ABSTRACT
The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.
Subject(s)
Mouth Mucosa/radiation effects , Tissue Engineering , Basement Membrane/radiation effects , Cell Adhesion/radiation effects , Cell Culture Techniques , Cell Differentiation/radiation effects , Cell Proliferation/radiation effects , Collagen Type IV/analysis , Collagen Type IV/radiation effects , Connective Tissue/radiation effects , DNA Damage/radiation effects , DNA Repair/radiation effects , Desmoglein 3/analysis , Desmoglein 3/radiation effects , Epithelium/radiation effects , Female , Fibroblasts/radiation effects , Gamma Rays , Humans , Integrin alpha6/analysis , Integrin alpha6/radiation effects , Integrin beta4/analysis , Integrin beta4/radiation effects , Interleukin-1beta/analysis , Interleukin-1beta/radiation effects , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/radiation effects , Keratin-19/analysis , Keratin-19/radiation effects , Keratinocytes/radiation effects , Male , Middle Aged , Mouth Mucosa/cytology , Rad51 Recombinase/analysis , Rad51 Recombinase/radiation effects , Radiation Dosage , Transforming Growth Factor beta1/analysis , Transforming Growth Factor beta1/radiation effects , Tumor Suppressor p53-Binding Protein 1 , Vimentin/analysis , Vimentin/radiation effectsABSTRACT
The aim of the present study was to elucidate the impact of liver transplantation (LTX) on myeloid dendritic cell (MDC) homeostasis. We observed a threefold reduction of circulating CD1c(+) MDC immediately after LTX (n = 16; P < 0.01), and normalization between 3 and 12 months after LTX. This decline was not due to recruitment of MDC into the liver graft, as numbers of MDC in post-LTX liver graft biopsies were not increased compared to pre-LTX biopsies (n = 7). Moreover, no change in chemokine receptor expression on circulating MDC was observed, suggesting that their homing properties were not altered. Normalization of circulating MDC was associated with withdrawal of corticosteroid therapy, and not with changes in calcineurin inhibitor intake, indicating that corticosteroids are responsible for the observed changes in numbers of circulating MDC. During high-dose corticosteroid treatment early after LTX, circulating MDC showed a lowered maturation status with decreased expression of human leucocyte antigen D-related (HLA-DR) and CD86 compared to pre-LTX values (P < 0.01). However, when MDC from blood of LTX recipients were matured ex vivo, they up-regulated HLA-DR and co-stimulatory molecules to a comparable extent as MDC from healthy individuals. In addition, ex vivo matured MDC from both groups had equal allogeneic T cell stimulatory capacity. In conclusion, during the first months after LTX numbers and maturational status of circulating MDC are impaired significantly, probably due to a suppressive effect of corticosteroids on MDC. However, corticosteroid therapy does not imprint MDC with an intrinsic resistance to maturation stimuli.
Subject(s)
Dendritic Cells/pathology , Immunosuppressive Agents/pharmacology , Liver Transplantation/immunology , Myeloid Cells/pathology , Adolescent , Adult , Aged , Biopsy , Cell Count , Cell Differentiation/drug effects , Cell Survival/drug effects , Dendritic Cells/drug effects , Female , Homeostasis/drug effects , Humans , Liver Transplantation/pathology , Macrophages/drug effects , Macrophages/pathology , Male , Middle Aged , Myeloid Cells/drug effects , Postoperative Period , Receptors, Chemokine/bloodABSTRACT
New techniques for reconstructing large defects of the floor of the mouth include the use of cultured mucosal substitutes. The purpose of this study was to compare dispase and thermolysin for keratinocyte isolation. Keratinocyte yield per surface area of rabbit buccal mucosa was assessed by histology, cytokeratin 13 (CK13) staining, seeding efficiency analysis and cell diameter quantification. Surface areas of cultured mucosa were calculated. Histology showed that treatment by thermolysin resulted in incomplete separation of epidermis from dermis. Also, the absolute number of keratinocytes/cm(2) isolated mucosa, cell yield, cell size and seeding efficiencies was higher in the dispase group. A 3.45-fold larger graft could be reconstituted using dispase. The use of dispase, rather than thermolysin, to isolate cells from buccal mucosa is concluded to be favourable.
Subject(s)
Cell Separation/methods , Endopeptidases/pharmacology , Keratinocytes/drug effects , Mouth Mucosa/cytology , Thermolysin/pharmacology , Animals , Cell Count , Colony-Forming Units Assay , Dermis/cytology , Dermis/drug effects , Epidermal Cells , Epidermis/drug effects , Keratin-13/metabolism , Keratinocytes/cytology , Keratinocytes/metabolism , Rabbits , Skin, ArtificialABSTRACT
The efficacy of anti-viral intravenous immunogobulins (anti-HBs Ig and anti-CMV Ig) in preventing acute rejection after liver transplantation was assessed in a retrospective analysis, and correlated to their effects on immune cells in vitro. HBs Ag-positive liver graft recipients (n = 40) treated prophylactically with anti-HBs Ig had a significantly lower incidence of acute rejection compared with recipients without viral hepatitis (n = 147) (12% vs. 34%; p = 0.012), while the incidence of rejection in HCV-positive recipients (n = 29) was similar to that in the control group. Treatment with anti-CMV Ig (n = 18) did not protect against rejection. In vitro, anti-HBs Ig suppressed functional maturation of and cytokine production by human blood-derived dendritic cells (DC) at concentrations similar to the serum concentrations reached during anti-HBs Ig treatment of liver graft recipients. In addition, anti-HBs Ig inhibited allo-antigen- and lectin-stimulated proliferation of peripheral T cells. Anti-CMV Ig suppressed functional DC-maturation and alloantigen-stimulated T-cell proliferation, but not lectin-driven T-cell proliferation. In conclusion, anti-HBs Ig protects against acute rejection after liver transplantation, probably by functional inhibition of the two principal immune cells involved in allograft rejection, DC and T cells.
