Recombinant IgA production: single step affinity purification using camelid ligands and product characterization.

Immunoglobulins of isotype A (IgA) mediate a key role in mucosal immunity and are promising new immunotherapeutic candidates, but difficulties in obtaining enough material often hamper their in vivo exploration. We established recombinant Chinese hamster ovary (CHO) cells which stably expressed two IgA1 antibodies under serum-free conditions. The two cell lines achieved significantly different specific productivities of 16 pg per cell and day and 100 times less, a common phenomenon in recombinant antibody expression which challenges the production and purification process. Polymeric IgA in crude culture supernatants was assembled with J chain and showed expected specificity. We employed an immobilized camelid anti-human alpha-chain VHH ligand and isolated both recombinant IgAs at high purity and yield in a single chromatographic step. The described method was irrespective of the light chain and specificity and may be used as a generic capture step for the isolation of any IgA. Results were compared with a multistep purification process consisting of an affinity step based on immobilized jacalin followed by anion exchange and hydrophobic interaction chromatography.

Deletion of the α immunoglobulin chain membrane-anchoring region reduces but does not abolish IgA secretion.

Class switching and plasma cell differentiation occur at a high level within all mucosa-associated lymphoid tissues. The different classes of membrane immunoglobulin heavy chains are associated with the Igα/Igß heterodimer within the B-cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membranes have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA-class BCR through a 'αΔtail' targeted mutation, deleting the Cα immunoglobulin gene membrane exon. This allowed us to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail(+/+) B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA(+) B lymphocytes. By contrast, in secretions the secretory IgA was very low, indicating that J-chain-positive plasma cells producing secretory IgA overwhelmingly differentiate from previously class-switched membrane IgA(+) memory B cells. In addition, although mucosa-associated lymphoid tissues are a major site for plasma cell accumulation, αΔtail(+/+) mice showed that the gut B-cell lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the membrane IgA BCR.

Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation.

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

[Expression of major histocompatibility complex class I-related chain A antibody in patients with end-stage renal disease and its clinical implications].

OBJECTIVE: To study the frequency of major histocompatibility complex class I-related chain A (MICA) antibody in patients with end-stage renal disease (ESRD). METHODS: Luminex flow cytometry and beads loaded with 11 MICA antigens were used to identify the MICA antibody and evaluate the antibody specificity in 110 patients with ESRD. RESULTS: The positivity rate of MICA antibody was 40% (12/30) in PRA-positive patients, significantly higher than the rate of 17.5% (14/80) in PRA-negative patients (chi(2)=6.120, P=0.013). MICA-specific antibodies against 10 of the 11 MICA antigens were detected in 26 MICA antibody-positive patients, and 26.92% of the MICA antibody-positive patients had antibodies with single-specificity and 73.08% had polyspecific antibodies. Three MICA antibody-positive patients with cadaveric kidney transplantation showed good function of the graft without acute rejection 2 months after the operation. CONCLUSION: The positivity rate of MICA antibody is significantly higher in PRA-positive patients, suggesting a strong correlation between MICA and PRA positivity. The MICA antibodies are polyspecific and probably consist of IgM and IgG. These data can be used as prospective data for these ESRD patients considering potential renal transplantation, and may facilitate further investigation of the association of MICA with renal transplantation.

[Problem of J-chain of immunoglobulins].

Molecules of secretory immunoglobulins (Ig) of classes A and M (sIgA and sIgM) play the main role in protection of mucosae from pathogenic factors. The apparatus of synthesis of these molecules represents the most powerful part of the immune system. One of the key elements of the sIgA and sIgM is J-chain. It represents an acid polypeptide of molecular mass of about 15 kDa composed of 137 amino acid residues including 8 cysteine residues and one site of N-glycosylation. The primary structure of the J-chain is unique: attempts to ascribe it to any family of known proteins so far have failed. The J-chain is inserted into the sIgA and sIgM molecules to form disulfide bonds with C-terminal sites of alpha- or mu-chains. It is necessary for formation of IgA dimers and IgM pentamers, for reception of these molecules by epithelial cells, binding of secretory component to them, and for transfer of sIgA and slgM molecules onto mucosal surfaces and into secrets of endocrine glands. The J-chain has been revealed in the cytoplasm of the early T- and B-lymphocyte precursors not producing Ig. The J-chain is detected in the human embryonic liver cells earlier than the expression of the mu-chain gene begins. Study of mice with knockout of J-chain B-lymphocytes-producents has shown their block of function of T-helpers providing formation of immunologic memory. Comparison of J-chain genes of mammals, amphibians, reptiles, and cartilaginous fishes has shown the degree of interspecies homology of these proteins to vary from 33% to 70%. The J-chain genes were revealed in representatives of all vertebrate classes except for cyclostomes and bony fishes. In 1996, data were published about the presence of the J-chain genes-homologs in invertebrates, tunicates, and cyclostomes. No papers reproducing or confirming these data have been published. On the contrary, in the literature an opinion appeared that indicate necessity to revise the notion about the presence of J-chain in invertebrates. The main unsolved issues on the J-chain involve the tertiary structure of this protein, its relation to some particular protein family, its functions in cells of the T- and B-lymphocytic differentiation lineages as well as its evolutionary age.

