide hormone that binds to the sole iron channel ferroportin (Fpn), and blocks iron efflux by degrading Fpn, trapping iron stored in the cell. Dysregulation of hepcidin by genetic mutations affecting hepcidin expression in hepatocytes is responsible for hereditary hemochromatosis where too little hepcidin leads to iron-overload disease, and iron refractory iron deficiency anemia (IRIDA) where hepcidin expression is inappropriately high leading to anemia. Hepcidin itself is regulated by iron and the inflammatory cytokine IL-6. Thus, hepcidin is dysregulated in most inflammatory diseases and chronic infections. Interest in developing diagnostic applications for hepcidin in iron disorders is high. Objective: We developed a high-affinity monoclonal antibody (mAb) to hepcidin and used it to produce a competitive ELISA (C-ELISA) similar to our first RUO test (Ganz et al. 2008). We determined the performance characteristics using CLIA analytical method validation guidelines. The Intrinsic Hepcidin IDx Test is used for serum and Li-Heparin plasma. We determined the reference range in adult first time blood donors (n=292) and studied an iron deficient (ID) sub-set of the donors. Methods: We developed mouse anti-human hepcidin monoclonal antibodies and coated, blocked, and dried the plates in a temperature and humidity controlled incubator. We used highly purified synthetic hepcidin as reference standard and a biotinylated hepcidin analog as the competitive tracer for the C-ELISA (Peptides International, Louisville, KY). The Intrinsic Hepcidin IDx Test is fully automated on the Biomek FX platform. Serum from first time blood donors (n=292) was obtained, and ferritin, transferrin, plasma iron, CRP, and hepcidin were measured. The reference range for hepcidin was determined and the normal iron status range determined by eliminating iron deficient (ID) donors or those shown to be iron overloaded. Receiver operator characteristic curves (ROC) were plotted to determine the utility of hepcidin and to identify optimal cutoffs of hepcidin as a test of iron deficiency. Results: The mAb used in the Intrinsic Hepcidin IDx Test is specific for human hepcidin. The analytical measurement range (AMR) is 4 - 200 ng/ml. The intra-assay precision CVs range between 7% (at 12 ng/ml) and 4% (at 99 ng/ml). The between-day precision CVs range between 7% (12 ng/ml) and 9% (100 ng/ml). The mean spike recovery across the AMR is 3%. Hepcidin was stable at 4oC for up to 7 days and if specimens are stored at -20oC, for at least 6 months. Hepcidin concentrations determined in blood donors were highly correlated with those measured using our polyclonal RUO C-ELISA (r = 0.95, p<0.001). The reference range is 4.4-54.1 ng/ml for women and 6.1-91.2 ng/ml for men. Clear gender differences were observed: hepcidin was higher in men (median 28.5 ng/ml, n = 143) than in women (median 13.1 ng/ml, n = 149). In addition, the ID group had lower hepcidin (median 6.7 ng/ml) than the normal iron status group (median 21.4 ng/ml, p<0.0001). The area under the ROC curve for hepcidin compared with various ferritin concentrations was greater than 0.90. An optimal hepcidin cutoff less than 10 ng/ml was determined to predict ID with a correct classification rate of greater than 86%. Conclusion: We developed and validated a sensitive, accurate and reproducible C-ELISA, the Intrinsic Hepcidin IDx test, using CLIA validation guidelines. We determined the hepcidin reference ranges by analyzing first-time blood donor serum. We found a significant difference in median hepcidin values between genders. Also, our data show that hepcidin was lower in donors with ID compared to iron replete, healthy donors. Disclosures Gutschow: Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment. Han:Intrinsic Lifesciences.: Employment, Equity Ownership. Ostland:Intrinsic LifeScienc s: Employment, Equity Ownership. Olbina:Intrinsic LifeSciences: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership.