Criterios para comparar modelos de segmentación

A total of 19 tissue samples (NG = 6 and GH = 1 3 ) were prepared for histological

examination. Prior to immunohistochemical analysis being carried out, one section (5 pm thick) from each biopsy sample was stained with H and E to assess the characteristic histological features of the NG and GH tissues and also to determine the

The sections were then immimostained for KGF antigen using a primary goat polyclonal antibody against human KGF (Table 2.1), followed by the application of a biotin- conjugated swine anti-goat antibody. Immunoreactivity was detected using ExtrAvidin peroxidase and visualised with DAB substrate (section 2.2.2). The sections were then mounted and viewed under the light microscope, as described in section 2.2.1.

The H and E staining of the NG tissues showed a relatively thin, keratinised stratified squamous epithelium associated with short rete pegs extending towards the CT (Figure 3.1 A). The CT formed the major portion of the NG tissue specimens. In marked contrast to the NG tissues, the epithelium in the GH tissues was thickened with elongated rete pegs and formed the major part of the specimen (Figure 3.1 B). Nuclei were seen in the cells of the comifled layer in 7 (54%) of the 13 GH samples. The CT was more cellular than NG tissues.

Immunohistochemical staining following incubation with antibody against KGF showed no brown-coloured enzyme reaction product in the NG tissue sections, indicating that this antigen was either not expressed or expressed at only very low levels in this tissue (Figure 3.2). Control sections were clearly negative, thus there was no difference between sections of NG which were treated with KGF-specific antibody and those, which received no antibody. In marked contrast, a very prominent brown staining was observed in the GH tissue, throughout the CT and in some regions of the epithelium (Figure 3.3). The staining in the CT was diffuse with no clear association with any particular cell type. However, the staining appeared to be more intense towards the interface between the CT and epithelium i.e. proximal to the basement membrane.

B

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Comified layer Granular layer Spinous cell layer Basal layer

Basement membrane Rete pegs

:a^ _{Granular layer}Comified layer

Spinous cell layer

Basement membrane

Basal layer Rete pegs

Figure 3.1. H and E stained sections of normal and hyperplastic gingiva. A. NG

tissue showing a relatively thin, keratinised epithelium with shallow rete pegs. B. CsA- induced GH tissue. Note the thickened epithelium with elongated rete pegs {original magnification x 300).

B

mmmm

Rw#mm

mamsm

Figure 3.2. Immunohistochemical analysis of KGF in normal gingival tissues. A;

Control NG section, received no anti-KGF antibody. B. NG section incubated with KGF-specific antibody. Note the absence of brown-coloured enzyme reaction product, indicating that KGF is not expressed in the NG tissue (original magnification x 350).

m -

' - # n ?

% , •

Com ified layer Granular layer Spinous cell layer Basal layer

Basement membrane

Rete pegs

Figure 3.2. Immunohistochemical analysis of KGF in normal gingival tissues.

C. High power view of NG section shown in B. Note no brown-coloured enzyme reaction product, indicating KGF is not expressed in NG tissue (original magnification

In addition to the CT, KGF was detected in the basal, spinous, granular and comified layers of the GE. However, the relative level of KGF appeared to be greatest in the comified layer, gradually decreasing towards the basal layer. In marked contrast to the CT, the staining in the epithelial cells was frequently intracellular as well as surface- associated (Figure 3.3 C and D). Nevertheless no detectable difference was noted in respect of staining intensity and expression pattem of KGF in GH samples obtained from patients on the different medications.

G E ^

Car y! /

B

Figure 3.3. Immunohistochemical analysis of KGF expression in hyperplastic

gingival tissues. A. Control GH section, received no primary antibody. B GH section

incubated with KGF-specific antibody. The brown colour indicates the presence of KGF. Note the prominent staining in the CT especially adjacent to the basement membrane (BM). Arrows in epithelium (GE) indicate the presence of KGF in the comified layer

(original magnification x 350).

