Association of complement factor B allotypes and serum biomarkers in rheumatoid arthritis patients and their relatives.

The aim of the study was to investigate the allotypic variability of complement factor B (BF) in patients and relatives with rheumatoid arthritis (RA) and its association with serological biomarkers and clinical features of the disease. BF allotypes were determined by high-voltage agarose gel electrophoresis in serum samples of 180 patients with RA, 198 relatives and 98 controls from Southern Brazil. Anticyclic citrullinated peptide (anti-CCP), antimutated citrullinated vimentin (anti-MCV) and IgA-rheumatoid factor (RF) were determined by ELISA and IgM-RF by latex agglutination in all samples. No significant differences were found in the allotypic variants of BF between patients with RA, relatives and controls, nor associations with gender and age of RA onset. BF*S07 allotype was significantly associated with extra-articular manifestations (EAMs; Secondary Sjögren Syndrome, pneumonitis, rheumatoid nodules) in patients with RA (P = 0.02; OR = 6.62). Patients with phenotype BF F had lower positivity for anti-MCV biomarker (P = 0.02; OR = 0.22) and those with allotype BF*S had higher prevalence of this autoantibody (P = 0.02; OR = 3.77). An increased frequency of RF-IgA was detected in relatives of patients with RA with BF FS07 phenotype (P = 0.02; OR = 7.78). Complement BF variability did not influence the development of RA in the studied patients, but BF variants may act as markers of disease prognosis, such as development of EAMs, corroborating with the role of the alternative pathway in the pathogenesis of RA.

Accuracy and coverage assessment of Oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (OryCun2.0) and localization of the IGH locus to chromosome 20.

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.

Association of FCRL3 genotypes with susceptibility of Iranian patients to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex disease, the hallmark of which is synovial joint inflammation. The substantial contribution from genetic factors in susceptibility to RA has been well-defined. The Fc receptor-like3 (FCRL3) gene is one of the genes that have recently shown a significant association with RA. To determine the possible role of FCRL3-169 C/T and FCRL3-110 A/G gene polymorphisms in the development of RA in Iranian patients, 320 RA patients and 302 healthy subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism. No significant difference was found in genotype and allele frequencies of FCRL3-169 C/T between patients and controls. In contrast, at position -110 A/G, the frequency of the AA genotype and A allele was significantly decreased in RA patients compared to controls (p = 0.005). After Bonferroni correction for multiple testing, no significant correlations between FCRL3-169 C/T and -110 A/G polymorphism and laboratory and clinical features of the patients was observed. In conclusion, the results of this study showed a significant association between FCRL3-110 A/G polymorphism and susceptibility to RA.

Alloantibodies to human platelet glycoprotein antigens (HPA) and HLA class 1 in a cross section of Nigerian antenatal women.

The prevalence of antibodies to human platelet antigens (HPA) and human leukocyte antigens (HLA) class 1 antigens among Nigerian pregnant women has not been reported in our country. This study was therefore aimed at screening the obstetric population for evidence of alloimmunization due to human platelet and HLA class 1 antigens. One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP 11b/111a 12 (8.3%), GP 1a/11a 35 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9 (6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-HPA-1a,-3a,-4a (4.2%), anti-HPA-1b,-3b,-4a (4.2%), anti-HPA-30 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%). A high prevalence rate of human platelet arid cytotoxic antibodies has been observed in our obstetric population. There is need to establish platelet serology laboratory for the proper antenatal and postnatal management of pregnant mothers in this region.

Rapid LC-MS screening for IgG Fc modifications and allelic variants in blood.

A new method for simultaneously screening allelic variants and certain Fc modifications on endogenous human IgG1 and IgG2 directly from blood samples is described. The IdeS endoproteinase was used to cleave IgG in serum to generate Fc, which, after denaturation, was analyzed directly as monomeric Fc (Fc/2) by LC-MS to identify the haplotype(s) present in each individual. The relative levels of IgG isotype and haplotype ratios were generated along with the profile of the major Fc glycans and several other modifications associated with each IgG1 or IgG2 haplotype. Since only minute quantities (5 µL) of blood are required and analysis can be highly automated, this approach lends itself to screening large populations. We demonstrate its utility in examining possible correlations between Fc properties and allelic variants. IgG1 core fucosylation, which significantly impacts antibody dependent cellular cytotoxicity (ADCC), showed an asymmetric distribution, with a small number of individuals showing unexpectedly high core afucosylation levels. In these individuals, IgG2 afucosylation levels were normal. Finally, a new IgG1 allotype, previously not characterized, was identified using this analytical methodology.

