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Protective and Destructive Immunity in the Periodontium: Part 2—T-cell-mediated Immunity in the Periodontium
Y.-T.A. Teng
Lab. of Molecular Microbial Immunity, Eastman Department of Dentistry, Eastman Dental Center, Box-683, 625 Elmwood Ave., and Centre for Oral Biology, Dept. of Microbiology and Immunology, School of Medicine and Dentistry, The University of Rochester Medical Center, Rochester, NY 14620, USA; andy_teng{at}urmc.rochester.edu
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Figure 2. Diagram showing T-cell expression cloning strategy to identify critical virulence antigens associated with periodontal immunity in vivo. Genomic DNA of Aa was partially digested and size-fractionated (0.5–4.0 kb) before being ligated into the pTrcHis-C vector. Ligated plasmids were transformed into E. coli Top10 strain by electroporation and then distributed onto selective plates. The size of the genomic library was estimated at  106 clones/µg DNA insert. Periodontal CD4+ T-cells were purified from Aa-HuPBL-NOD/SCID mice (Teng et al., 2000) and then expanded by rhIL-2 in vitro. Later, the CD4+ T-cells were transfected with a linearized DNA vector of an IL-2-inducible reporter construct with a lacZ sequence (NF-AT-lacZ). Hygromycin-resistant T-cells were then selected and expanded in hrIL-2. Thus, T-cell activation can be measured and visualized as lacZ expression in single cells or in bulk cultures. Controls included the sham- and lacZ-only transfected CD4+ T-cells, which did not yield any positive clones from screening. To screen bacterial antigens recognized by CD4+ T-cells, we plated E. coli clones from the Aa genomic library and added IPTG in vitro to induce protein expression. In parallel, autologous HuPBL-derived monocytes/macrophages were prepared and cultured overnight with rhIFN- to up-regulate HLA. CD4+ T-cells from Aa-HuPBL-NOD/SCID mice, after being transfected with NF-AT-lacZ, described above, were then added to the co-cultures for overnight incubation. The lacZ(+) cells were visualized by being stained with buffered X-gal. The positive wells giving blue signals were sequentially subjected to further screening for confirmation by the use of fewer clones (i.e., 5–10) per well, until a single positive clone was identified (for details, see Teng and Hu, 2003).
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Journal of Dental Research, Vol. 85, No. 3,
209-219 (2006)
DOI: 10.1177/154405910608500302
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and TNF-
vs. IL-10 and TGF-β cytokines. There may exist cross-talk or feedback regulation between the interacting cytokines in the micro-environment that modulate or influence the resulting RANKL-mediated periodontal osteoclastogenesis and tissue inflammation or anti-inflammatory processes associated with tissue repair (i.e., via fibroblasts). Note that the overall contribution of periodontal/mucosal resident cells or mesenchymal tissues to RANKL/OPG-mediated osteoclastogenesis is not described here, due to the lack of in vivo data.