The Fya, Fy6 and Fy3 epitopes of the Duffy blood group system recognized by new monoclonal antibodies: identification of a linear Fy3 epitope

Four new anti-Duffy murine monoclonal antibodies (MAbs): two anti-Fy6 (MIMA-107 and MIMA-108), one anti-Fya (MIMA-19) and one anti-Fy3 (MIMA-29) were characterized. Identification of epitopes by means of synthetic peptides (Pepscan) showed that the anti-Fy6 reacted most strongly with peptides containing the sequence 19QLDFEDV25 of the Duffy glycoprotein, and less strongly with peptides containing LDFEDV (MIMA-107) or LDF only (MIMA-108). The anti-Fya recognized epitope 38DGDYGA43 containing the Gly42 residue, which defines the Fya blood group antigen. MIMA-29 is the first anti-Fy3 reactive with a linear epitope 281ALDLL285 located in the fourth extracellular domain (ECD4, loop 3) of the Duffy glycoprotein. The four new antibodies extend the list of six anti-Fy MAbs formerly characterized by Pepscan analysis that allow some general conclusions. Fine specificities of various anti-Fya, or anti-Fy6 are not identical, but all of them recognize linear epitopes located around, respectively, Gly42 or between two potential N-glycosylation sites at Asn16 and Asn27. Anti-Fy3 recognize either a linear epitope located in ECD4, or a conformational epitope that includes amino acid residues of ECD4 and of other ECDs.     

Study of Anti-Fya in Five Black Fy(a-b-) Patients

Fya antibodies were detected in 5 Fy(a-b-) black patients during a 5-year period. The Duffy phenotype was confirmed using anti-Fya, anti-Fyb, and anti-Fy3 by an adsorption-elution technique. These 5 patients had sickle cell disease and had received multiple blood transfusions. The antibodies were all found during the investigation of a hemolytic transfusion reaction or failure to achieve the desired red cell increments after transfusion.     

A New Anti-Erythrocyte Antibody in the Duffy System: Anti-Fy4

Abstract. A new Duffy antibody, designated anti-Fy4, was found to react with all samples of type Fy(a-b-), some of type Fy(a-b+) and Fy(a+b-), and none of type Fy(a+b+).     

Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

OBJECTIVE: The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium. METHODS: To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS: EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION: This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.     

The Duffy Blood Group Determinants: Their Role in the Susceptibility of Human and Animal Erythrocytes to Plasmodium knowlesi Malaria

Duffy blood group negative erythrocytes from blacks are refractory to invasion by Plasmodium knowlesi merozoites in vitro, and blacks with this genotype are resistant to infection by P. vivax in vivo. In order to evaluate in a direct manner the role of Duffy blood group determinants in invasion by P. knowlesi merozoites, we studied erythrocytes from three rare non-black Duffy negative individuals, Fy(a-b-), in whom the Duffy negative phenotype probably represents a mutation and not the introduction of the black Fy gene. These cells were resistant to invasion by P. knowlesi in vitro indicating that resistance to invasion is mediated by the FyFy genotype and not another closely linked factor. The erythrocyte receptors for invasion, however, may not be the Fya or Fyb Duffy antigens themselves, or at least not restricted to these determinants, since refractory Duffy negative human erythrocytes were invaded after treatment with trypsin or neuraminidase although these enzyme-treated cells still lacked Fy a and Fy b determinants. Furthermore, new world monkey erythrocytes and chymotrypsinized chimpanzee and kra monkey erythrocytes were invaded, although there was no serologic evidence of Fya or Fyb determinants on these cells.     

Anti-Fy5, an Antibody Disclosing a Probable Association between the Rhesus and Duffy Blood Group Genes

Abstract. A new antibody, anti-Fy5, has been found in the serum of a Negro Fy(a–b–) person. It reacts with Fy(a+) or Fy(b+) red cells of common Rh type, but not with Fy(a–b–) cells from Negroes, or with Rh_null cells. An example of Caucasian Fy(a–b–) red cells was Fy5 positive. U-negative or LW-negative red cells give the reaction expected of their Duffy type. The data suggest that the Fy5 determinant is formed by interaction of Rhesus and Duffy gene products.     

Duffy blood group and malaria

Very important progress has been made over the last years in understanding the Duffy blood group system and its complexity. The Duffy blood group antigen serves not only as blood group antigen, but also as a receptor for a family of proinflammatory cytokines termed chemokines, and as a receptor for Plasmodium vivax malaria parasites. The Duffy antigen has been termed the "Duffy Antigen Receptor for Chemokines" (DARC) or the Duffy chemokine receptor. DARC might playa role as a scanvenger on the red blood cell surface to eliminate excess of toxic chemokines produced in some pathologic situations [48].

