Identification of a new European rabbit IgA with a serine-rich hinge region.

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.

Novel prognostic molecular factors: a quantum leap in the field of chronic lymphocytic leukemia.

Cytogenetic lesions do not completely explain clinical heterogeneity of chronic lymphocytic leukemia (CLL). The 2016 revision of the World Health Organization classification 2008 indicated that molecular lesions of TP53, NOTCH1, SF3B1 and BIRC3 have potential clinical relevance and could be integrated into an updated risk profile. The negative clinical implications of TP53 disruptions are well constituted and patients with these mutations should be considered for novel, small molecule signal transduction inhibitors therapies. Mutations of NOTCH1, SF3B1 and BIRC3 are associated with poor prognosis. Patients with mutated SF3B1 or NOTCH1 genes present shorter time to first treatment compared to unmutated group. NOTCH1 mutations are related to a high risk of Richter's syndrome transformation, especially in case of TP53 disruptions' coexistence. Large studies on MYD88 mutations in CLL have not explained clearly their clinical importance.The aim of this paper is to provide a comprehensive review on novel molecular aberrations identified in CLL.

Assessment of the short-term toxicity of TiO2 nanofiber in Sprague Dawley rats.

Synthetic nanomaterials have many unique chemical and physical properties, mainly due to their high specific surface area and quantum confinement effect. Specifically, titanium dioxide (TiO2 ) nanomaterial has high stability, anticorrosive, and photocatalytic properties. However, there are concerns over adverse biological effects resulting from bioeffects. This study was to investigate adverse effects associated with acute ingestion of TiO2 nanofiber (TDNF). TDNF was fabricated via electrospinning method, followed by dissolution in water. Six- to seven-week-old male Sprague Dawley rats were exposed to a total of 0, 40, and 60 ppm of TDNF for 2 weeks via oral gavage. Serum total protein and weight gain during the course of this study displayed marginal concentration-dependent alterations. These findings were followed by a global gene expression analysis to identify which transcripts might be responsive to TNDF toxicity. Differentially expressed mRNA levels were dose-dependently higher in animals exposed to TNDF. The majority of the affected genes were biochemically involved in immune response and inflammation. We believe this is due to the fact that TNDF is unable to penetrate the cell and forms phagocytosis sites that trigger inflammatory and immune response. All results taken together, short-term ingestion of TNDF produced marginal effects indicative of inflammation. Finally, the broad gene expression data were validated through quantification of immunoglobulin heavy chain alpha (Igha). Igha gene was upregulated in treated groups, showing similar expression patterns to the global gene expression data.

Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-ß1 into a biologically active protein.

Transforming growth factor beta (TGF-ß) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-ß isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-ß3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-ß1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-ß1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-ß1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-ß1, and co-expression of human furin enabled the proteolytic processing of latent TGF-ß1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-ß1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.

The change in Ig regulation from children to adults disconnects the correlation with the 3'RR hs1.2 polymorphism.

BACKGROUND: In the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3' of the constant alpha gene (3'RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3'RR1 and 3'RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele. RESULTS: We have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells. CONCLUSIONS: These data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.

Activation of the Ig Iα1 promoter by the transcription factor Ets-1 triggers Ig Iα1-Cα1 germline transcription in epithelial cancer cells.

Immunoglobulins (Igs) are known to be synthesized and secreted only by B lymphocytes. Class switch recombination (CSR) is a key event that enables B cells to express Igs, and one of the crucial steps for CSR initiation is the germline transcription of Ig genes. Surprisingly, recent studies have demonstrated that the Ig genes are also expressed in some epithelial cancer cells; however, the mechanisms underlying how cancer cells initiate CSR and express Igs are still unknown. In this study, we confirmed that the Ig Iα1 promoter in cancer cell lines was activated by the Ets-1 transcription factor, and the activity of the Ig Iα1 promoter and Ig Iα1-Cα1 germline transcription were attenuated after knockdown of Ets-1 by specific small interfering RNAs (siRNA). Furthermore, the expression of Ets-1 and Igα heavy chain in cancer cells was dose dependently upregulated by TGF-ß1. These results indicate that activation of the Ig Iα1 promoter by the transcription factor Ets-1 is a critical pathway and provides a novel mechanism for Ig expression in non-B cell cancers.

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.

Caloric restriction modifies both innate and adaptive immunity in the mouse small intestine.

