The percentage of necrotic and non-necrotic granulomas positive for specific cytokines in patients with pleural tuberculosis.

<p>The presence of IL-12, IFN-γ, TNF-α and IL-4 positive cells was assessed by <i>in situ</i> hybridisation in granulomas from pleural biopsies. Positivity was graded according to the total number of cells staining positive in a specific granuloma (<25%, grade 1; 25–75%, grade 2 and >75%, grade 3).</p

Necrosis and expression of mixed Th1/Th2 and Th1 responses in granulomas of pleural tuberculosis patients.

<p>The percentage of granulomas with necrosis and expression of Th1 and Th2 cytokines were assessed microscopically. Groups include HIV positive (PLTB HIV+, n = 6) and HIV negative (PLTB HIV−, n = 6) pleural TB patients (n = 12). The percentage of necrosis is represented by solid bars, the percentage of Th1 response by clear bars and the percentage of a mixed Th1/Th2 response by striped bars. There was a significantly higher percentage of necrotic granulomas in HIV positive compared to HIV negative TB patients (Bootstrap test <i>p</i><0.01).</p

Spearman rank R correlation for cytokines in granulomas of pleural TB patients.

<p>IL-12, IFN-γ, TNF-α and IL-4 mRNA expression was examined by <i>in situ</i> hybridisation on granulomas from pleural biopsies. Statistically significant correlations are noted with * with <i>p</i>-values in brackets.</p

Sequences of cytokine riboprobes used in <i>in situ</i> hybridisation.

<p>Sequences of cytokine riboprobes used in <i>in situ</i> hybridisation.</p

The percentage of granulomas positive for specific cytokines in HIV negative and HIV positive pleural tuberculosis patients.

<p>IL-12, IFN-γ, TNF-α and IL-4 mRNA expression was examined by <i>in situ</i> hybridisation on granulomas from pleural biopsies. Positivity was graded according to the total number of cells staining positive in a specific granuloma (<25%, grade 1; 25–75%, grade 2 and >75%, grade 3).</p

Cytokine mRNA expression in HIV positive and negative pleural tuberculous granulomas.

<p>Paraffin-embedded tissue was stained using ISH for mRNA expression. Representative sections of pleural needle biopsies from HIV positive (A–D) patients are shown. (A) Staining with the anti-sense IL-12 probe shows that over 75% of cells are positive for IL-12 mRNA expression. Magnification 400×. (B) Staining with the anti-sense IFN-γ probe shows that over 75% of cells are positive for IFN-γ mRNA expression. Magnification 400×. (C) Staining with the anti-sense TNF-α probe shows that over 75% of cells are positive for TNF-α mRNA expression. Magnification 400×. (D) Staining with the anti-sense IL-4 probe shows that over 75% of cells are positive for IL-4 mRNA expression. Magnification 400×. Representative sections of pleural needle biopsies from HIV negative (E–H) patients are shown. (E) Staining with the anti-sense IL-12 probe shows that 25%–75% of cells are positive for IL-12 mRNA expression. Magnification 100×. (F) Staining with the anti-sense IFN-γ probe shows that 25%–75% of cells are positive for IFN-γ mRNA expression. Magnification 100×. (G) Staining with the anti-sense TNF-α probe shows that 25%–75% of cells are positive for TNF-α mRNA expression. Magnification 100×. (H) Staining with the anti-sense IL-4 probe shows that less than 25% of cells are positive for IL-4 mRNA expression. Magnification 100×.</p

A granuloma map used to assess the slides from patients with granumatous diseases.

<p>This example represents the type of granuloma map used to assess the tissue biopsies of patients with pleural TB for the absence or presence of granulomas and type (necrotic or non-necrotic) of granuloma. Once the granuloma was identified, ISH was used to stain the tissue for mRNA cytokine expression. Magnification 25×.</p

Positive and negative control staining in human tissue.

<p>Paraffin-embedded tissue was stained using ISH for mRNA expression. Negative and sense probe controls were repeated each time lung tissue was stained for cytokine expression. In the first control (A) lung granulomatous tissue was not stained with probe. The absence of brown staining proves that there was no nonspecific staining. Magnification 200×. In the second control (B) anti-sense probe was applied to tissue known not to produce cytokines (sarcoma). The absence of brown staining proves that there was no nonspecific staining. Magnification 400×. In the third control (C) granulomatous lung tissue was stained with sense probe. The absence of brown staining represents specificity. Magnification 200×. In the last control (D) granulomatous lung tissue was stained with the anti-sense β-actin probe. Brown staining indicates the presence of non-degraded mRNA. β-actin expression is diffusely positive. Magnification 200×.</p