Human immunoglobulin allotypes are antigenic determinants (or "markers") determined serologically, classically by hemagglutination inhibition, on the human immunoglobulin (IG) heavy and light chains. The allotypes have been identified on the gamma1, gamma2, gamma3, and alpha2 heavy chains (they are designated as G1m, G2m, G3m, and A2m allotypes, respectively), and on the kappa light chain (Km allotypes). Gm-Am allotypes are inherited in fixed combinations, or Gm-Am haplotypes, owing to the linkage of the human IGHC genes (IGHG3, IGHG1, IGHA1, IGHG2, IGHG4, IGHE, and IGHA2 from 5' to 3' in the IGH locus on chromosome 14). Gm and Am allotypes have been one of the most powerful tools in population genetics and very instrumental in molecular characterization of the human IGHC genes (gene conversion, copy number variation, gene order). They represent a major system for understanding immunogenicity of the polymorphic IG chains, in relation with amino acid and conformational changes. The correlation between G3m allotypes and amino acid changes has been possible with the sequencing of many alleles of the IGHG3 gene, from individuals from different populations and with known allotypes. In this chapter, we integrate genetics and sequence data and provide an updated overview of the Gm-Am haplotypes and Km allotypes. We propose, for the first time, a complete elucidation of the G3m allotypes, illustrated by the "IMGT G3m allele butterfly" concept that allows a graphical representation of the G3m alleles (variants of a gene expressing a given set of allotypes). Knowledge of allotypes is important in antibody engineering and humanization of monoclonal antibodies to improve immunotherapy.

Chromogranin A expression is heritable in humans, and both plasma chromogranin A concentration and its releasable adrenal and sympathetic neuronal pools are augmented in established essential (hereditary) hypertension. To evaluate chromogranin A further as a simpler or "intermediate phenotype" in the complex trait of hypertension, we studied chromogranin A expression in the spontaneously hypertensive rat (SHR), a rodent model of essential hypertension. Both plasma (p < 0.0001) and adrenal medullary (p = 0.003 to p < 0.0001) chromogranin A were elevated in the SHR, even at the earliest stages (3-4 weeks of age). In the adult adrenal gland, both chromogranin A (p=0.005) and norepinephrine (p=0.011) were increased in the SHR, while dopamine beta-hydroxylase activity was diminished (p < 0.0001). Chromogranin A mRNA expression was also elevated in the SHR adrenal medulla (p = 0.017). Differences in chromogranin A processing were not noted between SHR and Wistar Kyoto control (WKY) rats. In an SHR x WKY genetic intercross, control of the adrenal chromogranin A phenotype by a single major locus was suggested by comparison of phenotypic variance of the F2 vs F1 generations, and by bimodal frequency histogram (3:1 ratio), confirmed by maximum likelihood analysis (chi2 = 74.6, p < 0.000001) in the F2 generation. However, microsatellite alleles at a surrogate locus (Ighe) 12.7 cM from chromogranin A (Chga), on rat chromosome 6, failed to co-segregate with blood pressure in an F2 generation (F = 0.06, p = 0.94). In another rodent model of hereditary hypertension, the genetically hypertensive mouse (BPH/2), adrenal chromogranin A (p=0.018) and norepinephrine (p = 0.004) were actually diminished. We conclude that over-expression of chromogranin A is a variable feature of mammalian genetic hypertension. In one rodent model (the SHR), over-expression of chromogranin A is largely controlled by a single genetic locus, but the chromogranin A locus itself is not directly linked to determination of the blood pressure elevation of the SHR.

Deletion mapping analyses have been employed to order the heavy chain variable region (VH) gene families in three inbred murine strains. These nine VH gene families have been positioned with respect to the J558 and 3660 VH families in A/J (Ighe) as follows: 3609-J558-(J606,VGAM3-8,S107)-3660-(X24,Q52,7183 )-DH. Maps generated with respect to J558 in the BALB/c (Igha) and C57BL/6 (Ighb) strains are consistent with these results. The organization of the VH complex produced by deletion mapping is quite different from the accepted map generated by other methods, particularly in that J558 is more DH distal and 3660 is more DH proximal than previously thought. The order presented here is compatible with VH rearrangement frequencies suggesting preferential utilization of DH-proximal VH gene segments. Our data also indicate that interspersion of some VH family members may be a common feature of the murine VH complex since the 3609 VH family is interdigitated in the three strains and a Q52 VH gene segment is interspersed in C57BL/6.