Nonhuman primate IgA: genetic heterogeneity and interactions with CD89.

Nonhuman primates are extremely valuable animal models for a variety of human diseases. However, it is now becoming evident that these models, although widely used, are still uncharacterized. The major role that nonhuman primate species play in AIDS research as well as in the testing of Ab-based therapeutics requires the full characterization of structure and function of their Ab molecules. IgA is the Ab class mostly involved in protection at mucosal surfaces. By binding to its specific Fc receptor CD89, IgA plays additional and poorly understood roles in immunity. Therefore, Ig heavy alpha (IGHA) constant (C) genes were cloned and sequenced in four different species (rhesus macaques, pig-tailed macaques, baboons, and sooty mangabeys). Sequence analysis confirmed the high degree of intraspecies polymorphism present in nonhuman primates. Individual animals were either homozygous or heterozygous for IGHA genes. Highly variable hinge regions were shared by animals of different geographic origins and were present in different combinations in heterozygous animals. Therefore, it appears that although highly heterogeneous, hinge sequences are present only in limited numbers in various nonhuman primate populations. A macaque recombinant IgA molecule was generated and used to assess its interaction with a recombinant macaque CD89. Macaque CD89 was able to bind its native ligand as well as human IgA1 and IgA2. Presence of Ag enhanced macaque IgA binding and blocking of macaque CD89 N-glycosylation reduced CD89 expression. Together, our results suggest that, despite the presence of IgA polymorphism, nonhuman primates appear suitable for studies that involve the IgA/CD89 system.

The B cell receptor promotes B cell activation and proliferation through a non-ITAM tyrosine in the Igalpha cytoplasmic domain.

In addition to the tyrosines of the Igalpha and beta immunoreceptor tyrosine-based activation motifs (ITAMs), the evolutionarily conserved Igalpha non-ITAM tyrosine 204 becomes phosphorylated upon antigen recognition by the B cell receptor (BCR). Here we demonstrate that splenic B cells from mice with a targeted mutation of Igalpha Y204 exhibited an isolated defect in T cell-independent B cell activation, proliferation, and antibody response upon BCR engagement, yet normal BCR capping, antigen internalization, antigen presentation, and T cell-dependent antibody production. Mutant B cells, present in normal numbers, exhibited unimpaired BCR-induced spleen tyrosine kinase (Syk) phosphorylation but reduced B cell linker protein (BLNK) phosphorylation, calcium flux, and nuclear factor kappaB (NFkappaB), c-jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. These results suggest that Igalpha non-ITAM tyrosine 204 promotes a distinct cellular response, namely T-independent B cell proliferation and differentiation via phosphorylation of the adaptor BLNK.

Cloning and characterization of the dromedary (Camelus dromedarius) neonatal Fc receptor (drFcRn).

The full length cDNA of the dromedary neonatal Fc receptor (drFcRn) alpha chain was isolated and found that it is similar to the neonatal Fc receptor (FcRn) of other species with a high overall similarity to ruminant FcRn alpha chains. The drFcRn/Fc contact residues are highly conserved and predicted to bind both conventional (IgG1) and heavy chain (IgG2a, IgG3) antibodies. Using immunohistochemistry, we detected its expression in the hepatocytes and in epithelial cells of portal bile ductuli and also in the mammary gland acini and ducti. Remarkably, Ser313, that was identified to be crucial for apical to basolateral transcytosis, is substituted in the drFcRn alpha chain. The full length of the dog and orangutan FcRn alpha chains was also identified from databases. Analyzing the phylogenetic relatedness of this gene we found that dromedary clustered together with artiodactyls, dog is located between artiodactyls and primates, where the orangutan was branched, reflecting the accepted evolutionary relationships.

