The Discovery of Chicken Foxp3 Demands Redefinition of Avian Regulatory T Cells.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

Identification of an Activating Chicken Ig-like Receptor Recognizing Avian Influenza Viruses.

Chicken Ig-like receptors (CHIRs) represent a multigene family encoded by the leukocyte receptor complex that encodes a variety of receptors that are subdivided into activating CHIR-A, inhibitory CHIR-B, and bifunctional CHIR-AB. Apart from CHIR-AB, which functions as an Fc receptor, CHIR ligands are unknown. In the current study, we used a panel of different BWZ.36 CHIR reporter cells to identify an interaction between specific CHIRs and avian influenza virus (AIV). The specificity of the CHIR-AIV interaction was further demonstrated using CHIR fusion proteins that bound to AIV-coated plates and were able to reduce the interaction of reporter cells with AIV. There was no difference in binding of CHIR to different AIV strains. Furthermore, CHIR fusion proteins reduced AIV-induced in vitro activation of NK cells obtained from lungs of AIV-infected animals, as judged by the lower frequency of CD107+ cells. Because the original CHIR reporter lines were generated based on sequence information about extracellular CHIR domains, we next identified a full-length CHIR that displayed similar binding to AIV. The sequence analysis identified this CHIR as a CHIR-A. Neuraminidase treatment of coated CHIR-human Ig proteins reduced binding of trimeric H5 proteins to CHIR. This suggests that the interaction is dependent on sialic acid moieties on the receptor. In conclusion, this article identifies AIV as a ligand of CHIR-A and describes the functional consequences of this interaction.

[Conservative treatment of an aseptic abscess syndrome with splenic abscesses in Crohn's disease]. / Konservative Therapie eines aseptischen Abszesssyndroms mit Milzabszessen bei Morbus Crohn.

A 24-year old woman with a history of Crohn's disease developed bloody diarrhea and multiple abdominal abscesses, daily fever, leukocytosis, and elevated CRP several months after her immunosuppressive therapy with azathioprine was stopped. Recurrent abscess punctures did not detect any pathogenic germs and neither clinical nor serological response was achieved by administration of different antimicrobial therapies. Additionally, new splenic abscesses arose despite ongoing therapy. Under the suspicion of the rare aseptic abscess syndrome, representing an auto-inflammatory, extra-intestinal manifestation of Crohn's disease, the antimicrobial therapy was stopped and an intravenous therapy with prednisolone was initiated. As soon as therapeutic response was achieved, an additional anti-TNF therapy with Infliximab was started and subsequently the intraabdominal and splenic abscesses disappeared.The knowledge of the aseptic abscess syndrome, which is characterized by (a) sterile abscesses with neutrophilic granulocytes, (b) negative blood cultures, (c) lack of response to antimicrobial treatment, and (d) rapid clinical improvement after initiation of prednisolone therapy with subsequent response in imaging, may avoid unnecessary operations like splenectomy in the present case. The exact pathophysiology of the aseptic abscess syndrome is unknown but, with regard to the sterile aspirates, an auto-inflammatory cause has been suggested. Data of a French case collection demonstrate that this syndrome may be present more frequently than expected in patients with chronic inflammatory bowel diseases. Up to now, this syndrome has not been described in German literature.

Multimodal and sequential treatment improves survival in patients with hepatocellular carcinoma.

Background and aims Therapy of hepatocellular carcinoma (HCC) mainly depends on tumor stage and liver function. The aim of this study was to identify additional predictors of overall survival in HCC patients with a particular attention to multimodal therapies. Methods Six hundred and seven consecutive HCC-patients treated in a tertiary center between 1988 and 2011 were retrospectively analyzed. Multivariate analysis was performed by logistic and Cox-regression, overall survival was analyzed by Kaplan Meier statistics. Results In comparison to unimodal therapies, multimodal treatment increased overall survival in BCLC-A patients from 16 to 26 months (p < 0.001), in patients with BCLC-B stage from 9.5 to 16 months (p < 0.001), in BCLC-C patients from 6 to 18 months (p < 0.001), and in stage BCLC-D from 2 to 8 months (not significant). Survival increased throughout all Child Pugh scores, and patients experienced benefits from multimodal therapy irrespective of alfa-fetoprotein levels. Comparing the time span 1988 - 1999 with 2000 - 2011, the rate of multimodal/sequential treatment increased from 12.3 % to 30 % (p < 0.001), and the overall survival of all (treated and non-treated) patients increased from 7 months (1988 - 1999) to 10 months (2000 - 2011, p < 0.001). In multivariate analysis, multimodal treatment was shown to be an independent predictor for overall survival besides elevated alfa-fetoprotein, Child Pugh score, and BCLC stage. Conclusion Multimodal therapies increase overall survival in HCC patients and should be considered in patients with HCC if practicable.