DP reactive antibody in a zero mismatch renal transplant pair.

This study describes molecular basis for positive B-cell flow crossmatch in a zero mismatch (0MM) transplant pair. Our end-stage renal disease patient was B-cell flow crossmatch positive with a 0MM deceased donor. DNA from the donor and patient were further typed by a high-resolution method. The patient's sera were tested for anti-HLA reactivity by single antigen bead using a Luminex platform. The patient and donor were found to be HLA identical except for a single DP allele MM (DPB1*0601). As there was no single antigen bead coated with DPB*0601, analysis of the amino acid residues of reactive and nonreactive DPB1 alleles was conducted. The results showed that all reactive alleles carried the amino acid D-E at residue 55 and 56 of DPB1. The MM allele DPB1*0601 also carries the DE 55-56 epitope. We conclude that positive B-cell flow crossmatch was likely the result of single MM in the DP locus.

[Diversity of wild and domestic pig populations estimated by a set of serum allotypes].

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens.

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.

Allelic variation at the VHa locus in natural populations of rabbit (Oryctolagus cuniculus, L.).

The large interallelic distances between the three rabbit Ig V(H)a lineages, a1, a2 and a3, suggest that the persistence time of the V(H)a polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the V(H) loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with V(H)a locus-specific alloantisera, showed either cross-reactivity ("a-positive" variants) or no reaction at all ("a-blank"). Using RT-PCR, we determined 120 sequences of rearranged V(H) genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the V(H) genes transcribed in a-positive rabbits are closely related to the V(H)1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use V(H) genes that, although clearly related to the known V(H)a genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit V(H)a residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the V(H)a4 and the V(H)a1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit V(H)a locus exist, one of which seems to be endemic in the Iberian range.

Successful management of a P-incompatible pregnancy using double filtration plasmapheresis.

The presence of PP1Pk antibody is considered a main cause of repeated spontaneous abortions in early pregnancy. Because PP1Pk antibody generated in maternal blood may disturb the feto-maternal environment, plasmapheresis is supposed to be a beneficial method of lowering PP1Pk antibody levels in women with blood group-incompatible pregnancies. Double filtration plasmapheresis (DFPP) was used to treat feto-maternal P-incompatible pregnancy. The successful use of DFPP to treat a pregnant Japanese woman is presented. This method is a useful treatment for group P women who have not been able to deliver a live infant.

B1 cells contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig allotype chimeric mice.

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.

Fcgamma receptor polymorphisms determine the magnitude of in vitro phagocytosis of Streptococcus pneumoniae mediated by pneumococcal conjugate sera.

Fcgamma receptors show two genetically determined polymorphisms: the biallelic FcgammaRIIa-R131 and -H131 polymorphism and the NA1/NA2 FcgammaIIIb polymorphism. Using 10 pre- and postconjugate vaccination sera from adults, we analyzed in vitro phagocytic capacities of three different combinations of polymorphonuclear leukocyte FcgammaR allotypes: those homozygous for the H131 and NA1 allotype, those homozygous for the R131 and NA2 allotype, and those heterozygous for both receptors. For pre- and postvaccination sera, mean phagocytosis levels for the homozygous H131/NA1 allotype were 4 -fold higher than for the homozygous R131/NA2 allotype. There was a strong and significant correlation between IgG2 ELISA antibody titers and phagocytosis levels for the homozygous H131/NA1 Fcgamma receptor allotype and the heterozygous allotype but not for the homozygous R131/NA2 allotype. There was no relation between IgG1 ELISA titer and phagocytosis level. Apparently the IgG2 antibodies induced are functionally the most important. This may explain the large effect of Fcgamma receptor polymorphisms on in vitro phagocytosis of pneumococci mediated by conjugate antisera.

Preparation of IgG subclass allotypes from polyclonal IgG.

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(-n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion-exchange and protein A-Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(-n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(-n) molecules is of special interest as no genetic marker has been found to identify this allotype.

Relation of ex vivo stimulated blood cytokine synthesis to post-traumatic sepsis.