Immunofixation reveals an apparent alpha heavy chain caused by precipitation of fibrinogen with IgA antiserum.

BACKGROUND: Fibrinogen present in serum specimens can interfere with the interpretation of serum protein electrophoresis. We report here the unexpected precipitation of fibrinogen by an IgA antiserum used in immunofixation electrophoresis. METHODS: Immunofixation electrophoresis of plasma combined with ethanol precipitation, serial dilution of plasma, and fibrinogen adsorption of the antiserum were used to investigate the apparent immunoreactivity of a commercial source of IgA antiserum to fibrinogen. RESULTS: Fibrinogen immunoreactivity by IgA antiserum was observed at fibrinogen concentrations > or =0.93 g/l. Ethanol precipitation of plasma removed fibrinogen and prevented the immunofixation of the protein by the IgA antiserum. Adsorption of the IgA antiserum with human fibrinogen removed its ability to precipitate fibrinogen, demonstrating cross-reactivity between the IgA antiserum and fibrinogen. CONCLUSIONS: Commercial sources of antiserum used for immunofixation electrophoresis may contain antibodies with specificity towards proteins typically absent from serum such as fibrinogen and can produce clinically misleading results.

Identification of allelic polymorphism in the caprine IGHA gene.

Variation in the immunoglobulin heavy alpha chain (IGHA) constant region has been reported in a number of species. In this study, the IGHA gene was investigated in goats using PCR-single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences were identified from 111 Boer and Angora goats. Either one or two sequences were detected in individual goats, and all the sequences shared high homology to the published ovine and bovine IGHA sequences. These sequences were predicted to encode three amino acid sequences, two with a longer hinge region and one with a shorter hinge region. The variation reported here may affect the structure of the hinge and hence the function of IgA.

An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion.

The Ig heavy (H) chain plays a pivotal role in the regulation of primary B cell development through its association with a variety of other proteins including Igalpha and Igbeta, the surrogate light chain components and bona fide L chains, to form transmembrane signaling complexes. Little is known about how alterations in the structure of the H chain variable region influence association with these proteins, or the signaling capacity of the complexes that form. Here we describe a line of VH 'knockin' mice in which the transgene-encoded VH region differs by eight amino acid residues from the VH region in a VH knockin line we previously constructed and characterized. The transgenic H chain locus in the line of mice we characterized earlier efficiently promotes H chain allelic exclusion and all phases of primary B cell development, resulting in the generation of mature B1, marginal zone (MZ) and follicular (FO) B cell compartments. In contrast, the transgenic H chain locus in the new line fails to enforce allelic exclusion, as evidenced by the majority of peripheral B cells expressing two H chains on their surfaces. Moreover, this locus inefficiently drives bone marrow B lymphopoiesis and FO B cell development. However, this H chain locus does promote MZ B cell development, from precursors that appear to be generated during fetal and neonatal life. We discuss these data in the context of previous findings on the influence of Ig H chain structure on primary B cell development.

Physical activity and stress resistance: sympathetic nervous system adaptations prevent stress-induced immunosuppression.

A physically active lifestyle incurs many health benefits. One recently recognized benefit of regular moderate exercise is stress reduction. The current review develops the hypothesis that physical activity may prevent stress-induced suppression of the immune system and suggests an immunophysiological mechanism (sympathetic nervous system constraint) for this effect.

Immunoproliferative small intestinal disease (IPSID): a model for mature B-cell neoplasms.

