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Cyclosporin A Specifically Affects Nuclear PLCß₁ in Immunodepressed Heart Transplant Patients with Gingival Overgrowth

¹ Department of SAU&FAL, University of Bologna,
² Department of Dental Science, University of Bologna,
³ ITOI-CNR, Unit of Bologna, c/o IOR, Bologna, Italy; and
⁴ UCO of Dental Sciences, DMUN, University of Trieste, Via Stuparich, 1, 34129, Trieste, Italy;

View larger version (32K): [in a new window]   Figure 1. Expression of the PLCß1 isoform during differentiation of primary human gingival fibroblasts. (A) Cells underwent lysis as described in MATERIALS & METHODS, and were subjected to SDS-PAGE, blotted, and immunoprobed with anti-PLCß1 antibody. A 20-µg quantity of protein was loaded for each lane. The results shown are representative of at least 3 independent determinations. (B) PLCß1 expression in purified nuclei from primary cultures of different fibroblasts populations. A 20-µg quantity of protein was loaded for each lane. (C) ß tubulin levels were used for normalization. The immunochemical analysis by Western blot on total homogenate and nuclear fraction does not give information on the precise localization of the enzyme, while, at the TEM level, the intranuclear and the intracytoplasmic distributions of the PLCß1 can be determined with high accuracy, and quantitative variations of the label distribution can be further investigated.

View larger version (62K): [in a new window]   Figure 2. Fibroblasts from patients undergoing CsA-immunosuppressive therapy without any clinical signs of gingival overgrowth (noGO). (a,b) Immunocytochemistry of PLCß1 at the TEM level of FCS-deprived (2a; UN = untreated cells; HC = heterochromatin; IC = interchromatin; N = nucleolus; bar = 0.5 µm) and CsA-treated cells (2b; +CsA; bar = 0.5 µm). The analysis was performed on thin sections of cell monolayers, partially dehydrated, and embedded in LR White resin at low temperature, a procedure that ensures a reliable antigen detection. Due to the absence of osmium fixation and to the characteristics of the hydrophilic resin, the membranes are not electrondense. (c) Quantitative evaluation of the nuclear labeling density was performed with the Mann-Whitney U test (UN; n = 10) (+CsA; n = 10). Values are the means of examined samples ± SD. No significant difference was observed (power = 0.74).

View larger version (60K): [in a new window]   Figure 3. Fibroblasts from healthy patients (HP) who never received any treatment with CsA. TEM micrographs of FCS-deprived fibroblasts (3a; UN = untreated cells; HC = heterochromatin; IC = interchromatin; N = nucleolus; bar = 0.5 µm) and CsA-treated ones (3b; +CsA; bar = 0.5 µm), both revealing scarce labeling related to interchromatin domains. (c) Quantitative evaluation of the nuclear labeling density was performed with the Mann-Whitney U test (UN; n = 10) (+CsA; n = 10). Values are the means of examined cases ± SD. No significant difference was observed (power = 0.71).

View larger version (118K): [in a new window]   Figure 4. Fibroblasts from patients undergoing CsA immunosuppresive therapy and showing pGO or fGO. TEM micrographs of fibroblasts from not-clinically-enlarged gingival sites (pGO1; a,b; UN = untreated cells, +CsA = after CsA administration; HC = heterochromatin; IC = interchromatin; bar = 0.5 µm), from hypertrophic areas (pGO2; d,e; N = nucleolus; bar = 0.5 µm) of the same patient and from gingival biopsies of a patient exhibiting a full-mouth GO (fGO; g,h; bar = 0.5 µm). Untreated (UN; a,d,g) fibroblasts reveal scarce labeling, while CsA-treated cells (+CsA; b,e,h) show an intense labeling of nuclear domains, particularly localized in the interchromatin areas. (c,f,i) Quantitative evaluation by the Mann-Whitney U test of the nuclear label of untreated fibroblasts (UN; n = 10) and after CsA administration to the culture medium (+CsA; n = 10); values are the means of examined samples ± SD. A significant increase of PLCß1 nuclear labeling (P < 0.05; Mann-Whitney U test) was observed in treated fibroblasts with respect to untreated cells in all three cases examined.