The present studies characterize at the clonal level the repertoire of lipopolysaccharide-responsive murine B lymphocytes committed to the production of antibodies reactive with denatured DNA. This repertoire is vast in normal mice as 1-5% of total mitogen-induced antibody-forming cell clones secreted denatured DNA-reactive antibodies when the splenocyte donors were CBA (Ighj), BALB/c (Igha), C57BL/6 (Ighb), CBA nu/nu, and C57BL/6 nu/nu athymic mice. The autoimmune NZB (Ighe) strain did not display elevated proportions of anti-denatured DNA antibody-forming cell precursors. Cross-reactions shown by CBA anti-denatured DNA antibodies suggest that many antibodies might derive significant binding energy from interaction with the bases or similar hydrophobic moieties. Cross-reactions with other tested polynucleotides were frequent, but cross-reactions with phospholipids and phosphocholine were undetectable. Most anti-DNA antibodies bound preferentially or exclusively to single-stranded denatured DNA as compared to double-stranded native DNA. The frequency of anti-denatured DNA antibody-forming cell precursors among CBA peritoneal cells was not elevated. Fluorescence-activated cell sorter-selected Ly-1-positive NZB splenic B cells were not enriched, and Ly-1 negative B cells were not depleted of anti-DNA antibody-forming cell precursors. These results show that antibody-forming cell precursors specific for denatured DNA are not restricted to the Ly-1 positive B-cell subset.

Anthelmintics are currently the most common method of worm control. The emergence of worms with multiple-drug resistance and issues of residues in the food chain make alternative parasite control measures a priority. To develop improved and sustainable methods for controlling Haemonchus contortus such as genetic selection of resistant sheep, a better understanding of the host-parasite relationship is required. A trial was undertaken using sheep surgically implanted with abomasal fistulas to enable sequential biopsy of the abomasal mucosa during trickle infection with two strains of H. contortus. These were ivermectin-resistant CAVR and ivermectin-sensitive McMaster. From a gross parasitology perspective, this approach enabled the effect of developing immunity to be observed on both the establishment and maturation of two CAVR doses within and between groups. Since the only difference in parasite treatment between the groups was the staggering of the two CAVR doses, microarray results from biopsies taken on the same day in different groups were combined and compared between different biopsy dates to observe differential gene transcription over time. Differential gene transcription was detected by comparing transcription in our array data between different biopsy dates using a low P value screen (P<0.01) and by compiling a list of 82 immunoparasitology-related genes and examining transcription in this list with a higher P value screen (P<0.05). Our microarray data were validated in silico by comparison with intelectin 2, trefoil factor 3, calcium activated chloride channel and mucin 5 from other gene transcription studies and with phenotypic data such as the response by gammadelta T cells and immunoglobulins to H. contortus. The first four genes are involved in non-specific responses to infection and mucosal healing. These were upregulated at the early time points and intelectin 2 remained prominent throughout the trial. As the trial progressed, immunoglobulin genes became strongly upregulated. These included IgCgamma IgG2a heavy chain constant region, IGHE immunoglobulin heavy constant epsilon and IGHM immunoglobulin heavy constant mu.

Since the initial characterization of IgE by Ishizaka et al. (1966), IgE was described in several mammalian species. In horses, a single gene encoding the IgE heavy chain constant region (IGHE gene) exists per haploid genome and several allelic variants of the equine IGHE gene were found. IgE occurs in its soluble form in equine serum and physiological concentrations of total IgE are around 1000-fold higher in normal horse than in normal human serum. Maternal IgE is enriched in the colostrum and transferred to the neonatal foal after birth. Foals do not produce detectable concentrations of endogenous IgE for several months after birth. IgE is also found on the surface of a small percentage of equine peripheral blood cells including basophils, and subpopulations of B-cells and monocytes, and on mast cells in various tissues such as the skin, and the submucosa of the airways and intestine. Both, the high- and low-affinity IgE receptor genes are identified in the horse suggesting binding of soluble IgE from the circulation to these receptors. Horses naturally develop type I hypersensitivities. IgE-mediated mechanisms were implicated in the pathogenesis of several allergic diseases in horses since almost 30 years. The findings were mainly based on the induction of immediate skin reactions after intradermal testing with allergen extracts. With the development of the first monoclonal antibodies to equine IgE within the past years, more insights into the pathogenesis of allergic diseases could be obtained. Today, various techniques are available to detect soluble IgE and the sensitization of mast cell or basophils with IgE in horses. An IgE-mediated allergic etiology is confirmed for skin hypersensitivity. The causing role of IgE in other diseases such as recurrent airway obstruction (RAO) still remains controversial. More recent studies did not confirm an IgE-mediated pathogenesis of RAO. This suggested that the disease is a chronic inflammatory condition with some indication for an involvement of delayed-type hypersensitivity mechanisms. In summary, our knowledge about the role of IgE during the immune response of the horse improved tremendously during the past decade. New IgE-specific tools and technologies are likely to uncover additional aspects of IgE-mediated mechanisms in equine health and disease.