Immunoproliferative small intestinal disease (IPSID) was recently added to the growing list of infectious pathogen-associated human lymphomas. Molecular and immunohistochemical studies demonstrated an association with Campylobacter jejuni. IPSID is a variant of the B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), which involves mainly the proximal small intestine resulting in malabsorption, diarrhea, and abdominal pain. Geographically, IPSID is most prevalent in the Middle East and Africa. IPSID lymphomas reveal excessive plasma cell differentiation and produce truncated alpha heavy chain proteins lacking the light chains as well as the first constant domain. The corresponding mRNA lacks the variable heavy chain (V(H)) and the constant heavy chain 1 (C(H)1) sequences and contains deletions as well as insertions of unknown origin. The encoding gene sequence reveals a deletion of V region and parts of C(H)1 domain. Cytogenetic studies demonstrated clonal rearrangements involving predominantly the heavy and light chain genes, including t(9;14) translocation involving the PAX5 gene. Early-stage IPSID responds to antibiotics (30%-70% complete remission). Most untreated IPSID patients progress to lymphoplasmacytic and immunoblastic lymphoma invading the intestinal wall and mesenteric lymph nodes, and may metastasize to a distant organ. IPSID lymphoma shares clinical, morphologic, and molecular features with MALT lymphoma, lymphoplasmacytic lymphoma, and plasma cell neoplasms.

Basal Igalpha/Igbeta signals trigger the coordinated initiation of pre-B cell antigen receptor-dependent processes.

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igalpha/Igbeta that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of V(H)(D)J(H) recombination, and induced kappa light (L) chain recombination and cytoplasmic kappa L chain protein expression. Interestingly, Igalpha/Igbeta-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRalphabeta expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igalpha/Igbeta-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.

Neuraminidase treatment abrogates the binding abnormality of IgA1 from IgA nephropathy patients and the differential charge distribution of its alpha-heavy chains.

We have studied the interaction of the Gal-GalNAc-reactive champedak lectin-C with neuraminidase-treated and untreated IgA1 from IgA nephropathy patients. The binding ability of the lectin to untreated IgA1 from IgA nephropathy patients was significantly lower as compared to the untreated IgA1 from normal controls. This differential lectin-binding capacity was abrogated when the experiment was performed on neuraminidase-treated sera. Treatment of the serum IgA1 with neuraminidase also abrogated the differential charge distribution between the alpha-heavy chains of IgA nephropathy patients and normal controls.

[Production of monoclonal antibodies to T-independent type 2 antigens with OptiMem-Iscove medium]. / Poluchenie monoklonal'nykh antitel k T-nezavisimym antigenam 2 tipa s ispol'zovaniem credy OptiMem-Iscove .

To obtain monoclonal antibodies to T-independent antigens of type 2, having low immunogenicity and incapable of inducing the appearance of memory cells, the use of the medium OptiMem-Iscove (1:1) with 10% of fetal serum, glutamine (50 mM) and antibiotics (100 units/ml) is proposed. The main advantage of this medium is the possibility of cloning cells without the use of feeders. The method has been approved in the process of obtaining monoclonal antibodies (McAb) to TI-2 antigens, both bacterial (S3) and synthetic (PVP), as well as to McAb to T-dependent antigen (alpha-chains of human IgA).

Secretory IgA epitopes in basal tears of extended-wear soft contact lens wearers and in non-lens wearers.

PURPOSE: To determine secretory IgA epitopes in tears of extended-wear soft contact lens wearers and non-wearing controls. METHODS: We developed enzyme-linked immunosorbent assays (ELISA) to determine the tear concentrations of two epitopes of secretory IgA, the IgA alpha-chain and the secretory component. These epitopes were measured in basal tears of 20 individuals in 6 nights of extended wear of etafilcon A soft contact lenses and in 19 non-lens-wearing individuals. RESULTS: Levels of IgA alpha-chain immunoreactivity were significantly decreased in the lens-wearing group compared to non-lens wearers. However, the level of secretory component immunoreactivity was not significantly different between groups. IgA alpha-chain and secretory component immunoreactvity were highly correlated; however, some samples showed a marked variation between these two values. CONCLUSION: Tear concentrations of sIgA epitopes are significantly reduced in extended-wear contact lens wearers, and may contribute to the increased susceptibility to ocular infection seen in this group.

A peptide derived from a polyreactive monoclonal anti-DNA natural antibody can modulate lupus development in (NZBxNZW)F1 mice.

In lupus-prone (NZBxNZW)F1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti-DNA antibodies. While these D23 Id+ antibodies were able to regulate anti-DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti-DNA antibodies and furthermore could participate in the lupus-like syndrome. To explore further the role of the D23 Id+ antibodies, we injected young B/W mice with a peptide corresponding to the VH CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA-reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti-DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.