Analysis of immunoglobulin transcripts in the ostrich Struthio camelus, a primitive avian species.
ABSTRACT: Previous studies on the immunoglobulin (Ig) genes in avian species are limited (mainly to galliformes and anseriformes) but have revealed several interesting features, including the absence of the IgD and Ig? encoding genes, inversion of the IgA encoding gene and the use of gene conversion as the primary mechanism to generate an antibody repertoire. To better understand the Ig genes and their evolutionary development in birds, we analyzed the Ig genes in the ostrich (Struthio camelus), which is one of the most primitive birds. Similar to the chicken and duck, the ostrich expressed only three IgH chain isotypes (IgM, IgA and IgY) and ? light chains. The IgM and IgY constant domains are similar to their counterparts described in other vertebrates. Although conventional IgM, IgA and IgY cDNAs were identified in the ostrich, we also detected a transcript encoding a short membrane-bound form of IgA (lacking the last two C(H) exons) that was undetectable at the protein level. No IgD or ? encoding genes were identified. The presence of a single leader peptide in the expressed heavy chain and light chain V regions indicates that gene conversion also plays a major role in the generation of antibody diversity in the ostrich. Because the ostrich is one of the most primitive living aves, this study suggests that the distinct features of the bird Ig genes appeared very early during the divergence of the avian species and are thus shared by most, if not all, avian species.
Project description:Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.
Project description:Only three Ig isotypes, IgM, IgX, and IgY, were previously known in amphibians. Here, we describe a heavy-chain isotype in Xenopus tropicalis, IgF (encoded by C(phi)), with only two constant region domains. IgF is similar to amphibian IgY in sequence, but the gene contains a hinge exon, making it the earliest example, in evolution, of an Ig isotype with a separately encoded genetic hinge. We also characterized a gene for the heavy chain of IgD, located immediately 3' of C(mu), that shares features with the C(delta) gene in fish and mammals. The latter gene contains eight constant-region-encoding exons and, unlike the chimeric splicing of muC(H)1 onto the IgD heavy chain in teleost fish, it is expressed as a unique IgD heavy chain. The IgH locus of X. tropicalis shows a 5' V(H)-D(H)-J(H)-C(mu)-C(delta)-C(chi)-C(upsilon)-C(phi) 3' organization, suggesting that the mammalian and amphibian Ig heavy-chain loci share a common ancestor.
Project description:BACKGROUND:Immunoglobulin (Ig) D is largely localized to the upper airway and reacts with colonizing respiratory pathogens. OBJECTIVE:To determine whether chronic rhinosinusitis (CRS) is associated with increased IgD expression. METHODS:We performed immunofluorescent staining for cytoplasmic IgD, IgA, IgM, and surface plasma cell marker CD138 (syndecan-1) in sinus tissue of patients with CRS with and without nasal polyps (CRSwNP and CRSsNP, respectively) and control subjects without CRS (n = 6 each). Sinonasal mucus antibody levels of patients with CRSwNP or CRSsNP and control subjects were measured by enzyme-linked immunosorbent assay (n = 13, 11, and 9 subjects, respectively). Cells per square millimeter and antibody levels were compared by analysis of variance. Histopathology was performed with sinus tissue from subjects in the 3 groups (n = 6, 8, and 13 subjects respectively). RESULTS:Cells expressing cytoplasmic IgD exceeded those with cytoplasmic IgA and IgM and represented most CD138+ plasma cells in the lamina propria. The frequencies of IgD+ plasma cells were significantly higher in patients with CRSsNP and CRSwNP compared with control subjects (P < .01). Only patients with CRSwNP showed increased frequencies of IgM and IgA plasma cells (P < .01). In contrast to high plasma cell frequencies in tissues, the levels of secreted IgD were lower than those of IgA, IgM, and IgG but were highest in the CRSwNP group compared with the other groups (P < .05). CONCLUSION:IgD plasma cells are prominent in sinus tissues and are increased in CRS. That IgD protein also shows the lowest concentration of antibodies in secretions suggests that its activity might be targeted to the tissue rather than secretions.