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.

Immunoglobulins loci in mammals are well known to be organized within a translocon, however their origin remains unresolved. Four of the five classes of immunoglobulins described in humans and rodents (immunoglobulins M, G, E and A-IgM, IgG, IgE and IgA) were found in marsupials and monotremes (immunoglobulin D-IgD was not found) thus showing that the genomic structure of antibodies in mammals has remained constant since its origin. We have recently described the genomic organization of the immunoglobulin heavy chain locus in reptiles (IGHM, IGHD and IGHY). These data and the characterization of the IGH locus in platypus (Ornithorhynchus anatinus), allow us to elucidate the changes that took place in this genomic region during evolution from reptile to mammal. Thus, by using available genome data, we were able to detect that platypus IGH locus contains reptilian and mammalian genes. Besides having an IGHD that is very similar to the one in reptiles and an IGHY, they also present the mammal specific antibody genes IGHG and IGHE, in addition to IGHA. We also detected a pseudogene that originated by recombination between the IGHD and the IGHM (similar to the IGHD2 found in Eublepharis macularius). The analysis of the IGH locus in platypus shows that IGHY was duplicated, firstly by evolving into IGHE and then into IGHG. The IGHA of the platypus has a complex origin, and probably arose by a process of recombination between the IGHM and the IGHY. We detected about 44 VH genes (25 were already described), most of which comprise a single group. When we compared these VH genes with those described in Anolis carolinensis, we find that there is an evolutionary relationship between the VH genes of platypus and the reptilian Group III genes. These results suggest that a fast VH turnover took place in platypus and this gave rise to a family with a high VH gene number and the disappearance of the earlier VH families.

By several crossing studies it has been demonstrated that the MHC class-II genes of the RT1u haplotype, Iddm1, and the lymphopenia, Iddm2, are essential, but not sufficient for diabetes development in the BB rat. Using diabetic BB/OK and diabetes-resistant DA rats it has been shown that a third non-MHC gene, Iddm3, on chromosome 18 cosegregates with diabetes in the BB/OK rat subline. Because mapping results need not be consistent among different crosses, we genetically analysed a new cross population using diabetic BB/OK and diabetes-resistant SHR/Mol rats analysing 73 microsatellite markers. The genetic analysis of Iddm1 and Iddm2 homozygous [(BB/OK x SHR)F1 x BB/OK] first backcross hybrids (BC1) confirmed the action of Iddm3 and one predisposing non-MHC locus, Iddm4, near Ighe/D6Mgh2 on chromosome 6 and one protective locus, Iddm5r(esistance), detected around Igf2/Tnt on chromosome 1. From these novel findings it is concluded that the diabetogenic phenotype of the BB/OK rat subline is the result of the interaction of predisposing and protecting diabetes genes.

