*2.8. Assays for Ligands of Apoptosis*

The ligands of TNF-dependent apoptosis were quantitatively determined in serum, circulating leukocytes, cervical fluid, and cervical wall cells. The protein levels of sFAS, TRAIL, TNF-alpha, and IL-2 were measured by the ELISA method, using corresponding kits and following the manufacturer's instructions (BCM Diagnostics, Woodland, CA, USA).

Gene expression for sFAS and TRAIL in cervical biopsies was determined by quantitative real-time PCR, using TaqMan technology in accordance with the protocols of the European Anti-Cancer Program (EAC 2003). In brief, cervical tissue was soaked in an RNA-stabilising solution (RNA-later, Ambion, Austin, TX, USA) and stored at −20 ◦C for a period of not more than 3 months. The samples were homogenised on ice, and total RNA was isolated using an iPrepPureLink TM Total RNA kit (Invitrogen, Waltham, MA, USA). The primers for sFAS were: fwd: AAGCGGTTTACGAGTGACT; rev: TG-GTTCCAGGTATCTGCTTC [Itoh]. Primers for TRAIL were: fwd: TGAAATCGAAAGTAT-GTTTGGGAATAGATG; rev: TGACGAAGAGAGTATGA ACAGCCCCTGCTG [61,62].

#### *2.9. Statistics*

Statistical analysis was performed using the STATISTICA 6.0 program from StatSoft Inc. The reported values were treated as continuous. The normality of the data was checked using the Shapiro–Wilk test. Since the distribution of the data was significantly different from normal, non-parametric statistics were used. The results were expressed as the mean ± SD. In some cases, values were presented as median, lower and upper quartiles, and minimum and maximum. The Mann–Whitney U test was employed for comparison between independent groups of data. To evaluate the difference between connected data, the two-tailed Student's *t*-test was applied, and *p* values < 0.05 were considered to be significant.

Correlations between different oxidative markers, as well as between oxidative markers and ligands of TNF-alpha-induced apoptosis, were evaluated by the Pearson linear correlation coefficient. A *p* value < 0.05 was considered to be statistically significant. If necessary (comparison among three groups or more), *p*-values were adjusted for multiple comparisons, using the Bonferroni adjustment.

#### **3. Results**

#### *3.1. Local Cervical Oxidative and Nitrosative Markers in HPV-Infected Female Patients at Different Stages of Cervical Carcinogenesis*

The measurement of selected markers of oxidative and nitrosative stress in the cervical biopsies (Figure 1) revealed a significant increase in MPO activity in the CIN III group as compared to the CIN I-II HPV16/18 group, and to the low oncogenic HPV10/13 non-

dysplastic group. The levels of nitrotyrosine were significantly reduced in the CIN III group as compared to the CIN I-II and the HPV10 and HPV13 groups.

**Figure 1.** Markers of oxidative stress and antioxidant defence in cervical tissue of females infected with different types of human papillomavirus and at different stages of the disease. Sample size of groups: HPV10/13, n = 42; HPV16/18 CIN I and CIN II, n = 75; HPV16/18 CIN III, n = 45. \* *p* < 0.05 versus HPV10/13 group; ## *p* < 0.05 versus HPV16/18 CIN I-II group; \*\* *p* < 0.01 versus HPV10/13 and HPV16/18 CIN I-II groups.

In the CIN I-II group, a strongly significant (*p* < 0.01) negative correlation between nitrotyrosine and MPO activity levels was found, as well as a positive significant (*p* < 0.05) correlation between nitrate/nitrite levels and nitrotyrosine, as shown in Figure 2.

**Figure 2.** Correlations between redox parameters in cervical tissue of HPV-infected females with CIN I and CIN II (n = 75). (**A**) Correlation between MPO activity and nitrotyrosine content; (**B**) correlation between nitrotyrosine and nitrate/nitrite levels.

#### *3.2. Cervical and Circulating Ligands of TNF-Alpha Apoptosis in HPV-Infected Female Patients at Different Stages of Cervical Carcinogenesis*

The data on TNF-alpha-induced apoptosis ligands in the serum and the cervical fluid are shown in Figure 3. The TRAIL levels were found significantly (*p* < 0.01) reduced in the cervical fluid of the HPV16 and HPV18 groups, both with CINI-II dysplasia and with CIN III cancerous lesions. Non-significant changes could be observed among the study groups for the TNF-alpha and sFAS ligands. The analysis of the serum levels of the circulating ligands of TNF-alpha-induced apoptosis revealed significant differences between the CIN I-II dysplastic group and both the healthy donors and the low oncogenic HPV-infected groups for sFAS, IL-2, and TRAIL (*p* < 0.05), and even more marked (*p* < 0.01) in the case of TNF-alpha. The difference versus healthy donors was highly significant (*p* < 0.01) for all ligands in the CIN III group.

At the same time, the analysis of the mRNA expression of sFAS and TRAIL in cervical tissue (biopsies) showed a significant (*p* < 0.01) decrease in the expression of sFAS in the groups infected with high oncogenic HPV strains, both without dysplasia and with CIN I-II, as well as a marked (*p* < 0.01) reduction in TRAIL mRNA expression in the CIN I-II pre-cancerous group (Figure 4).

**Figure 3.** Serum (**a**) and cervical (**b**) ligands of TNF-alpha-induced apoptosis in females at different stages of HPV-associated carcinogenesis. Sample size of groups: HPV 10/13, n = 42; HPV 16/18 and healthy, n = 152; HPV 16/18 CIN I and CIN II, n = 75; healthy donors, n = 15. \* *p* < 0.05 vs. healthy donors and low oncogenic HPV infection; \*\* *p* < 0.01 vs. healthy donors and low oncogenic HPV infection.

**Figure 4.** *Cont*.

**Figure 4.** sFAS and TRAIL mRNA expression (arbitrary units) in cervical biopsies of females with HPV infection and of healthy donors. Sample size of groups: HPV 10/13, n = 42; HPV 16/18 and healthy, n = 152; HPV 16/18 CIN I and CIN II, n = 75; healthy donors, n = 15. \* *p* < 0.05 \*\* *p* < 0.01 vs. healthy donors, and low and highly oncogenic HPV infection.