Plasmodium vivax (P. vivax) causes approximately between 70 and 80 million cases of malaria per year and is the most amply distributed human malaria in the world [51]. Individuals with the Duffy-negative phenotype are resistant to P. vivax invasion, and the molecular mechanism that gives rise to the phenotype Fy(a – b – ) in black individuals has been associated with a point mutation ?33TC expressed in homozigosity in the FYB allele [5]. Despite P. vivax be widespread throughout the tropical and subtropical world, it is absent from West Africa, where more than 95% of the population is Duffy negative. Recently, this point mutation has been described in heterozigosity in the FYA allele in others malaria endemic regions [7, 8], and until now we do not know if it confers a certain degree of protection against P. vivax infection.     

Duffy blood group and malaria

Very important progress has been made over the last years in understanding the Duffy blood group system and its complexity. The Duffy blood group antigen serves not only as blood group antigen, but also as a receptor for a family of proinflammatory cytokines termed chemokines, and as a receptor for Plasmodium vivax malaria parasites. The Duffy antigen has been termed the "Duffy Antigen Receptor for Chemokines" (DARC) or the Duffy chemokine receptor. DARC might play a role as a scanvenger on the red blood cell surface to eliminate excess of toxic chemokines produced in some pathologic situations [48]. Plasmodium vivax (P. vivax) causes approximately between 70 and 80 million cases of malaria per year and is the most amply distributed human malaria in the world [51]. Individuals with the Duffy-negative phenotype are resistant to P. vivax invasion, and the molecular mechanism that gives rise to the phenotype Fy(a - b - ) in black individuals has been associated with a point mutation - 33TC expressed in homozigosity in the FYB allele [5]. Despite P. vivax be widespread throughout the tropical and subtropical world, it is absent from West Africa, where more than 95% of the population is Duffy negative. Recently, this point mutation has been described in heterozigosity in the FYA allele in others malaria endemic regions [7, 8], and until now we do not know if it confers a certain degree of protection against P. vivax infection.     

A Second Example of Anti-Fy51

Abstract. Anti-Fy5 has been found in the serum of a transfused Fy(a–b–) Negro woman. The antibody reacts with red cells that are Fy(a+) or Fy(b+) or both, but not with Fy(a–b–) cells or with cells of the Rh_null phenotype. Rh_null red cells of both amorph and regulator types are nonreactive with anti-Fy5. If interaction between Rhesus and Duffy gene products is necessary for Fy5 antigen synthesis, it must be the Rh gene itself and not the X¹r modifier that is involved.     

The Duffy protein: a malarial and chemokine receptor

A major advance towards understanding the Duffy blood group system has been achieved with the cloning of FY, a single-copy gene located in the 1q22->q23 region of chromosome 1. The product of FY Is an acidic glycoprotein (gp-Fy), which spans the plasma membrane seven times and has an exocellular N-terminal domain and an endocellular C-terminal domain. The system consists of four alleles, five phenotypes, and five antigens. FYA, FYB, FYB(ES), and FYB(WK) are the alleles; Fy(a+b-), Fy(a-b+), Fy(a+b+), Fy(a-b+(wK)), and Fy(a-b-), are the phenotypes, and Fy(a), Fy(b), Fy3, Fy5, and Fy6 are the antigens. Fy(a-b-), or Duffy-negative individuals, lack the Duffy protein on erythrocytes and are predominantly African and American blacks. They have the FYB(Es) allele with a mutation in the promoter region, which abolishes the expression of the protein in erythrocytes only. In the few cases of non-black Fy(a-b-) individuals, a nonsense mutation prevents the synthesis of gp-Fy. In Fy(a-b+(wk)) erythrocytes, the Fy(b) antigen is weakly expressed due to a reduced amount of the protein. The Fy5 antigen includes the Rh protein, and the Fy6 antigen is defined by a murine monoclonal antibody. Gp-Fy is produced in several cell types, including endothellal cells of capillary and postcapillary venules, epithelial cells of kidney collecting ducts, and lung alveoli, as well as PurkinJe cells of the cerebellum. The Duffy protein plays a role in inflammation and in malaria Infection. The protein is a member of the superfamily of chemokine receptors and is the receptor to which certain malarial parasites bind to invade red blood cells. The parasite-specific binding site, the binding site of chemokines, and the major antigenic domains are located in overlapping regions at the exocellular N terminus of the Duffy protein.     

血管紧张素Ⅱ诱导内皮细胞产生IL-8和Duffy抗原/趋化因子受体的研究

目的:探讨Duffy抗原/趋化因子受体(DARC)和IL-8在血管紧张素II(AngⅡ)致炎机制中可能的作用.方法:体外培养人脐静脉内皮细胞,分别用PBS(对照组)、AngⅡ(10-7 mol/L)诱导0～24 h,收集0、6、12、24 h 4个时间点的细胞培养上清和内皮细胞,采用ELISA法分别检测细胞培养上清和内...     