The IGHG (ImmunoGlobulin constant Heavy G chain) genes are situated close to the IGHE gene on chromosome 14q32, 5'mu, delta, gamma3, gamma1, alpha1, gamma2, gamma4, epsilon, alpha2, 3', in linkage disequilibrium. The polymorphism of gamma3, gamma1 and gamma2 genes, is investigated as alternative allotypes. They are inherited in a Mendelian fashion and are expressed randomly in allelic exclusion. The alternative and functionally different gamma3, gamma1 and gamma2 gene variants, are found in four IGHG haplotypes, coding 4 B-cell variants: IGHG*bfn (=B1-cells), IGHG*bf-n (=B2-cells), IGHG*gan (=B3-cells) and IGHG*ga-n (=B4-cells). The dominance of the IGHG2*n allele from the IGHG*bfn haplotype (=B1-cells) has been shown in repeated investigations, namely in patients with asthma and allergy with increased serum levels of IgE>> 600 ku/l and more often so in those with IgE>> 1,000 ku/l or IgG4>1 g/l, in childhood asthma patients with mean level of IgE = 1,762 ku/l and in allergen exposed individuals developing laboratory animal allergy. In children with non-atopy and mean IgE level = 9.5 ku/l there is instead a dominance of the alternative allotypes from the IGHG*ga-n (=B4-cells) with IGHG2*-n alleles. In a case-control study allergic children with a family history of allergy, clinically manifest allergy and/or positive SPT, the IGHG*bfn haplotype (=B1-cells) with the IGHG2*n allele dominates, with increased risk of atopy and the IGHG*bf-n haplotype (=B2-cells) with the IGHG2*-n allele is infrequent with low risk, probably protective against atopy. The phenotypic expressions of the IGHG*bfn haplotype (=B1 cells) and IGHG*bfn/*bfn diplotypes (B1/B1-cells) are increased IgG2*n allotype together with increased IgE serum levels and IgE sensitisation in agreement with atopy. The alternative IGHG*ga-n/*ga-n diplotype (B4/B4-cells) express low IgG1*a- and IgG2*-n allotypes, together with low IgE and non-IgE sensitisation, in agreement with non-atopy. Together these studies have given us a greater understanding of the involvement of IGHG genes, IGHG coded B-cells and immunochemical and functional variants of IgG molecules describing different forms of asthma and allergy, which will improve diagnoses and treatment.

We report on the analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51× whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG, and IGHE sequences in another. Some IGKV, IGLV, and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3-Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa, and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.

Most genetic studies of bronchial asthma deal with IgE responsiveness. The manner by which allergens trigger IgE production and activate mast cells suggests that several genetic loci may be involved. Several reports of candidate genes include chromosome 6 and HLA antigens, chromosome 14q11 and the alpha chain of the T cell receptor, chromosome 11q32 and the beta chain of the high-affinity IgE receptor and chromosome 5 and the gene cluster for IL-4, respectively. In addition, the immunoglobulin heavy chain G (IGHG) genes on chromosome 14q32 have been associated with both atopic and non atopic bronchial asthma in children. In order to further investigate the role of IGHG genes in asthmatic children, the phenotypes of patients with homozygous but alternative IGHG genes were investigated. IGHG gene expression of patients with childhood asthma was determined by serum Gm allotypes with a quantitative competitive indirect ELISA method. The groups consisted of 24 children with the homozygous G3m(b/b)-G1m(f/f)-G2m(n/n) and 16 with the alternative G3m(g/g)-G1m(a/a)-G2m(-n/-n) genes. The two different genotypes were investigated for serum IgE (PRIST), serum IgG subclass levels (radial immunodiffusion), Gm allotype levels (competitive ELISA), IgA and IgM levels (radial immunodiffusion), peripheral blood eosinophils, specific IgE antibodies (skin prick test, SPT, or radioallergosorbent test, RAST), number of peripheral blood CD lymphocyte markers (flow cytometry) and serum IL-4 and IFN-gamma levels (ELISA). Comparison of the two genotypes in children with bronchial asthma revealed significantly increased IgE (p < 0.001), increased specific IgE (p < 0.001), as investigated by SPT or RAST (n = 10 allergens tested), increased number of peripheral blood eosinophils (p < 0.01), increased serum IgG1(f/f)(p < 0.001), IgG2(n/n) (p < 0.001) and IgG3(b/b)(p < 0.01) levels, and decreased CD8 given in percent of the total number of peripheral lymphocytes, (p < 0.02) in the G3m(b/b)-G1m(f/f)-G2m(n/n) genotype. The asthmatic children with the G3m(g/g)-G1m(a/a)-G2m(-n/-n) genes instead showed low IgE levels, practically no specific IgE antibodies, a lower number of peripheral blood eosinophils, lower IgG1(a/a), IgG2(-n/-n) and IgG3(g/g) serum levels and higher CD8 lymphocyte numbers. The results show that the IGHG3(b/b)-IGHG1(f/f)-IGHG2(n/n) genes are in linkage disequilibrium with allergen-specific high-responding IGHE genes and present the atopic phenotype of bronchial asthma, while the IGHG3(g/g)-IGHG1(a/a)-IGHG2(-n/-n) genes present the nonatopic phenotype of childhood asthma. The two genotypes with different amino acid epitopes of their constant heavy gamma1, gamma2 and gamma3 chains presented qualitatively different IgG1, IgG2 and IgG3 molecules, respectively, and also different serum IgG1, IgG2 and IgG3 levels, together with different numbers of peripheral blood eosinophils and CD8 lymphocytes. The two IGHG genotypes represent different pathways of human immune regulation. An association of atopic IGHG genotype with other candidate genes for atopy could be suggested.