Although caloric restriction (CR) apparently has beneficial effects on the immune system, its effects on the immunological function of the intestinal mucosa are little known. The present study explored the effect of CR on the innate and adaptive intestinal immunity of mice. Balb/c mice were either fed ad libitum (control) or on alternate days fed ad libitum and fasted (caloric restriction). After 4 months, an evaluation was made of IgA levels in the ileum, the gene expression for IgA and its receptor (pIgR), as well as the expression of two antimicrobial enzymes (lysozyme and phospholipase A2) and several cytokines of the intestinal mucosa. CR increased the gene expression of lysozyme and phospholipase A2. The levels of IgA were diminished in the ileum, which apparently was a consequence of the reduced transport of IgA by pIgR. In ileum, CR increased the gene expression for most cytokines, both pro- and anti-inflammatory. Hence, CR differentially modified the expression of innate and adaptive immunity mediators in the intestine.

Effect of moderate exercise on IgA levels and lymphocyte count in mouse intestine.

The aim of the present study was to determine the effect of moderate exercise on the production and secretion of IgA in mouse duodenum, on lymphocyte levels in the lamina propria, and on gene expression encoding for cytokines that regulate the synthesis of α-chain of IgA and the expression of pIgR in the lamina propria. Two groups of young Balb/c mice were fed ad libitum, one sedentary and the other with an exercise program (swimming) for 16 weeks. IgA levels in the duodenum were quantified by ELISA; the number of IgA containing cells as well as B cells, CD4(+) and CD8(+) T cells in the duodenal mucosa was determined by immunohistochemistry; gene expression was analyzed by real-time PCR, and the expression of proteins by Western blotting. Because of physical training, in the duodenum there was a decrease in the number of IgA producing cells, but an increase in the levels of IgA. Additionally, exercise increased the expression of the genes encoding for IL-4, IL-6, IL-10, TNF-α and TGF ß, cytokines that regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine. Thus, the increased IgA found in the duodenal lumen is probably due to the increased production of IgA in the LP and the increased transport of the pIgA-pIgR complex across epithelial cells. Possibly the increased S-IgA levels in the bile also contribute to the change in IgA levels.

[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.

Transcription of a productively rearranged Ig VDJC alpha does not require the presence of HS4 in the IgH 3' regulatory region.

V gene assembly, class switch recombination, and somatic hypermutation are gene-modifying processes essential to the development of an effective Ab response. If inappropriately applied, however, these processes can mediate genetic changes that lead to disease (e.g., lymphoma). A series of control elements within the Ig H chain (Igh) locus has been implicated in regulating these processes as well as in regulating IgH gene transcription. These include the intronic enhancer (Emu) and several elements at the 3' end of the locus (hs1,2, hs3a, hs3b, and hs4) known collectively as the 3' regulatory region. Although it is clear that the Emu plays a unique role in V gene assembly, it has not been established whether there are unique functions for each element within the 3' regulatory region. In earlier studies in mice and in mouse cell lines, pairwise deletion of hs3b and hs4 had a dramatic effect on both class switch recombination and IgH gene transcription; deletion of an element almost identical with hs3b (hs3a), however, yielded no discernible phenotype. To test the resulting hypothesis that hs4 is uniquely required for these processes, we induced the deletion of hs4 within a bacterial artificial chromosome transgene designed to closely approximate the 3' end of the natural Igh locus. When introduced into an Ig-secreting cell line, an Igalpha transcription unit within the bacterial artificial chromosome was expressed efficiently and the subsequent deletion of hs4 only moderately affected Igalpha expression. Thus, hs4 does not play a uniquely essential role in the transcription of a productively rearranged Ig VDJCalpha transcription unit.

Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells.

Generally, only B lymphocytes express immunoglobulin. Recently, we found the expression of Ig alpha heavy chain in human epithelial cancer cells unexpectedly. We first detected Ig VDJ-Calpha and Ialpha-Calpha transcripts in multiple cancer cell lines. Further, the configuration of the Ig heavy chain genomic locus was analyzed in human cancer cells. We found that cancer cells have the recombination VDJ region, but bear Ig Salpha region in germline configuration, which is different from Ig expression pattern in B cells. And human epithelial cancers possess the essential effectors including RAG-1 and RAG-2, but not activation induced cytidine deaminase (AID) protein. These provide further proofs for Ig alpha expression. In addition, we found that human cancer cells not only express the protein of Ig alpha chain, but also secrete the protein in secretory IgA (SIgA) pattern. Importantly, diverse CDR3 recombinations were found in human cancer cells of different epithelial origin. Since IgA is the key immunoglobulin which contributes to local immunity of mucous membrane, the aberrant expression of Ig alpha heavy chain might increase our further comprehension to development and immunity of cancers.

