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Suppression of VH, C gamma and C mu gene products in rabbits by maternal immunization against IgM allotype and detection of somatic recombinant molecules in these animals.

Suppression of the mu-chain allotype Ms16 was obtained in young heterozygous Ms16/Ms17 rabbits by immunizing the Ms17 mother against the paternal Ms16 allotype. Examination of the concentration of the VH and CH allotypes of paternal origin (a2,Ms16 and e14) in the offspring, revealed not only Ms16 suppression but also VH and C gamma allotypic suppression. The degree of suppression for the C gamma markers (4-25-fold decrease) was much less than that for the C mu or VH markers (50-150-fold decrease). The excess C gamma marker (e14) was found to be present for the major part on molecules possessing the VH allotype derived from the homologous allelic chromosome. The pattern of persistence of molecules with the suppressed C gamma marker in the serum is consistent with the idea that these molecules arise by somatic recombination and that the gene order is VH, C mu and C gamma.

Animals↗

Surface immunoglobulin of rabbit peripheral blood lymphocytes: detection of allotypes by fluorescence and rosetting.

Specific anti-allotype reagents were prepared to detect heavy and light chain allotypes on surface immunoglobulin-positive (sIg+) cells from a3a3b4b4, a2a2b5b5 homozygous and a2a2b4b5 heterozygous rabbits. Total sIg+ peripheral blood lymphocytes (PBL) were detected with fluorescein-labeled goat anti-light chain and sheep anti-rabbit Ig reagents. The total sIg+ cells detected with these reagents and the sum of a and b allotypes individually measured with specific fluorescent anti-allotype reagents (a2 + a3 or b4 + b5) were measured more or ess the same when measured by fluorescence-activated cell sorter (FACS) II flow microfluorometry. The data provided no evidence for single cells expressing both allelic allotypes (allelic inclusion). We found that FACS and resetting techniques were generally equally sensitive. However, we detected a greater proportion of total b4+ plus b5+ cells by rosetting than by fluorescence in some PBL preparations from heterozygous b4b5 rabbits. This was not seen with artificial mixtures of b4b4 and b5b5 cells. The nature of these cells is not yet known. Conceivably, they were not scored as lymphocytes by light-scatter analysis on the FACS and hence were not counted, but were indistinguishable from lymphocytes by microscopy.

Animals↗

Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits.

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.

Animals↗

Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression.

Embryonally induced allotype suppression in M-1,G-1 heterozygous chickens was stable for at least 18 months after hatching. Suppression was established rapidly since injection of antigen only 4 days after anti-IgM-1 antiserum failed to abrogate its effect. Injected chickens had undetectable serum levels (i.e. less than 40 micrograms/ml) of the suppressed IgM-1 and low (0.3-0.6 mg/ml) levels of the linked IgG-1 allotype. This correlated with a complete depletion of cells bearing the relevant IgM-1 allotypes and a compensatory increase in the alternative nonsuppressed IgM-1 allotype-bearing cells in the spleen, peripheral blood and bursa. Cell transfer studies suggested that suppression could not be attributed to allotype-specific suppressor cells.

Animals↗

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus).

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (K1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109-111); 2) the four positions N-terminal to the interdomain disulfide bond (167-170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.

Alleles↗

The susceptibility to insulin-dependent diabetes mellitus is associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3,4 heterozygotes.

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P = 6 x 10(-6)): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P = 0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P = 0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C4↗

Mouse IgA allotypes have major differences in their hinge regions.