The number of immunoglobulin heavy chain (IGH) constant genes (IGHC) varies among mammals. To annotate the porcine IGHC genes, we sequenced the entire IGHC-containing genomic region from a single porcine haplotype. The resulting contiguous sequence included in 5' the IGH diversity (D) gene cluster and in 3' TMEM121, which flank the IGHC cluster in the human genome, suggesting that we had obtained the entire genomic region containing porcine IGHC. This region was about 190-kb long, in good agreement with those of other mammals. The porcine IGHC cluster contained 10 genes, IGHM, IGHD, six IGHG genes, IGHE and IGHA. The porcine IGHG genes formed a cluster between IGHD and IGHE, with IGHG3 considered as the most ancient IGHG gene, located at the beginning of the IGHG cluster. Furthermore, the porcine sequence contained two IGHG5 and two IGHG6 genes, but no IGHG genes for IgG2 and IgG4, suggesting flexibility within the IGHG cluster. We also recorded structural differences in the switch regions of the IGHC genes that may be important in their transcription. This haplotype can serve as a reference for future studies on other haplotypes and for functional analysis of porcine immunoglobulin (IG) isotypes.

In the immune response of BALB/c mice (Igha) to Pneumococcus the majority of antibodies express the idiotype of the myeloma protein TEPC 15 (T15). In contrast mice of the A/J strain (Ighe) do not express this idiotype. Using (BALB/c X A/J)F1, F2 or backcross mice it could be shown that in allotype heterozygous animals (Igha/e) Pneumococcus pneumoniae preferentially stimulates B cells expressing a heavy chain (H) encoded by genes in the BALB/c H chain gene complex. Phosphorylcholine (PC)-specific hybridoma lines were established from BALB/c and A/J spleen cells and idiotypically analyzed using monoclonal antibodies (mAb) specific for the T15 idiotopes 32/65, 10/13, 16/13 or 21A5. Whereas the majority of the BALB/c PC-binding mAb express these idiotopes, only some of the A/J mAb are positive for one or the other of the idiotopes formed by the variable (V) regions of the H and the light chain of the myeloma protein T15. However, 80% of the A/J PC-binding hybridoma proteins were bound by the anti-idiotopic mAb 21A5. This mAb is specific for a determinant partially formed by the C alpha and partially by the V regions of the myeloma protein T15. The mRNA of one of these T15- A/J PC-binding hybridoma lines was sequenced. VH and V kappa were identical with sequences found for BALB/c T15-like antibodies. The sequence of the D segment was structurally very different. The importance of the D segment in the dominant expression of the T15 idiotype is discussed.

Stimulation of small, resting, splenic B cells with bacterial lipopolysaccharide (LPS) induces proliferation, differentiation to plasma cell formation, and the expression of immunoglobulin heavy chain (IgH). When this is combined with agents which crosslink surface Ig, differentiation and the induction of surface immunoglobulin are suppressed even though proliferation proceeds. We find that anti-mu antibodies suppresses Ig gene expression of transfected mu constructs, even if either the membrane or secretory segments have been deleted. We examined the effects of anti-mu treatment on the IgH enhancer (IgHE) attached to a heterologous test gene (CAT). Indeed the IgH enhancer alone was subject to anti-mu suppression, while the SV40 enhancer was insensitive. To determine what was responsible for suppression of enhancer function by anti-mu we examined nuclear extracts from stimulated splenic B cells for the presence of sequence-specific DNA binding activities to various sites within the enhancer. We found two specific differences--an induction in mu E5 binding activity, and a reduction in octamer transcription factor 2 (OTF2) binding activity, after anti-mu treatment. Analysis of these cells by in situ immunofluorescence with anti-OTF2 antibodies suggests that the nuclear localization of OTF2 in anti-mu treated cells may change, as well as its absolute level.