The cytokine production in endotoxin stimulated blood of patients immediately after polytrauma with high risk for developing sepsis or multi organ failure was analysed. Forty patients sustaining traumatic injury with >/=317 pts according to the Injury Severity Score (ISS), 10 of whom developed severe sepsis (ACCP/SCCM conference 1992) were included in the study. Levels of interleukin 8 (IL-8), IL-6 and tumour necrosis factor (TNF) were measured by ELISA in endotoxin-stimulated whole blood and IL-10 and IL-6 in serum. The allotype for the bi-allelic Nco I restriction length polymorphism in the TNF locus was determined for each patient.Two to four hours after polytrauma endotoxin-stimulated synthesis of TNF and IL-6 was found to be reduced in whole blood from patients compared to healthy donors, whereas no such differences were found for IL-8 synthesis. At this time, however, the patients who developed sepsis at a later stage (day 4-6) showed significantly (P<0.05) enhanced IL-8 synthesis in endotoxin stimulated whole blood in comparison to healthy donors. The IL-6 and TNF production of their blood was significantly enhanced compared to patients with uncomplicated recovery. Ninety per cent of the patients developing sepsis were of the TNFB2/TNFB2 allotype, whereas this was the case for only 30% of the non-septic group. Assessment of endotoxin-stimulated cytokine synthesis may provide a prognostic indicator for patients at high risk for developing a sepsis syndrome.

Clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy.

OBJECTIVE: To describe the clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy (IIM) and to compare these with the features of sporadic IIM. METHODS: Clinical signs and symptoms, autoantibodies, HLA-DRB1 and DQA1 alleles, and GM/KM phenotypes were compared among 36 affected and 28 unaffected members of 16 unrelated families in which 2 or more blood relatives developed an IIM. In addition, findings in patients with familial IIM were compared with those in 181 patients with sporadic IIM. The families included 3 pairs of monozygotic twins with juvenile dermatomyositis, 11 families with other siblings or relatives with polymyositis or dermatomyositis, and 2 families with inclusion body myositis. RESULTS: The clinical features of familial IIM were similar to those of sporadic IIM, although the frequency of myositis-specific autoantibodies was lower in familial than in sporadic IIM. DRB1*0301 was a common genetic risk factor for familial and sporadic IIM, but contributed less to the genetic risk of familial IIM (etiologic fraction 0.35 versus 0.51 in sporadic IIM). Homozygosity at the HLA-DQA1 locus was found to be a genetic risk factor unique to familial IIM (57% versus 24% of controls; odds ratio 4.2, corrected P = 0.002). CONCLUSION: These findings emphasize that 1) familial muscle weakness is not always due to inherited metabolic defects or dystrophies, but may be the result of the development of IIM in several members of the same family, and 2) multiple genetic factors are likely important in the etiology and disease expression of familial IIM, as is also the case for sporadic myositis, but DQA1 homozygosity is a distinct risk factor for familial IIM.

Identification of rabbit immunoglobulin latent Ckappa1 allotype genes alters the concept of allelic inheritance.

In the rabbit, expression of immunoglobulin Ckappa1 light chain genes is believed to be under allelic control. Conventionally, four nominal allotypic variants, b4, b5, b6 and b9 have been shown to be co-dominantly expressed at the Ckappa1 gene locus. Analogously, the heavy chain allotypes, VHa1, VHa2 and VHa3, found in the V region, are also believed to be inherited co-dominantly. However, after our earlier discovery of non-allelic or latent allotypes in the serum and on cell surfaces. we subsequently reported that cDNA sequences for latent b5 and b6 were identical to nominal b5 and b6, respectively (Ishaq et al., 1990). The latent b5 cDNAs were from two homozygous b4,b4 rabbits; the latent b6 cDNA was found in a heterozygous b4,b9 rabbit. The cDNA sequences had been obtained from lymph nodes and spleens of rabbits which had been infected with Trypanosoma brucei in order to induce latent allotypes more consistently. In this article, employing spleen DNA from three different T. brucei-infected rabbits, (one, heterozygous b4,b9; two others, homozygous, b4,b4), we initially detected two bands by Southern analysis after Hind III digestion using a 624 base pair Ckappa1 b4 probe derived from a b4,b4 rabbit. However, the probe was non-specific allotypically as it hybridized to b5, b6 and b9 Ckappa1 DNA. Therefore, in order to search for the latent genes, we used allotype-specific oligonucleotides for b5, b6 and b9 to probe DNAs from both normal and T. brucei-infected rabbits by Southern blotting. At the outset, employing a b4 oligonucleotide probe, we detected a single 5.8 Kb segment in two b4,b4 rabbit DNAs after Bg1 II digestion. The findings, using the 624 base pair Ckappa1 b4 probe and the b4 oligomer, agreed with earlier data reported by others. Subsequently, we tested kidney, liver and spleen DNAs from one of these and other rabbits for genomic latent b5, b6 and b9 using these specific oligomeric probes. For each latent allotype, Southern analysis revealed latent-allotype specific DNA segments in the genome. After cosmid cloning and sequencing, latent kappa1, b5, b6 and b9 genes were found to be identical in their coding regions with their nominal counterparts. The genes contained at the 5' end the PyPyXPyAG RNA splice acceptor site found in immunoglobulin and many eukaryotic genes. as well as the termination codon TAG, together with AATAAA and the T-rich sites responsible for cleavage-polyadenylation in the untranslated region downstream from the 3' end. Single cosmid clones representing the b5, b6 and b9 genes were mapped for restriction sites which resulted in identifying putative Jk and enhancer regions. The results thus indicate that latent allotype genes are potentially functional. The data provide evidence that allotypes are not strictly controlled by allelic genes but must be regulated by an hierarchical mechanism which provides for synthesis of allelic allotypes mainly (10-20 mg/ml) together with non-allelic allotypes at lower concentrations (2-20 microg/ml) following activation of the latent genes. These results lay to rest the belief that Ckappa1 latent allotypes are the products of scrambled genes or idiotypic mimicry. Importantly, we now have the possibility of investigating the factors leading to latent allotype gene expression, the Vk and Jk regions associated with the genes, and therefore whether antibody diversity is expanded. We do not know, nor do we imply, that latent allotypes are present in all rabbits. However, since the four conventional Ckappa1 allotypes are present in the genome of several of our tested rabbits, and are presumably functional. we are faced with the probability that rabbit allotypes under certain conditions may in fact behave as isotypes and not allotypes.