D

1

Comified layer Granular layer

Spinous cell layer

Basal layer

Basement membrane

Spinous cell layer Basement membrane

Basal layer

Figure 3.3. Immunohistochemical analysis of KGF expression in hyperplastic

gingival tissues. High power view of the section shown in B. Note intracellular staining

in the cells of the epithelial layers (C) and diffuse staining in the CT with greater intensity subjacent to the basement membrane (D) (original magnification x 700).

3.1.2 M easurement of KGF by ELISA

In the previous section, the immunohistochemical analysis revealed the up-regulation of KGF in GH compared with NG tissues. To verify the immunohistochemical findings, a quantitative analysis of KGF was carried out on the tissue extracts using ELISA. The total amount of protein was measured in tissue lysates of 7 NG and 17 GH biopsy samples using the Bradford reagent with BSA as a standard, as described in 2.2.3.1. A representative standard curve of BSA generated with concentrations between 0.2 and 2.0 mg/ml is shown in Figure 3.4. The total protein in each sample was calculated from the equation obtained after plotting absorbance values against the known BSA concentration (section 2.2.3.1). y = 0.146X + 0.004 = 0.999 0.3 0.2- ■e

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Figure 3.4. A standard curve of the Bradford assay. The Bradford assay was carried out using BSA as a standard, A5 7 0 nm was measured and subsequently absorbance

values plotted against the known concentrations of BSA. The curve was approximated as linear, the equation obtained is shown on top of the graph, where y = absorbance,

Subsequently, KGF was measured in the tissue lysates by ELISA using a pair of KGF- specific monoclonal antibodies and rKGF as a standard between 0.3 and 5.0 ng/ml for each individual experiment, as described in section 2.2.S.2. Figure 3.5 shows a representative standard curve o f rKGF. The concentration of KGF in each sample was calculated using the equation obtained by plotting absorbance values against the known concentrations of rKGF. The amount of KGF was calculated per mg total protein in each sample. y = 0.217% + 0.005 = 1.000 1.25 1.00 - 0> _{0 . 7 5 -}

I

Figure 3.5. A standard curve generated with rKGF. ELISA assay was carried out using rKGF as a standard, A4 5 0 measured and plotted against the known concentrations

of KGF. The curve was approximated as linear, the equation obtained is indicated on top of the graph, where y = absorbance, x = concentration of KGF, and r^ = linear relationship between absorbance and rKGF.

The distribution of KGF obtained by ELISA in each of the NG and GH tissue samples is shown in Figure 3.6. The average amount of KGF in NG tissue samples was 7.0 ± 0.9 (range 1.0 to 12.0) ng/mg total protein. In marked contrast to the NG tissue samples, the amount of KGF in the GH samples was much higher, with an average amount of 68.0 ± 5.03 (range 6.0 to 116.0) ng/mg total protein. Thus the level of KGF in GH tissue samples was approximately 10-fold greater than in the NG tissue samples.

150 c S

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Figure 3.6. Distribution of KGF (ng/mg total protein) in normal and hyperplstic

gingival tissues. Tissue lysates were prepared from NG and GH tissues and KGF

measured by ELISA. Horizontal lines and bold numbers in brackets indicate the average amount of KGF. Significance levels compared with NG: *** P < 0.001 (very highly significant).

A wide variation in the amount o f KGF (range 6.0 to 116.0 ng/mg total protein) was noted among the GH tissue samples. KGF in 5 of the 17 GH tissue samples was less than 50 ng/mg total protein, while it was between 50 to 100 ng/mg total protein in 8 o f the 17

samples and the remaining 4 samples contained more than 100 ng/mg total protein. It is notable that 4 of the 5 samples containing less than 50 ng/mg total protein were obtained from patients receiving combined therapy with CsA and NIF, while the fifth one was obtained from a patient receiving CsA only.

Since the data were not normally distributed, they were analysed non-parametrically, using the Mann-Whitney U test. The difference in the amount of KGF in GH and NG tissue samples was found to be very highly significant (p < 0.0 0 1).