Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert single-strand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either S mu and MSH2 or UNG and MSH2 cannot undergo CSR.     

The regulation of immunoglobulin E class-switch recombination

Immunoglobulin E (IgE) isotype antibodies are associated with atopic disease, namely allergic rhinitis, asthma and atopic dermatitis, but are also involved in host immune defence mechanisms against parasitic infection. The commitment of a B cell to isotype class switch to an IgE-producing cell is a tightly regulated process, and our understanding of the regulation of IgE-antibody production is central to the prevention and treatment of atopic disease. Both those that are presently in use and potential future therapies to prevent IgE-mediated disease take advantage of our existing knowledge of the specific mechanisms that are required for IgE class switching.     

T-independent activation-induced cytidine deaminase expression, class-switch recombination, and antibody production by immature/transitional 1 B cells

Inflammation elicits a splenic lymphopoiesis of unknown physiologic significance but one that juxtaposes developing B cells and exogenous Ag. We show that immature and transitional 1 (immature/T1) B cells constitutively express activation-induced cytidine deaminase and B lymphocyte-induced maturation protein 1 in amounts that support accelerated plasmacytic differentiation and limited class-switch recombination. In vivo, activation of immature/T1 B cells by TLR ligands or bacterial vaccine rapidly induces T1 cells to divide, proliferate, and secrete IgM, IgG, or IgA Ab; in vitro, proliferation and differentiation are substantially enhanced by B cell-activating factor. We propose that inflammation-induced extramedullary lymphopoiesis represents a specialized mechanism for innate Ab responses to microbial pathogens.     

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5′ to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged γ2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged γ2b gene contained the 5′ flanking region of the γ3 gene (S_γ3 region) which are linked to the 5′ flanking region of the γ2b gene (S_γ2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line γ2b genes, which include the S_γ2b and S_γ3 regions. Both _γ2b and S_γ3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S_γ1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S_γ1, S_γ2b and S_γ3 repeating units share significant homology with each other. The Sμ region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S_γ regions. These results suggest that the recombination responsible for class switch from μ to γ or from a γ to another γ, may be facilitated directly or indirectly by homology of repeating sequences in S regions.     

Enhancement of suppressor T cell activity by injection of anti-IFN-gamma monoclonal antibody

In the present study, the contribution of IFN-gamma to the generation of helper activity in mice was investigated by use of anti-mouse IFN-gamma rat mAB (AN 18.17.24). This mAb was alum precipitated and injected i.p. before or after carrier priming. Results show that spleen cell helper activity is markedly inhibited by anti-IFN-gamma mAb injection. This inhibition is time and dose dependent, and counteracted by IFN-gamma administration. Thus, the anti-IFN-gamma mAb appears to inhibit helper cell activity by neutralization of the IFN-gamma required for the antibody response. Moreover, AN 18.17.24 mAb injection results in increased activation of Lyt-2+ T cells which markedly suppress Th activity. These findings altogether indicate that besides the activation of macrophages and Th, IFN-gamma seems to exert a negative interference in suppressor T lymphocyte circuits and, as a consequence, to inhibit immunosuppression.     

Control of Cre recombination by regulatory elements from Xer recombination systems

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.     

Enhancement of mRNA nuclear transport by promoter elements

This report describes studies on the kinetics of herpes thymidine kinase (TK) mRNA transport in X. laevis oocytes as studied by microinjection and microdissection. We show that TK mRNA nuclear transport in this cell can be dramatically altered by introduction of specific DNA sequences into the nucleus. Introduction of DNA sequences encompassing the promoter results in enhancement, in trans, of a transport process that can deliver previously synthesized RNA to the cytoplasm. The report suggests that the promoter of a eukaryotic gene can functionally interact with the mechanism involved in mRNA nuclear transport.     