In the human, the order of the immunoglobulin heavy chain constant region (Ig CH) genes is the following: 5'-M-D-G3-G1-EP1-A1-GP-G2-G4-E-A2-3'. Extensive multigene deletions have been described in the Ig CH locus, some of these encompassing up to 160 kb. To date six different multigene deletion haplotypes have been identified, designated I to VI according to the chronological order of their being found: deletion I (del G1-EP1-A1-GP-G2-G4), II (del EP1-A1-GP), III (del A1-GP-G2-G4-E), IV (del EP1-A1-GP-G2-G4), V (del GP-G2-G4-E-A2), VI (del G1-EP1-A1-GP-G2). Individuals were found either homozygous for one type of deletion or heterozygous for two different deletions, mainly (17 cases out of 18) in the Mediterranean area. So far, deletions I and II have been found in Tunisia, deletions III, IV and V in Italy, and deletion VI in Sweden. In this paper, we show that a Tunisian, T17, previously reported as being homozygous for a deletion of type IV, is, in fact, homozygous for a deletion that encompasses A1-GP-G2-G4-E. Therefore T17 is the first case of a deletion of type III reported in the Tunisian population. Molecular analysis demonstrates that the T17 deletion occurred between highly homologous regions located downstream of IGHEP1 and IGHE, respectively. In contrast to the EZZ deletion, the recombination occurred near or in the switch regions, since the homologous regions involved in the deletion extend over 4.5 kb of DNA and encompass the I alpha 1-S alpha 1 and I alpha 2-S alpha 2 regions, respectively.

It has been suspected for many years that cattle possess two functional IgH gene loci, located on Bos taurus autosome (BTA) 21 and BTA11, respectively. In this study, based on fluorescence in situ hybridization and additional experiments, we showed that all functional bovine IgH genes were located on BTA21, and only a truncated μCH2 exon was present on BTA11. By sequencing of seven bacterial artificial chromosome clones screened from a Hostein cow bacterial artificial chromosome library, we generated a 678-kb continuous genomic sequence covering the bovine IGHV, IGHD, IGHJ, and IGHC genes, which are organized as IGHVn-IGHDn-IGHJn-IGHM1-(IGHDP-IGHV3-IGHDn)3-IGHJn-IGHM2-IGHD-IGHG3-IGHG1-IGHG2-IGHE-IGHA. Although both of two functional IGHM genes, IGHM1 and IGHM2, can be expressed via independent VDJ recombinations, the IGHM2 can also be expressed through class switch recombination. Likely because more IGHD segments can be involved in the expression of IGHM2, the IGHM2 gene was shown to be dominantly expressed in most tissues throughout different developmental stages. Based on the length and identity of the coding sequence, the 23 IGHD segments identified in the locus could be divided into nine subgroups (termed IGHD1 to IGHD9). Except two members of IGHD9 (14 nt in size), all other functional IGHD segments are longer than 30 nt, with the IGHD8 gene (149 bp) to be the longest. These remarkably long germline IGHD segments play a pivotal role in generating the exceptionally great H chain CDR 3 length variability in cattle.

Highly atopic individuals, with marked allergy, have extremely elevated total plasma IgE levels. To determine if atopy could be associated with structural alterations involving the IGHE gene of the immunoglobulin heavy chain constant region, the genomic DNA from five atopic individuals was examined. We describe here the identification of a deletion of approximately 120kb, including the IGHA1, IGHGP, IGHG2, AGHG4, and IGHE genes of the IGH constant region, in one atopic patient. This deletion arose de novo from a maternally derived chromosome. The deletion, although apparently not the primary cause of the atopic phenotype of this patient, could be indirectly responsible for the phenotype by exposing aberrant immunoglobulin-regulating elements within the paternally derived IGH constant region.

BACKGROUND

Asthma is a heterogeneous disease for which a strong genetic basis has been firmly established. Until now no studies have been undertaken to systemically explore the network of asthma-related genes using an internally developed literature-based discovery approach. This study was to explore asthma-related genes by using literature-based mining and network centrality analysis.

METHODS

Literature involving asthma-related genes were searched in PubMed from 2001 to 2011. Integration of natural language processing with network centrality analysis was used to identify asthma susceptibility genes and their interaction network. Asthma susceptibility genes were classified into three functional groups by gene ontology (GO) analysis and the key genes were confirmed by establishing asthma-related networks and pathways.

