Sean P. Bradley

Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination.

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Sμ segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but find very few mutations in GL Sγ3 and Sγ1 regions in activated B cells. These data suggest that switch recombination initiates in the Sμ segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Sμ segments differ, suggesting different repair mechanisms. Mutations in recombined Sμ regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Sμ do not. Additionally, induction conditions affect mutation specificity within the GL Sμ segment. Mutations are most frequent near the S–S junctions and decrease rapidly with distance from the junction. Finally, we find that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S–S junctions, indicating that the recombining DNA ends are accessible to end‐processing enzyme activities.

Gene silencing in the endocrine pancreas mediated by short-interfering RNA

Objectives: RNA interference as mediated by short-interfering RNA (siRNA) offers a nonviral means to silence genes in tissue; however, few data exist about gene therapy using siRNA in pancreas tissue. To determine if siRNA treatment could silence an endogenous gene in pancreatic islets, we developed a murine model using the endocrine pancreas. Methods: The insulin 2 (Ins2) gene was targeted with siRNA, and quantitative RT-PCR, fluorescent microscopy, and FACS were used to measure transcript levels and siRNA cellular uptake and transfection efficiency. Isolated pancreatic islets were transfected with siRNA in vitro using a liposomal delivery method in a dose titration (50-400 nM) or pooled from BALB/c mice having received siRNA (100 μg) via hydrodynamic tail vein injection. Results: The Ins2 transcript level was significantly reduced by 55% in vitro with FACS data showing a transfection efficiency over 45% with the 400 nM concentration. In vivo delivery of siRNA to pancreatic islets revealed a 33% reduction in Ins2 mRNA levels, although siRNA was able to be detected in 19% of isolated islet cells. Conclusion: We have successfully used RNA interference to silence an endogenous tissue-specific gene (Ins2) in pancreatic islets when transfected in vitro or administered in vivo.

The Histone Methyltransferase Suv39h1 Increases Class Switch Recombination Specifically to IgA

Ab class (isotype) switching allows the humoral immune system to adaptively respond to different infectious organisms. Isotype switching occurs by intrachromosomal DNA recombination between switch (S) region sequences associated with CH region genes. Although isotype-specific transcription of unrearranged (germline) CH genes is required for switching, recent results suggest that isotype specificity is also determined by the sequences of downstream (acceptor) S regions. In the current study, we identify the histone methyltransferase Suv39h1 as a novel Sα-specific factor that specifically increases IgA switching (Sμ-Sα recombination) in a transiently transfected plasmid S substrate, and demonstrate that this effect requires the histone methyltransferase activity of Suv39h1. Additionally, B cells from Suv39h1-deficient mice have an isotype-specific reduction in IgA switching with no effect on the level of germline Iα-Cα transcripts. Taken together, our results suggest that Suv39h1 activity inhibits the activity of a sequence-specific DNA-binding protein that represses switch recombination to IgA.

Genetic expression profile during acute cellular rejection in clinical intestinal transplantation.

Small bowel transplantation is a successful treatment for irreversible intestinal failure. Acute cellular rejection (ACR) represents the major cause of graft loss. However, little is known regarding the mechanisms of ACR and no data exist on the genetic response occurring during ACR. We report a genetic expression profile determined using oligo-microarrays of the intestinal graft during an ACR episode. Mucosal biopsies were obtained from the graft at weeks 0, 1, 2, 3, 6, and 52 posttransplant. We observed a statistically significant increase in transcript levels from 51 genes between biopsy samples from before ACR compared with during ACR. Functional analysis of these genes revealed an interferon (IFN)-alpha signature associated with a type I IFN immune response from dendritic cells or association with cellular proliferation and division. These genetic data support that dendritic cell activation was ongoing during ACR and suggest that IFNalpha production as a potential immunosuppressive target for transplantation.

Does activation-induced deaminase initiate antibody diversification by DNA deamination?

Activation-induced deaminase (AID) is expressed only in germinal center B cells. There, it is required for somatic hypermutation, gene conversion and class switch recombination of antibody variable region segments, three processes that diversify antibodies during immune responses. Although AID has homology to RNA-editing enzymes, three recent reports suggest it could initiate the diversification processes by deaminating cytidine residues within the antibody genes themselves.