Six IgA allotypes are serologically identifiable in inbred mice. The sequences of the PCR-amplified C alpha 1, C alpha 2 and C alpha 3 exons from the genomic DNA of mice of four previously unsequenced allotypes already have been compared with those of BALB/c and of a wild mouse, Mus pahari, in the literature. Sporadic differences, including several that may encode the known allotypic determinants, are found throughout the three exons, but major differences occur in the hinge. The hinge is longest in DBA/2 ( Igh-2(c)) mice, having an extra codon compared with that of BALB/c ( Igh-2(a)) and B10.A ( Igh-2(b)) mice. It is two codons shorter in CE ( Igh-2(f)) and four shorter in M. pahari, AKR and NZB (both Igh-2(d)) mice, but the position of the missing codons in the latter two strains is offset from that in M. pahari. The hinges in BALB/c ( Igh-2(a)) and DBA/2 ( Igh-2(c)) differ most from each other and from the other three allotypes, which are fairly closely related. Both BALB/c and DBA/2 have O-linked glycosylation sites, but they are in different positions in the hinge. Compared with BALB/c ( Igh-2(a)), B10.A( Igh-2(b)) has two extra Cys residues in the hinge, while DBA/2 ( Igh-2(c)), AKR/NZB ( Igh-2(d)) and CE ( Igh-2(f)) each have one. The differences in hinge length may have arisen by mismatching of highly repetitive portions of its sequence during meiotic recombination. Possible effects of the differences in hinge length and composition on the behavior of the mouse IgA allotypes are discussed.

Amino Acid Sequence↗

Selective suppression of allotype expression induced in vitro: maintenance of suppression following adoptive transfer.

The question of the ultimate fate of lymphocytes subjected to treatment with anti-allotype antibody (Ab) has been investigated by means of an adoptive transfer system that uses noninbred rabbits matched for major histocompatibility antigens and mismatched for allotype. Suppression of b4 immunoglobulin (Ig) production was induced by incubating lymphocytes from b4b5 rabbits with anti-b4 in culture. Transfer of b4-suppressed cells to newborn recipients of allotype b6b6 resulted in stable chimerism of mixed donor-recipient allotypes, in which b4 Ig production remained suppressed. In recipients of non-Ab-treated cells, b4 Ig production predominated over b5, as had been the case in the intact donor. No evidence for stimulation of b4 Ig synthesis was seen, even when lymphocytes and serum from 1-week-old recipients were examined. When lymphoid cells of antigen-primed b4b5 donors were treated with anti-b4 in vitro and transferred, Ab production of the b4 type was specifically suppressed, with compensatory over-production by Ab-forming cells of the b5 type. The results reported here indicate that although anti-allotype Ab is not directly cytotoxic, a significant proportion of the b4-committed cells were irreversibly inactivated as a result of Ab pulse treatment.

Animals↗

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity.

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.

Alleles↗

Immunoglobulin allotypes and IgG subclass antibody response to Aspergillus fumigatus in cystic fibrosis patients.

BACKGROUND: A majority of patients with cystic fibrosis (CF) become colonised with Aspergillus fumigatus (Af.), but only a minority develops allergic bronchopulmonary aspergillosis (ABPA). ABPA is associated with increased levels of specific immunoglobulin G (IgG) anti-Af. antibodies with a characteristic IgG subclass distribution. We examined whether this characteristic immune response was under the influence of GM and KM allotypes, which are genetic markers (antigenic determinants) on gamma- and kappa-light chains, respectively. METHODS: Sera from 233 CF patients were typed for seven GM determinants and two KM determinants. The types were correlated to IgG subclass anti-Af. antibody levels and to the presence or absence of Af. colonisation as well as ABPA. RESULTS: The IgG2 antibody level was significantly higher in heterozygous GM (1,2,17 23 5,21 and 1,3,17 23 5,21) compared to homozygous GM allotypes (p = 0.02). Patients with the same allotypes tended to have higher IgG1 (p = 0.051). In patients with ABPA, being heterozygous for G1M and G3M was linked to higher IgG4 and lower IgG3 as compared to the other genotypes. The KM markers did not influence the antibody levels. The allotype GM(3 23 5), associated with atopic bronchial asthma, tended to make a relatively larger group in ABPA patients compared to non-ABPA and patients not colonised with Af. (p = 0.09). CONCLUSIONS: An influence of the GM allotypes on the immune response to Af. and on the development of ABPA in patients with CF is suggested.

Adolescent↗

Characterization of the b99 allotype of rabbit immunoglobulin kappa 1 light chain.