RESULTS

Three hundred and twenty-six genes related with asthma such as IGHE (IgE), interleukin (IL)-4, 5, 6, 10, 13, 17A, and tumor necrosis factor (TNF)-alpha were identified. GO analysis indicated some biological processes (developmental processes, signal transduction, death, etc.), cellular components (non-structural extracellular, plasma membrane and extracellular matrix), and molecular functions (signal transduction activity) that were involved in asthma. Furthermore, 22 asthma-related pathways such as the Toll-like receptor signaling pathway, hematopoietic cell lineage, JAK-STAT signaling pathway, chemokine signaling pathway, and cytokine-cytokine receptor interaction, and 17 hub genes, such as JAK3, CCR1-3, CCR5-7, CCR8, were found.

CONCLUSIONS

Our study provides a remarkably detailed and comprehensive picture of asthma susceptibility genes and their interacting network. Further identification of these genes and molecular pathways may play a prominent role in establishing rational therapeutic approaches for asthma.

In a previous study, we showed that the immunoglobulin heavy-chain (IgH) enhancer (IgHe) is near or in an initiation zone of chromosomal DNA replication, which is preferentially active in B cells (K. Ariizumi, Z. Wang, and P. W. Tucker, Proc. Natl. Acad. Sci. USA 90:3695-3699, 1993). This suggests the existence of a functional relationship between IgHe-mediated transcription and DNA replication. To test this theory, we utilized simian virus 40 (SV40) DNA replication as a model of chromosomal replication. IgHe or its operationally divisible domains (5'-En, core, and 3'-En) were introduced into SV40 minichromosomes (IgHe-SV40). Results of replication assays with IgHe-SV40 replicons indicated that the 5'-En and 3'-En activated or suppressed SV40 DNA replication regardless of the presence of SV40 enhancers or promoters in these replicons. The activity did not reside in IgHe core sequences. The results suggested that the 5'- and 3'-En regulated SV40 replication through direct interaction with the origin, not through suppression at the SV40 enhancer and/or promoter. In an effort to identify elements within the 5'-En motif that contributed to this effect, we found that the E site, but not microE5 and microE2 boxes, upregulated DNA replication. Our results provide another possible regulatory function for the 5'-En and 3'-En domains besides transcriptional suppression of IgHe.

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse ( Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5' end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.

The primary antigen-specific antibody response of various strains of mice to TEPC-15/PnC immune complexes has been examined. We found that both BALB/c and C3H mice were good responders to the PnC antigen; however, C3H mice were low responders, whereas BALB/c mice were high responders to the TEPC-15/PnC complexes. Using congenic strains on the C3H and BALB/c background, we have shown that the response to the complexes is not restricted by gene products of the H-2 complex or by the Igh (allotype) locus. However, responsiveness may be controlled by genes linked to the Igh locus, since we have shown that strains that are Ighj, Ighd, and Ighf are low responders, whereas strains that are Igha, Ighb, and Ighe are high responders to the immune complex. Moreover, responsiveness correlates with the expression of the T15 Id as measured using the anti-T15 monoclonal antibody, AB1-2. Thus, strains such as BALB/c, BALB.B, BALB.K, and CB-20, which express high levels of T15 (AB1-2) Id in their PFC response to PnC are relatively high responders to TEPC-15/PnC complexes, whereas C3H, C3H.SW, and C3H-OH, which express low levels of the T15 (AB1-2) Id, are low responders to the complexes. Finally, we found that BALB/c mice are high responders to complexes formed with T15+ antibodies, whereas they are low responders to complexes formed using T15- antibodies. The results suggest that the antigen-specific response to these immune complexes is Id-restricted.

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ>> mIgγ1>> mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

The orthologous immunoglobulin C epsilon 1 gene (IGHE) of the common chimpanzee, pygmy chimpanzee, orangutan, white-handed gibbon, and Japanese macaque was assigned to the human chromosome 14 homologue in each species and regionally mapped by fluorescence in situ hybridization to PTR15q32 (common chimpanzee), PPA15q32 (pygmy chimpanzee), PPY15q32 (orangutan), HLA17qter (white-handed gibbon), and MFU7q29 (Japanese macaque). The gene localized to the terminal region of the chromosome in each species, and so this probe provides a new telomeric DNA marker for nonhuman primates.