Evaluation of combined Gd-EOB-DTPA and gadobutrol magnetic resonance imaging for the prediction of hepatocellular carcinoma grading.

BACKGROUND: Tumor biopsy is not essential for the diagnosis of hepatocellular carcinoma (HCC); however, grading remains important for the prognosis. PURPOSE: To investigate whether combined Gd-EOB-DTPA and gadobutrol liver magnetic resonance imaging (MRI) can predict HCC grading. MATERIAL AND METHODS: Thirty patients (66.6 ± 7.3 years) with histologically confirmed HCC (grade 1, n = 5; grade 1-2, n = 6; grade 2, n = 13; grade 2-3, n = 2; grade 3, n = 4) underwent two liver MRIs, one with gadobutrol and one with Gd-EOB-DTPA, on consecutive days. Blinded to grading, two radiologists reviewed the gadobutrol and Gd-EOB-DTPA images in consensus with respect to: (i) HCC hyper-/iso-/hypointensity in the arterial, portal-venous/delayed, and Gd-EOB-DTPA hepatocellular phase; and (ii) morphologic tumor features (encapsulated growth, vessel invasion, heterogeneity, liver capsule infiltration, satellite metastases). RESULTS: A significant correlation with grading was not found for either the combined dynamic information of all gadobutrol phases (r = -0.187, P = 0.331) or all the Gd-EOB-DTPA phases (r = 0.052, P = 0.802). No correlation with grading was found for a combination of arterial and hepatocellular phase in Gd-EOB-DTPA MRI (r = 0.209, P = 0.305), a combination of both arterial phases (gadobutrol and Gd-EOB-DTPA) with the Gd-EOB-DTPA hepatocellular phase (r = 0.240, P = 0.248), or a combination of all available gadobutrol and Gd-EOB-DTPA phases (r = 0.086, P = 0.691). For all gadobutrol information (dynamic phases and morphology; r = 0.049, P = 0.801) and for all Gd-EOB-DTPA information (r = 0.040, P = 0.845), no correlation with grading was found. Hepatocellular Gd-EOB-DTPA phase iso-/hyperintensity never occurred in grade 3 HCCs. CONCLUSION: Histological HCC grading cannot be predicted by combined Gd-EOB-DTPA/gadobutrol MRI. However, Gd-EOB-DTPA hepatocellular phase iso-/hyperintensity was never detected in grade 3 HCCs.

Vγ9 and Vδ2 T cell antigen receptor genes and butyrophilin 3 (BTN3) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (Vicugna pacos).

Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.

The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes.

In most mammals, the MHC class I molecules are polymorphic and determine the specificity of peptide presentation, whereas the transporter associated with antigen presentation (TAP) heterodimers are functionally monomorphic. In chickens, there are two classical class I genes but only one is expressed at a high level, which can result in strong MHC associations with resistance to particular infectious pathogens. However, the basis for having a single dominantly expressed class I molecule has been unclear. Here we report TAP1 and TAP2 sequences from 16 chicken lines, and show that both genes have high allelic polymorphism and moderate sequence diversity, with variation in positions expected for peptide binding. We analyze peptide translocation in two MHC haplotypes, showing that chicken TAPs specify translocation at three peptide positions, matching the peptide motif of the single dominantly expressed class I molecule. These results show that coevolution between class I and TAP genes can explain the presence of a single dominantly expressed class I molecule in common chicken MHC haplotypes. Moreover, such coevolution in the primordial MHC may have been responsible for the appearance of the antigen presentation pathways at the birth of the adaptive immune system.

Chicken γδ T cells proliferate upon IL-2 and IL-12 treatment and show a restricted receptor repertoire in cell culture.

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8-, and TCR1lowCD8- subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8- subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function.

Characterization of a novel chicken γδ TCR-specific marker.