Project description:Nematode infections may induce immune-modulatory effects and influence host-immune responses to other pathogens. The aim of the study was to investigate whether a mixed nematode-infection influences non-specific and vaccine-induced humoral immunity against Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), and Avian Metapneumovirus (AMPV) in already vaccinated hens of a dual-purpose (Lohmann Dual, LD) or a layer genotype (Lohmann Brown Plus; LB). Until 17 weeks-of-age, LD (<i>n</i> = 70) and LB (<i>n</i> = 109) hens were vaccinated against major bacterial and viral diseases and coccidiosis. At 24 weeks-of-age, the hens received either a placebo or an oral inoculation of 1,000 infectious eggs of <i>A. galli</i> and <i>H. gallinarum</i>. Plasma total immunoglobulin (Ig) isotypes (IgY, IgM, IgA) levels and vaccine-induced antibody titers against NDV, IBV, and AMPV were determined from 2 to 18 weeks post-infection (wpi). Infections had no suppressing effect on total Ig isotypes IgY, IgM, and IgA as well as on vaccine-induced antibody titers against NDV, IBV, and AMPV (<i>P</i> > 0.05). Overall, LB hens had higher levels of IgY, IgM, and IgA than those of LD hens (<i>P</i> < 0.05). There were no differences between IBV titers of the two genotypes (<i>P</i> > 0.05). Independent of infection status of the hens, NDV titers were higher in LB hens than in LD hens at wpi 2 (<i>P</i> < 0.05), but not in following weeks (<i>P</i> > 0.05). Uninfected LD hens had lower AMPV titers than their infected counterparts at 6 and 14 wpi (<i>P</i> < 0.05). Regardless of nematode infection, LD hens revealed a higher risk of responding weak to vaccination against NDV (odds ratio = 5.45; <i>P</i> = 0.021) and AMPV (odds ratio = 13.99, <i>P</i> < 0.001) than did LB hens (<i>P</i> > 0.05). We conclude that nematode infections have no adverse effects on non-specific and vaccine-induced humoral immunity in either genotype. LB hens have higher levels of total immunoglobulin isotypes than LD hens. Except for IBV, vaccine-induced humoral immune responses show a dependency on genotype. Dual-purpose hens show lower responsiveness to vaccinations against NDV and AMPV, possibly due to factors associated with increased body fat reserves in this genotype.
Project description:The ability of immunoglobulin (Ig) to recognize pathogens is critical for optimal immune fitness. Early events that shape preimmune Ig repertoires, expressed on IgM+ IgD+ B cells as B cell receptors (BCRs), are poorly defined. Here, we studied germ-free mice and conventionalized littermates to explore the hypothesis that symbiotic microbes help shape the preimmune Ig repertoire. Ig-binding assays showed that exposure to conventional microbial symbionts enriched frequencies of antibacterial IgM+ IgD+ B cells in intestine and spleen. This enrichment affected follicular B cells, involving a diverse set of Ig-variable region gene segments, and was T cell-independent. Functionally, enrichment of microbe reactivity primed basal levels of small intestinal T cell-independent, symbiont-reactive IgA and enhanced systemic IgG responses to bacterial immunization. These results demonstrate that microbial symbionts influence host immunity by enriching frequencies of antibacterial specificities within preimmune B cell repertoires and that this may have consequences for mucosal and systemic immunity.
Project description:Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.
Project description:In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.
Project description:Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified ? and ? junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.
Project description:Immunoglobulin (Ig) A nephropathy (IgAN) is the most common glomerulonephritis, which is characterized by the deposition of IgA antibody in the glomerulus. Systematic dissection of immune composition may contribute to a better understanding of the alternations in the immune system in IgAN. To this end, here we applied immune repertoire sequencing technology for parallel analysis of the complementary determining region 3 (CDR3) of all B cell receptors, including all five antibody subtypes (IgA, IgG, IgM, IgE and IgD), in 13 patients with IgAN and 7 healthy individuals. A significant decrease in CDR3 length was observed in the IgAN group. In particular, the JH6 family was significantly increased in IgAN. Amino acid usage was also altered in IgAN. Shannon, Simpson, Gini and Diversity 50 indices also revealed significant differences in the diversity of IgG, IgM and IgA antibodies as compared with controls. The proportions of IgA and IgG were increased, whereas IgM was decreased in IgAN. Moreover, a greater number of CDR3 sequences was shared between patients with IgAN. These findings suggest that the BCR immune repertoire is dramatically altered in IgAN, as characterized by shortened CDR3 length, as well as decreased overall diversity of CDR3.
Project description:In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection. To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis with or without previous oral vaccination was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of transcription were identified. The most inducible genes encoded avidin (AVD), extracellular fatty acid binding protein (EXFABP), immune responsive gene 1 (IRG1), chemokine ah221 (AH221), trappin-6-like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and ?? T-lymphocytes, we found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221, TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42 of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization of the course of S. Enteritidis infection in chickens.