A previously undetected allotypic specificity, A99, was found in a wild rabbit (Oryctolagus cuniculus) population in Portugal. The A99 allotype cross-reacted with anti-b6 and anti-b96 sera, but carried its own antigenic determinants and was therefore distinct from the known allotypes of the b series. The A99 determinants were located on the kappa 1 light chain of IgG molecules and were inherited as allotypic determinants of the b series. Taken together, these data provide evidence that A99 is encoded by an allele at the b locus, tentatively designated b99. The great similarity between the b6, b96 and b99 allotypes suggests that they derived from a common ancestor.

Animals↗

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5.

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).

Amino Acid Sequence↗

Gm (23) allotypes and Fcgamma receptor genotypes as risk factors for various forms of periodontitis.

OBJECTIVES: Given the diversity of the distribution of the Gm (23) allotypes and FcgammaR genotypes in different ethnic groups, it was our purpose to examine their clinical significance in periodontitis in Taiwan. MATERIAL & METHODS: Genomic DNA of 50 patients with chronic periodontitis (CP), 30 patients with generalized aggressive periodontitis (G-AP) and 74 healthy controls were harvested. The Gm (23) allotypes were determined by radial immunodiffusion test, and the FcgammaR IIa (CD32) and IlIb (CD16) genotypes were determined by polymerase chain reaction-based allele-specific oligonucleotide hybridization. RESULTS: The overall carrier rate of the Gm (23+) allotype was higher than 85%, and the Gm (23-) allotype was statistically over-represented in patients with CP compared to the controls. There were no differences in the distributions of the three genotypes of FcgammaR IIa and IIIb among the three tested groups. The frequency of the R131 allele of the FcgammaR IIa polymorphisms was higher in G-AP than in CP when R/H allelic frequencies (p = 0.01) were examined by the chi2 test. CONCLUSION: The Gm (23-) allotype might be a potential risk factor for CP. Although the R131 allele of FcgammaR IIa occurred more frequently in G-AP than in CP, its clinical significance could not be justified in this study.

Adult↗

Human immunoglobulin allotypes: previously unrecognized determinants and alleles defined with monoclonal antibodies.

The highly polymorphic system of serologically defined genetic markers on human IgG heavy chains (Gm allotypes) is second only to the HLA complex in terms of the large number of determinants, alleles, and haplotypes that can be used for analyses of disease associations and other genetic studies. However, present typing methods are based on the use of anti-Gm antisera that are derived mainly from fortuitously immunized human donors, often requiring processing before use, and must be used in a hemagglutination-inhibition assay that cannot be used in typing for isoallotypic determinants (currently termed "non-markers"). In studies presented here, we describe an allotyping system that utilizes monoclonal antibodies in a "sandwich" modification of the solid-phase radioimmunoassay, which is capable of reliable quantitative typing of allotypic, isoallotypic, and isotypic immunoglobulin determinants. We show that these highly reproducible, easily disseminated, and essentially inexhaustible reagents can be used for rapid, sensitive, and quantitative Gm typing. Using this system we define two previously unrecognized Gm determinants, one of which, found to date only in Caucasians, is different from all known Gm markers and thus defines previously unrecognized alleles and haplotypes. The other determinant co-segregates with the conventional G3m(b1) marker but is distinct from that marker on serological grounds. The successful preparation of mouse monoclonal antibodies that detect human Gm allotypic differences and the development of an assay system capable of typing isoallotypic as well as allotypic determinants opens the way to further dissection and application of this rich genetic system.

Alleles↗

T-cell regulation of antibody responses: demonstration of allotype-specific helper T cells and their specific removal by suppressor T cells.