Chickens are a species with a high number of γδ T cells in various tissues. Despite their abundance, γδ T cells are poorly characterized in chickens, partially due to a lack of specific reagents to characterize these cells. Up until now, the TCR1 clone has been the only γδ T cell-specific monoclonal antibody (mAb) in chickens and additional reagents for γδ T cell subsets are needed. In order to address this issue, new mAb were generated in our laboratory by immunizing mice with in vitro cultured γδ T cells. In an initial flow cytometric screen a new mAb, clone "8D2", displayed an interesting staining pattern that mirrored γδ TCR up- and downregulation in the γδ T cell line D4 over time, prompting us to characterize this antibody further. We compared the expression of the unknown 8D2 epitope in combination with TCR1 staining across various primary cells. In splenocytes, peripheral blood lymphocytes and intestinal epithelial cells, 8D2 consistently labeled a subset of TCR1+ cells. To determine, whether specific γδ T cell receptors were recognized by 8D2, we sorted γδ T cells according to their 8D2 and TCR1 expression and analyzed their TCR V(D)J gene usage by TCR profiling. Strikingly, sorted 8D2+ cells preferentially expressed Vγ3 genes, whereas the TCR Vγ genes used by TCR1+ 8D2- cells were more variable. γδ TCR in 8D2+ cells were most frequently comprised of gamma chain VJ genes TRGV3-8 and TRGJ3, and delta chain VDJ genes TRDV1-2, TRDD2, TRDJ1. To confirm binding of 8D2 to specific γδ TCR, the preferentially utilized combination of TRG and TRD was expressed in HEK293 cells in combination with CD3, demonstrating surface binding of the 8D2 mAb to this Vγ3 γδ TCR-expressing cell line. Conversely, HEK293 cells expressing either Vγ1 or Vγ2 TCR did not react with 8D2. In conclusion, 8D2 is a novel tool for identifying specific Vγ3 bearing γδ T cells.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

The chicken SLAM family.

The signaling lymphocytic activation molecule (SLAM) family of receptors is critically involved in the immune regulation of lymphocytes but has only been detected in mammals, with one member being present in Xenopus. Here, we describe the identification, cloning, and analysis of the chicken homologues to the mammalian SLAMF1 (CD150), SLAMF2 (CD48), and SLAMF4 (CD244, 2B4). Two additional chicken SLAM genes were identified and designated SLAMF3like and SLAM5like in order to stress that those two receptors have no clear mammalian counterpart but share some features with mammalian SLAMF3 and SLAMF5, respectively. Three of the chicken SLAM genes are located on chromosome 25, whereas two are currently not yet assigned. The mammalian and chicken receptors share a common structure with a V-like domain that lacks conserved cysteine residues and a C2-type Ig domain with four cysteines forming two disulfide bonds. Chicken SLAMF2, like its mammalian counterpart, lacks a transmembrane and cytoplasmic domain and thus represents a glycosyl-phosphatidyl-inositol-anchored protein. The cytoplasmic tails of SLAMF1 and SLAMF4 display two and four conserved immunoreceptor tyrosine-based switch motifs (ITSMs), respectively, whereas both chicken SLAMF3like and SLAMF5like have only a single ITSM. We have also identified the chicken homologues of the SLAM-associated protein family of adaptors (SAP), SAP and EAT-2. Chicken SAP shares about 70 % identity with mammalian SAP, and chicken EAT-2 is homologous to mouse EAT-2, whereas human EAT-2 is much shorter. The characterization of the chicken SLAM family of receptors and the SAP adaptors demonstrates the phylogenetic conservation of this family, in particular, its signaling capacities.

Identification of a chicken CLEC-2 homologue, an activating C-type lectin expressed by thrombocytes.

Receptors on natural killer (NK) cells are classified as C-type lectins or as Ig-like molecules, and many of them are encoded by two genomic clusters designated natural killer gene complex (NKC) and leukocyte receptor complex, respectively. Here, we describe the analysis of an NKC-encoded chicken C-type lectin, previously annotated as homologue to CD94 and NKG2 and thus designated chicken CD94/NKG2. To further elucidate its potential function on NK cells, we produced a specific mab by immunizing with stably transfected HEK293 cells expressing this lectin. Staining of various chicken tissues revealed minimal reactivity with bursal, or thymus cells. In peripheral blood mononuclear cell and spleen, however, the mab reacted with virtually all thrombocytes, whereas most NK cells in organs such as embryonic spleen, lung and intestine were found to be negative. These findings indicate that the gene may not resemble CD94/NKG2, but rather a CLEC-2 homologue, a claim further supported by sequence features such as an additional extracellular cysteine residue and the presence of a cytoplasmic motif known as a hem immunoreceptor tyrosine-based activation motif, found in C-type lectins such as Dectin-1, CLEC-2, but not CD94/NKG2. The biochemical analyses demonstrated that CLEC-2 is present on the cell surface as heavily glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. These analyses reveal that the chicken NKC may not encode NK cell receptor genes, in particular not CD94 or NKG2 genes, and identifies a chicken CLEC-2 homologue.

High prevalence of significant liver fibrosis and cirrhosis in chronic hepatitis B patients with normal ALT in central Europe.