Serologic allogeneic chimerism.

BACKGROUND: At least some transplanted livers secrete soluble human leukocyte antigens (sHLA) of donor phenotype into the body fluids of recipients. The individuals in whom this phenomenon occurs are by definition serologic allogeneic chimeras. Because an allogeneic transplanted liver may induce tolerance to itself and other organs in animals of the donor strain, and because maintenance of a soluble antigen in the circulation of any animal in sufficient quantity for a sufficient period generally leads to tolerance, this phenomenon may be biologically important. This study was performed to determine how common this phenomenon is and whether it occurs after transplantation of organs other than the liver. METHODS: We studied 445 serum samples obtained from transplant recipients (liver, n=12; kidney, n=18; and heart, n=8) before and at various intervals after transplantation. All patients studied had allografts that had functioned for more than 1 year. We used an enzyme-linked immunosorbent assay to quantitate sHLA-A2 and sHLA-A1/A3/A11 (as a cross-reacting group). Donor and recipient combinations were selected in which measurable allotypes in donors were not present in recipients. In some instances, an additional allotype was present in a recipient but not in a donor. RESULTS: All liver transplant recipients had detectable donor sHLA in their serum samples after transplantation. In 72% of kidney and 50% of heart transplant recipients, donor sHLA was found persistently in serum samples obtained after transplantation. Interestingly, all heart transplant recipients of HLA-A3, but none of HLA-A2, had detectable donor sHLA in their serum samples, a finding that may be due to technical reasons. High and stable serum concentrations of donor sHLA characterize long-term stable allograft function. CONCLUSIONS: Donor sHLA is produced by all transplanted livers, most transplanted kidneys, and at least half of (but probably more) transplanted hearts. The hypothesis that donor sHLA may be tolerogenic to liver transplants can be expanded to include kidney and heart transplants.

Immunoglobulin levels in patients with carbohydrate-deficient glycoprotein syndrome type I.

BACKGROUND: The characteristic feature of carbohydrate-deficient glycoprotein syndrome (CDGS) type I, a multisystemic disease, is underglycosylation of many serum glycoproteins, such as transferrin. A few cases of severe infections during childhood have been reported and an underlying immunodeficiency has been suggested. Because of this and the fact that all immunoglobulin (Ig) isotypes are glycoproteins we analysed the Ig levels in patients with CDGS I. METHODS: The serum concentrations of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE, and the frequency of the G2m(23) allotype were measured by enzyme immunoassay in 15 patients with CDGS type I. RESULTS: Ten (67%) patients had an elevated level of at least one Ig, when compared to age-related reference ranges. No particular isotype was involved although a tendency towards high IgE levels was registered. The frequency of homozygous G2m(23)-negative CDGS patients (33%) was not different from that of blood donors (34%). CONCLUSION: We conclude that CDGS I patients have no major changes in the serum levels of any specific Ig isotype. The severe infections observed in some CDGS patients are therefore unlikely to involve any Ig deficiency. Our results do not exclude that Ig of patients with CDGS may have altered physiological functions because of abnormal glycosylation.