Qiang Pan

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies.

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.

Artiodactyl IgD: The Missing Link

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig δ genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cδ gene appears to have been duplicated from the Cμ gene >300 million yr ago. The ruminant μCH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, ∼20 million yr ago, inserted upstream of the δ gene hinge regions, and later modified by gene conversion. A short Sδ (switch δ) sequence resulting from the second duplication, is located immediately upstream of the bovine Cδ gene and directs regular μ-δ class switch recombination in the cow. The presence of Cδ genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

Alternative end joining during switch recombination in patients with Ataxia‐Telangiectasia

Ataxia‐Telangiectasia (A‐T) and Nijmegen breakage syndrome (NBS) are recessive genetic diseases with similar cellular phenotypes that are caused by mutations in the recently described ATM (encoding ATM) and NBS1 (encoding p95) genes, respectively. Both disorders are accompanied by immunodeficiency in a majority of patients, but the mechanism involved has as yet not beenestablished. We demonstrate that in cells from A‐T patients, the switch (S) recombination junctions are aberrant and characterized by a strong dependence on short sequence homologies and devoid of normally occurring mutations around the breakpoint. A low number of S fragments were generated in cells from NBS patients and showed only limited dependence on sequence identity and mutation frequencies were similar to those observed in normal controls. We propose that ATM and p95 are both involved in the final step(s) in class switch recombination with related, but disparate, functional roles. Thus, the general pathway involved in DNA repair also has a major influence on the immunoglobulin isotype switching process.

Regulation of Germline Promoters by the Two Human Ig Heavy Chain 3′ α Enhancers

The human IgH 3′ enhancers, located downstream of each of the two Cα genes, modulate germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The regulation of GL α1 and α2 promoters by different human 3′ enhancer fragments was investigated in cell lines representing various developmental stages. Both α1HS1,2 and α2HS1,2 fragments show equally strong enhancer activity on the GL α1 and α2 promoters in both orientations when transiently transfected into a number of mature B cell line (DG75, CL-01, and HS Sultan). However, there is no activity in a human pre-B cell line (NALM-6) nor a human T cell line (Jurkat). HS3 shows no enhancer activity by itself in any of the cell lines, whereas a modest effect is noted using HS4 in the three mature B cell lines. However, the combination of the α2HS3-HS1,2-HS4 fragments, which together form a potential locus control region, displays a markedly stronger enhancer activity than the individual fragments with a differential effect on the α1 and α2 promoters as compared with the γ3 promoter. Our results suggest that the human GL α promoter may be regulated by two independent pathways. One pathway is induced by TGF-β1 which directs IgA isotype switch through activation of the GL α promoter and no TGF-β1-responsive elements are present in the different 3′ enhancer fragments. The other route is through the human 3′ enhancer regions that cis-up-regulate the GL α promoter activity in mature B cells.

Duplications and deletions of the human IGHC locus: evolutionary implications

Abstract A limited number of deletions and duplications within the human immunoglobulin heavy chain constant locus (IGHC) has previously been reported. We studied the IGHC locus in about 500 individuals representing three major races of human, Negroid (Gambian), Mongoloid (Japanese and Chinese), and Caucasoid (Iranian and Swedish). The haplotype frequency of duplications is highest in the Mongoloid population (22%), followed by the Caucasian (10%) and Negroid (5%) populations. The corresponding frequency of deletions are 2, 1.5, and 3.5%, respectively. New types of multiple duplications were found in this study on different genetic (H haplotype and racial) backgrounds. The most common duplication, found in all populations studied, encompasses the IGHA1-IGHE genes. The only deletion common to all racial groups is an isolated deletion of the IGHG4 gene. Our data are consistent with the hypothesis that the Caucasoid-Mongoloid group diverged from the hominoid ancestor after development of the Negroid populations, with subsequent evolution within the respective groups thereafter.

Regulation of switching and production of IgA in human B cells in donors with duplicated α1 genes

IgA is the predominant immunoglobulin class synthesized in humans and can be subdivided into two subclasses, IgA1 and IgA2, each encoded by a separate gene and differentially expressed depending on age and anatomical localization of the producing cells. Duplication of the α1 gene is frequently observed in selected populations. As this duplication may serve to enhance IgA‐mediated immunity, we determined its effect on switching and production of IgA in human B cells. We developed a nested PCR strategy, involving sequencing the switch (S) α2 region, the only human S region not sequenced to date, to assess the proportion of cells switching to IgA1 and IgA2 in vivo. Our results show that there is no difference in the serum and salivary levels of IgA1 and IgA or rate of switching to IgA1 and IgA between normal donors and individuals carrying α1 gene duplications, suggesting involvement of a regulatory step in the production of IgA.

Targeting of human switch recombination breakpoints: implications for the mechanism of μ ‐ γ isotype switching

We recently characterized the allelic variants of the human Sγ4 region which makes it possible to accurately identity and map Sμ‐Sγ4 fragments from in vivo switched B cells. Twenty‐six fragments were identified and a comparison was made with all previously published Sμ‐Sγ sequences ( n = 82). Switch recombination outside the region flanking the Sγ repeat sequence is a rare event in vivo and differences previously observed in patterns between in vitro and in vivo switched B cells appear to be artefactual and due to constraints of the methods used. Furthermore, internal deletions in the switch regions are common, but do not appear to be involved in isotype stabilization. A slight preference for switching to the B (SNIP) site was observed, suggesting a limited importance of both the B and A (SNAP) in the switching process. Mutations can be identified on either one or both sides of the switch junction, showing involvement of an error‐prone process, and the pattern of mutations/substitutions at and around the junctions shows non‐random nucleotide replacements by the enzyme(s) involved which may help in its future identification.

An allotype‐associated polymorphism in the γ3 promoter determines the germ‐line γ3 transcriptional rate but does not influence switching and subsequent IgG3 production

The human IgG3 (b) allotype is associated with a high and the (g) allotype with a low mean serum level of IgG3 which is due to a low frequency of B cell switching in the latter. In the present study, we found a polymorphism in position ‐73 (C → A), located in the 4th NF‐κB site of the germ‐line (GL) γ3 promoter, resulting in a significant decrease of both the basal and induced activity in the (g) allotype‐associated promoter. Over‐expression experiments also showed that this polymorphism reduced the synergistic activation of the promoter by Stat6 + NF‐κB p50 / p65 or Stat6 + C / EBPγ. A low level of GL γ3 transcripts was also observed in individuals carrying the (g) allotype‐associated promoter region. However, an individual homozygous for a crossover between the promoter and switch region, i. e. with a (g) allotype‐associated promoter and a (b) allotype‐associated switch region, showed a normal level of switching and IgG3 serum level. This suggests that although the (g) allotype‐associated promoter is functionally inferior to that of the (b) allotype‐associated promoter, these differences do not affect switching and final production of IgG3 and that polymorphisms in the switch region are more important in controlling this process.