The indication for antiviral treatment of patients with chronic hepatitis B is based on serum HBV DNA levels, transaminases, and histological grade and stage. The relation of liver fibrosis and inflammation to ALT activity in chronic hepatitis B infection was investigated in a nonendemic, European setting. A total of 253 patients with chronic hepatitis B who had undergone liver biopsy at the Clinic of Gastroenterology, Hepatology, and Infectious Diseases, Düsseldorf,Germany over the past 19 years (1990­2009) were evaluated. Thirty-nine patients had persistently normal transaminases, 86 patients had ALT with 1­2 x ULN (upper limit of normal) and 128 patients had ALT >2 x ULN. Liver fibrosis or inflammation was defined as significant for stages or grades ≥ 2 according to the Desmet/Scheuer score. Significant liver fibrosis (F ≥ 2)was found in 36%, cirrhosis in 18%, and significant inflammation (G ≥ 2) in 27% of patients with normal transaminases. There was no difference in the stage of liver fibrosis and the frequency of cirrhosis between patients with normal and elevated transaminases. The most important factor associated with the presence of cirrhosis in multivariate analysis was age ≥ 40 years (P < 0.003). If concomitant factors like elevated GGT or male sex were furthermore present high prevalences of significant liver disease were found. The data indicate that, in a European setting, patients with chronic hepatitis B infection, and normal transaminases frequently have significant liver fibrosis or cirrhosis.Therefore, liver biopsy or liver stiffness measurement (LSM) should be performed in these patients to determine the stage of liver fibrosis.

Chicken IgY binds its receptor at the CH3/CH4 interface similarly as the human IgA: Fc alpha RI interaction.

Chicken IgY, the ancestral form of mammalian IgE and IgG, is recognized by the high-affinity FcY receptor CHIR-AB1, a member of the leukocyte receptor family. In this study, we have characterized the receptor ligand interaction site by consecutive truncations of the Fcv IgY domains and mutational analyses of selected residues. Using several fusion proteins that linked the human Cgamma2 and Cgamma3 domains with the Fcv IgY domains, a binding assay revealed that both the Fcv3 and Fcv4 domains were essential for the IgY CHIR-AB1 interaction. Sequence comparisons of chicken IgY with human IgA demonstrated that 11 of the 19 contact residues important for the IgA FcalphaRI interaction have been conserved in chicken IgY, although the overall amino acid identity is only 34%. Among the 19 amino acids at respective positions in IgY, the mutation of two residues in the Fcv3 and two in the Fcv4 domain completely abolished the IgY to CHIR-AB1 binding revealed by two independent assays. Three further mutations substantially altered the interaction. Molecular modeling on the Cv3 to Cv4 crystal structure revealed that all critical residues, although on two domains, are in close proximity. The importance of N-linked carbohydrates was demonstrated by the failure of the CHIR-AB1 interaction after mutation of the glycosylation site. The identification of the IgY Cv3/Cv4 interdomain region as critical for binding to CHIR-AB1 significantly enhances our understanding of the IgY receptor interaction and allows further conclusions regarding the FcR phylogeny.

The chicken leukocyte receptor complex encodes a family of different affinity FcY receptors.

Chicken Ig-like receptors (CHIR) form a large family in the leukocyte receptor complex on microchromosome 31 with inhibitory, activating, and bifunctional receptors. Recently, we characterized CHIR-AB1 as a high-affinity, primordial FcY receptor. Given that the CHIR family represents a multigene family, it is plausible that more than a single receptor binds to IgY. Therefore, after comparing CHIR-AB1-like sequences in databases, we cloned CHIR-AB1 homologues from two individual chickens representing the lines M11 and R11 with primers binding to highly conserved regions. In both lines this approach yielded 18 different CHIR-AB amino acid versions, with one sequence out of each line that was identical with the previously characterized B19 CHIR-AB1 Ig domain and two additional R11-M11 identical sequence pairs. All M11-derived CHIR-AB homologues were then expressed as soluble human Ig fusion proteins. Following standardization of the fusion protein concentration with an ELISA, the IgY, IgM, and IgA binding activities were determined by ELISA. Six fusion proteins recognized IgY, whereas none bound to IgM and IgA. The affinities of selected fusion proteins were determined using surface plasmon resonance yielding an equilibrium binding constant between 25 nM for high binders and 260 nM for low binders. Sequence comparisons and subsequent mutational analysis of selected residues identified five amino acids that are potentially involved in IgY binding. These results imply that multiple FcY receptors of variable affinity are encoded by the CHIR locus and that different chicken lines may express both unique as well as highly conserved FcY receptors.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.