Allotype suppressor T cells (Ts) generated in SJL X BALB/c mice specifically suppress production of antibodies marked with the Ig-1a allotype. The studies presented here show that allotypes Ts suppress by specifically removing helper T cell (Th) activity required to facilitate differentiation and expansion of B cells to Ig-1b antibody-forming cells. We show first that Ts and Th belong to different T-cell subclasses as defined by Ly surface antigens. Ts are Ly2+Lyl- and thus belong to the same subclass as cytotoxic precursor and effector cells; Th are Lyl+Ly2- cells and thus belong to the subclass containing cells which can exert helper functions and initiate delayed hypersensitivity reactions. Placing these cells in these two subclasses shows that Th are different from Ts and suggests that they play different roles in regulating antibody responses. The difference in these roles is defined by the evidence presented here showing that Ts attack Th and regulate the antibody response by specifically regulating the availability of Th activity. We show that in allotype suppressed mice, Ts which suppress Ig-1b antibody production have completely removed the Th activity of helping Ig-1b cells without impairing Th activity which helps other IgB B cells. These findings imply the existence of allotype-specific Th for Ig-1b cells (Ig-1b Th). We directly establish that Ig-1b cells require such help by showing that carrier-primed spleen cells from Iga/Iga congenic hybrids help Ig-1a B cells from hapten-primed Igb/Iga donors but do not help Ig-1b B cells from the same donor in the same adoptive recipient.

Animals↗

Anti-immunoglobulin antibodies. I. Expression of cross-reactive idiotypes and Ir gene control of the response to IgG2a of the b allotype.

The anti-allotype antibody response to the b allotypic form of IgG2a is regulated by major histocompatibility complex (MHC)-encoded immune response (Ir) genes. Mice of d, b, p, q, r, and s haplotypes make a strong anti-allotype response on immunization with the CBPC101 myeloma protein (IgG2ab), whereas mice of the k, m, a, a1, u, and z haplotypes made no, or a very poor, response. All responder strains produce anti-IgG2ab antibodies which share common idiotypes (Id) without relation to the allelic forms of the Ig heavy-chain-constant region genes that the responding mice possess. Isoelectric focusing analysis of the anti-allotype antibodies produced in various strains of mice showed that they are of limited heterogeneity and quite similar from strain to strain. Five out of six hybridoma products with specificity for CBPC101 allotype expressed cross-reactive idiotypes (IdX). Two of hybridoma products expressing IdX identify CH3-domain determinants, and one has been assigned a CH2-domain specificity.

Animals↗

Susceptibility to invasive Haemophilus influenzae type b disease and the immunoglobulin G2m(n) allotype.

There has been considerable controversy about the role of the immunoglobulin G2m(n) allotype and risk of invasive Haemophilus influenzae type b (Hib) disease. This allotype was studied in a large cohort of Finnish children (178) with invasive Hib disease. The G2m(n) allotype distribution was similar to that in the normal white Finnish population. No increased risk of Hib disease could be associated with the n-/n- genotype [i.e., lack of G2m(n) allotype]. Thus, the G2m(n) allotype does not seem to be a major determinant of susceptibility to Hib infection among white populations in industrialized countries.

Child↗

Immunoglobulin allotypes and genetic susceptibility to invasive Haemophilus influenzae type b and Staphylococcus aureus infections in South African children.

OBJECTIVE: The purpose of this study was to determine whether the G2m(n), G1m(f) and Km(3) immunoglobulin allotypes have any association with susceptibility to invasive Haemophilus influenzae type b (Hib) and Staphylococcus aureus (S. aureus) infections in children. METHODS: Direct enzyme-linked immunosorbent assays with commercially available monoclonal antibodies were established to quantitate G2m(n) and G1m(f) allotypes. A qualitative enzyme-linked immunosorbent assay with polyclonal rabbit anti-Km(3) antibody was established for Km(3) determination. RESULTS: The G2m(n) marker occurred in 34.4% of the mixed ancestry population and 2.9% of the Black population. There was a significantly decreased frequency of the G2m(n) allotype in mixed ancestry children with Hib meningitis (8.5%) and Hib osteomyelitis/septic arthritis and a decreased frequency of Km(3) in black and mixed ancestry children with Hib meningitis. The frequency of G2m(n), G1m(f) and Km(3) allotypes in patients with S. aureus osteomyelitis/septic arthritis were not significantly different from normal population frequency. CONCLUSIONS: This study shows a clear association between the absence of the G2m(n) allotype in mixed ancestry children and susceptibility to invasive infections caused by H. influenzae and an association between the absence of Km(3) and Hib meningitis in both black and mixed ancestry children.

Arthritis, Infectious↗