Enhancement of antibody production by lysophosphatidylcholine and alkylglycerol

Inflammation products of normal and cancerous tissues, lysophosphatidylcholine and dodecylglycerol, were tested for their adjuvant effect on the antibody response. Mice treated with these agents and immunized with sheep erythrocytes simultaneously or at 3 days posttreatment developed a greatly enhanced antibody production as demonstrated by the Jerne plaque assay. Mice immunized at 3 days postadministration of agents did not significantly produce enhanced antibody-secreting cells as compared with those of mice simultaneously immunized. Since the mechanism of macrophage activation by lysophospholipids requires contribution of B and T cells, BALB/c-nu/nu mice treated with these agents and subsequently immunized with sheep erythrocytes did not produce antibodies. However, conditioned medium of in vitro-treated BALB/c-nu/nu B cells efficiently transmitted a signal to untreated BALB/c +/+ T cells for enhanced macrophage ingestion activity. This observation suggests that lysophospholipid-activated macrophages and T cells efficiently transmitted antigenic signal to the antibody-producing B cell population. Therefore, we conclude that these lipid metabolites have dual beneficial effects for the host by enhancing phagocytosis and antibody production. Thus, lysophosphatidylcholine and dodecylglycerol have potential practical application as adjuvants that could be administered separately or in combination with antigens.     

Enhancement of monoclonal antibody production by lysine-containing peptides

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity. No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found. All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase.The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly. The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.     

Human genomic deletions mediated by recombination between Alu elements

Recombination between Alu elements results in genomic deletions associated with many human genetic disorders. Here, we compare the reference human and chimpanzee genomes to determine the magnitude of this recombination process in the human lineage since the human-chimpanzee divergence approximately 6 million years ago. Combining computational data mining and wet-bench experimental verification, we identified 492 human-specific deletions (for a total of approximately 400 kb) attributable to this process, a significant component of the insertion/deletion spectrum of the human genome. The majority of the deletions (295 of 492) coincide with known or predicted genes (including 3 that deleted functional exons, as compared with orthologous chimpanzee genes), which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees. Overall, we found that Alu recombination-mediated genomic deletion has had a much higher impact than was inferred from previously identified isolated events and that it continues to contribute to the dynamic nature of the human genome.     

Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease.

The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.     

Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.     

Enhancement of murine in vitro antibody formation by hyperthermia

The influence of hyperthermia on primary in vitro antibody formation by C57BL/6 splenocytes to a T-dependent antigen, sheep red blood cells (SRBC), and a T-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), was evaluated. Following heat treatment (39 or 40 °C), spleen cells demonstrated two- to fivefold increases in antibody production to SRBC. This enhancement of humoral immunity is critically dependent on the timing of hyperthermia administration relative to antigenic exposure. Examination of the kinetics for the SRBC response revealed that heat significantly lengthens the time period of antibody production. Although a number of parameters were examined, antibody production to TNP-LPS from hyperthermically treated cultures were comparable to control (37 °C treated) cultures. Finally, we have determined that the heat-induced increases in antibody formation to SRBC are mediated through T lymphocytes.     

Enhancement of hormonal activity with a monoclonal antibody specific to porcine growth hormone

Abstract

The potentiation of the biological effects of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. A monoclonal antibody (mAb), designated PS‐7.6, was raised against pGH and repeatedly shown to enhance the responsiveness of hypophysectomized (hypox) rats to pGH. As a result, animals receiving a combination treatment of pGH and mAb PS‐7.6 together gained significantly more weight than those receiving the same doses of pGH alone. The enhancing action of the mAb was a rapid process and its effective doses ranged from 0.1 to 2 mg/injection. The ability of the antibody to augment the hormonal activity persisted beyond the 5‐day treatment period and the differences in net weight gain between treated and control animals remained significant for 28 days. Results from treatment frequency studies further suggested that pGH when complexed with mAb PS‐7.6 required fewer injections and produced a greater efficacy than being administered alone. Therefore, present findings suggest that mAb PS‐7.6 may prove useful for not only improving the efficiency of pGH, but also developing a novel formulation for sustained pGH release.     

Enhancement of endotoxin activity by pulmonary surfactant

The modification of the activity of lipopolysaccharide (LPS) in the pulmonary lavage fluid (PLF) of guinea pigs was assessed by a chromogenic Limulus assay. The activity of the lPS bound to Escherichia coli or of LPS extracted from the bacteria was observed to increase significantly in PLF. This increase in activity was amplified after heating at 75°C for 5 min. Pulmonary surfactant (PS) obtained from PLF showed a similar increase in the activity of LPS, indicating that PS is most probably the key agent in this modification.