Mutations in the erythrocyte chemokine receptor (Duffy) gene: the molecular basis of the Fya/Fyb antigens and identification of a deletion in the Duffy gene of an apparently healthy individual with the Fy(a – b–) phenotype

Summary. The erythrocyte chemokine receptor, a receptor for Plasmodium vivax , carries the antigens of the Duffy blood group system. Sequence analysis of reticulocyte RNA from individuals of known Duffy phenotype showed that the Fy^a antigen differs from the Fy^b antigen as a result of a single nucleotide difference (A¹³¹ or G) encoding amino acid Gly⁴⁴ (Fy^a) or Asp (Fy^b) in the N-terminal extracellular domain of the glycoprotein. Evidence is presented for two different genetic backgrounds giving rise to the Fy(a - b-) phenotype. The most likely genetic mechanism in most individuals of the Fy(a - b–) phenotype is down-regulation of Duffy glycoprotein mRNA. However, the Duffy gene from a very rare Caucasian individual (AZ) with the Fy(a – b–) phenotype has a 14 base-pair deletion (nucleotides 287-301) resulting in a frameshift which introduces a stop codon and produces a putative truncated 118 amino acid protein. The occurrence of this mutation in an apparently healthy individual raises questions about the functional importance of the Duffy glycoprotein not only in normal erythrocytes but also in all human cells and tissues.     

Expression of DARC, CXCR3 and CCR5 in giant cell arteritis

OBJECTIVES: Leucocyte infiltration is the hallmark of vasculitis, chemokines being mainly responsible for leucocyte migration into inflamed tissues. The objective was to evaluate the local expression of chemokines and chemokine receptors in biopsies of patients with giant cell arteritis (GCA) compared with arteries from patients with polymyalgia rheumatica (PMR). We studied the expression of CCR5, CXCR3 and that of the Duffy antigen/receptor of chemokine (DARC), a chemokine internalizing receptor (interceptor), in parallel to the expression of the CCR5 ligand RANTES/CCL5. METHODS: Paraffin-embedded tissue sections from six patients with GCA and five patients with PMR were available for immunohistological analysis of chemokine receptor expression. RANTES/CCL5 mRNA was detected in tissue sections by in situ hybridization. RESULTS: In patients with biopsy-proven giant cell arteritis, CCR5 and CXCR3 were highly expressed by infiltrating leucocytes in involved tissue sections. Predominant clustering of CCR5+ and CXCR3+ leucocytes was found in the adventitia and was co-localized with the expression of CCL5/RANTES mRNA. Interestingly, we found marked expression of DARC on adventitial high endothelial venules in vasculitis lesions of patients with GCA, while in arteries from patients with PMR DARC was only expressed on a low number of vessels with flat lining endothelium. CONCLUSIONS: The co-localization of infiltrating CCR5+ and CXCR3+ leucocytes together with CCL5/RANTES and DARC in vasculitis lesions suggests a role for these chemokine receptors in leucocyte infiltration, possibly supported by DARC-mediated vascular presentation of chemokines.     

Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria

Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fy(a) or Fy(b), resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fy(a), compared with Fy(b), significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fy(a) had 41-50% lower binding compared with Fy(b) cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fy(a+b-) phenotype demonstrated a 30-80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fy(a+b-) phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.     

Identification of Human Norovirus GII.3 Blockade Antibody Epitopes

Human noroviruses are a common pathogen causing acute gastroenteritis worldwide. Among all norovirus genotypes, GII.3 is particularly prevalent in the pediatric population. Here we report the identification of two distinct blockade antibody epitopes on the GII.3 capsid. We generated a panel of monoclonal antibodies (mAbs) from mice immunized with virus-like particle (VLP) of a GII.3 cluster 3 strain. Two of these mAbs, namely 8C7 and 8D1, specifically bound the parental GII.3 VLP but not VLPs of GII.4, GII.17, or GI.1. In addition, 8C7 and 8D1 efficiently blocked GII.3 VLP binding with its ligand, histo-blood group antigens (HBGA). These data demonstrate that 8C7 and 8D1 are GII.3-specific blockade antibodies. By using a series of chimeric VLPs, we mapped the epitopes of 8C7 and 8D1 to residues 385–400 and 401–420 of the VP1 capsid protein, respectively. These two blockade antibody epitopes are highly conserved among GII.3 cluster 3 strains. Structural modeling shows that the 8C7 epitope partially overlaps with the HBGA binding site (HBS) while the 8D1 epitope is spatially adjacent to HBS. These findings may enhance our understanding of the immunology and evolution of GII.3 noroviruses.     