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.

Roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease.

AIM: To evaluate roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease (IPSID) and to study profiles of kappa (kappa) and lambda (lambda) light chains and IgA heavy chain. METHODS: The study consisted of 11 cases of IPSID and similar number of controls which included 11 of normal intestinal mucosa and 11 of high grade B cell lymphoma of ileum. The parameters analyzed included clinical profiles, biochemical and other laboratory investigations, radiologic and histological findings including immunohistochemistry. RESULTS: All IPSID cases had demonstrable serum IgA heavy chain and heavy mucosal plasma cell infiltration. According to Galian's histological staging, there were 4 patients with stage A and 7 with stage B. kappa and lambda light chains were over-expressed in 7 patients; 1 stage A patient had H pylori-positive active gastritis and eradication of H pylori led to disease remission. Stage A biopsies had higher expression for syndecan-1, while stage B had higher expression for bcl6 and p53. Syndecan-1, kappa and lambda light chains and IgA heavy chain showed inverse relationship with bcl6 and p53. All patients were treated with doxycycline. CHOP regime was added in 5 patients who developed frank lymphoma. Three died of the disease due to extensive organ infiltration. CONCLUSION: Certain immunomarkers like syndecan-1, kappa and lambda light chains and IgA heavy chain could be of much help in identifying early stage IPSID. Stage B IPSID showed higher expression for bcl6 and p53 than stage A IPSID. bcl6 and p53 expressions correlated with a more advanced disease stage and aggressive tumour behavior.

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.

Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal "blunt end-joining," but impaired end joining with partially complementary (1-3 bp) DNA ends. There was also an increased usage of microhomology at the mu-alpha switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (> or =10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia-mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

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.

Polymorphism of the IGHA gene in sheep.

Genetic variation in immunoglobulin A, the most abundant immunoglobulin in mammalian cells, has not been reported in ruminants. In this study, variation in the immunoglobulin heavy alpha chain constant gene (IGHA) of sheep was investigated by amplification of a fragment that included the hinge coding sequence, followed by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences, each characterized by unique SSCP banding patterns, were identified. One or two sequences were detected in individual sheep and all the sequences identified shared high homology to the published ovine and bovine IGHA sequences, suggesting that these sequences represent allelic variants of the IGHA gene in sheep. Sequence alignment showed that these sequences differed mainly in the 3' end of exon 1 and in the coding sequence of the hinge region. There was either a deletion or an insertion of two codons in the hinge coding region in these allelic variants. Codon usage in the hinge coding region was quite different from that in the non-hinge coding regions of the gene, suggesting different evolution of the IGHA hinge sequence. Three novel amino acid sequences of ovine IGHA were also predicted, and variation in these sequences might not only affect antigen recognition but also susceptibility to cleavage by bacterial or parasitic proteases.

Interallelic class switch recombination contributes significantly to class switching in mouse B cells.

Except for the expression of IgM and IgD, DNA recombination is constantly needed for the expression of other Ig classes and subclasses. The predominant path of class switch recombination (CSR) is intrachromosomal, and the looping-out and deletion model has been abundantly documented. However, switch regions also occasionally constitute convenient substrates for interchromosomal recombination, since it is noticeably the case in a number of chromosomal translocations causing oncogene deregulation in the course of lymphoma and myeloma. Although asymmetric accessibility of Ig alleles should theoretically limit its occurrence, interallelic CSR was shown to occur at low levels during IgA switching in rabbit, where the definition of allotypes within both V and C regions helped identify interchromosomally derived Ig. Thus, we wished to evaluate precisely interallelic CSR frequency in mouse B cells, by using a system in which only one allele (of b allotype) could express a functional VDJ region, whereas only interallelic CSR could restore expression of an excluded (a allotype) allele. In our study, we show that interchromosomal recombination of V(H) and Cgamma or Calpha occurs in vivo in B cells at a frequency that makes a significant contribution to physiological class switching: trans-association of V(H) and C(H) genes accounted for 7% of all alpha mRNA, and this frequency was about twice higher for the gamma3 transcripts, despite the much shorter distance between the J(H) region and the Cgamma3 gene, thus confirming that this phenomenon corresponded to site-specific switching and not to random recombination between long homologous loci.