Duffy antigen expression on reticulocytes does not alter following blood loss in an autologous donation model

Background  The Duffy blood group (Fy) antigen functions as the receptor whereby the malarial parasite Plasmodium vivax invades reticulocytes. In this study, we evaluated an autologous blood donation model to measure Fy expression during the anticipated response to blood loss.
Aims  This study aims to examine Fy expression following anticipated reticulocytosis in response to blood loss from autologous whole blood donation.
Method  Subjects were healthy blood donors presenting for planned collection of two or three autologous units. Whole blood (450 ml ± 10%) was collected and processed. Blood samples for Fy testing were obtained from the donations. These were assayed by flow cytometry by measuring binding of a phycoerythrin-labelled anti-Fy6 antibody and compared against reticulocyte numbers. Reticulocyte numbers were measured using thiazole orange. Results were compared from baseline (first donation) with samples at second and, if available, third, donations. Phenotyping for Fy a and b antigens was performed.
Results  Reticulocytes increased by a mean of 37% over baseline [0·93% (range 0·31–1·93) to 1·23% (0·32–3·51%)] following donation of two ( n = 32) or three ( n = 9) autologous whole blood units. Absolute reticulocyte count remained low. Mean and median Fy expression on mature red blood cells and reticulocytes did not change from baseline levels despite individual variation. No apparent relationship to serologically determined Fy a and/or b antigen status was present.
Conclusion  Baseline expression of Fy antigen on mature red blood cells and reticulocytes is quite variable between individuals, but appears not to be greatly affected by mild to moderate reticulocytosis following blood loss in an autologous blood donation model.     

Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization

Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.     

The coding sequence of Duffy blood group gene in humans and simians: restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy- negative individuals

The coding and untranslated flanking sequences of Duffy gene (FY) in humans and simians are in a single exon. The difference between the two codominant alleles, FY*A and FY*B, is a single change at nucleotide 306: guanidine is in FY*A and adenine is in FY*B. This produces a codon change that subsequently modifies the amino acid at position 43 of gpFy, the major subunit of the Duffy blood group protein complex. The glycine at this position in antigen Fya exchanges with aspartic acid in antigen Fyb. The guanidine at nucleotide 306 creates an additional Ban I restriction site in FY*A. Ban I digestion of DNA-PCR amplified products of FY*B and FY*A yields three and four fragments, respectively. Restriction fragment length polymorphism (RFLP) studies show that Fy(a+b-) and Fy(a-b+) whites are FY homozygous, that most Fy(a-b-) blacks have FY*B, and most Fy(a+b-) blacks are FY*A/FY*B heterozygous. In the black population a silent FY*B is very common, but a silent FY*A has not been found yet. On RNA blot analysis, the gpFy cDNA clone detected mRNA in the lung, spleen, and colon but not in the bone marrow of Duffy-negative individuals. Therefore, there is no null phenotype in Fy(a-b-) blacks. The gpFy homology between human and chimpanzee is 99% with a single residue change at position 116 (valine to isoleucine), whereas a 94% homology is found in squirrel and rhesus monkeys, and there is a 93% homology in aotus monkey when compared with humans. The N-terminal exocellular domain of simian gpFy helps to identify a set of amino acids critical for antibody and malarial parasite specificities.     

Integrin GPIIIa49-57 is the pivotal switch controlling platelet fragmentation

Unique autologous antibodies (Abs) against platelet integrin GPIIIa49-66 (CAPESIEFPVSEARVLED) have been detected in patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP), which is capable of inducing complement-independent platelet fragmentation through reactive oxygen species (ROS) release. However, the efficiency of inducing platelet fragmentation is inconsistent among the different patient Abs or similar rabbit polyclonal Abs against the region and the reason remains unclear. In this study, we developed a batch of murine monoclonal antibodies (mAbs) against different locus of GPIIIa49-66 region by hybridoma technology. All these mAbs are capable of binding to human platelets. Among these mAbs, clones 1E7 and 5A10 were identified to target the epitope of GPIIIa49-57 (CAPESIEFP, named P1); clones 1C1 and 1E5 target GPIIIa57-64 (PVSEARVL, named P2), and clones 4D5 and 5F8 target GPIIIa59-66 (SEARVLED, named P3). By incubation of human platelets with these mAbs, the platelet fragmentation induced by mAbs against P1 was 5–6 folds higher than that by the control mAb (6-fold for 5A10 and 5.6-fold for 1E7). However, platelet fragmentation induced by mAbs against P2 (1C1) and P3 (5F8) was only 1.9- and 1.1-fold higher than that by the control mAb, respectively. Thus, our data demonstrate that platelet integrin GPIIIa49-57 is the pivotal switch controlling platelet fragmentation.