The population of the study
CRC patients were those who attended several Gastroenterology and Hepatology Centers in the state of Selangor, Malaysia from March 2006 to December 2007, that underwent elective surgical resection of colorectal cancer. Adenoma patients were those who were referred to do colonoscopy due to various reasons and resection of intestinal polyps was done. Twenty seven men and 23 women with primary colorectal adenocarcinoma were included in this study prior to any chemotherapy. On the other hand, seven men and seven women with colorectal adenoma who had already undergone colonoscopical resection of adenomatous polyps were involved in this study. The medical history of the involved patients in this study was evaluated; no one had history of gastrointestinal disease or ulceration which might affect the seroprevalence of S. gallolyticus or B. fragilis. On the other hand, 30 age- and sex- matched control subjects were involved. They were referred to hospitals for doing colonoscopy for various reasons in whom normal colonic mucosa was confirmed and no other gastrointestinal disease or history of gastrointestinal diseases and ulcerations were found. Colonoscopical biopsy was taken from control subjects to compare the level of IL-8 and NFKB mRNA expression with that in mucosal colonic tissues from patients with adenoma and CRC. Moreover, blood samples were taken from the control group to calculate the seropositivity cutoff value for S. gallolyticus IgG antibodies. The resultant cutoff value was applied on CRC and adenoma patients as well as on age- and sex- matched 30 apparently healthy volunteers group (HV) who agreed to do colonoscopy in case results of ELISA would show high level of S. gallolyticus IgG antibodies. HV were medically examined and their medical records and history were reviewed and no major or gastrointestinal illness was found. Written consents were officially obtained from all participants in this study. The study was carried out in the scope of Helsinki declaration of ethical principles of medical research and permission was granted from the Ethics Committee of biomedical research of University Putra Malaysia.
Sampling and processing of specimens
Samples of 3 to 5 ml of blood for serum isolation were withdrawn from control and adenoma groups at time of colonoscopy, from HV group after taking the medical history, and from CRC patients 2-3 days before surgery. Regarding the histopathology, a set of steps was pursued under the supervision of a pathologist to minimize as could as possible the fixation-related loss of mRNA. These steps were minimal prefixation time of 1 hour, the use of cold 4% paraformaldehyde, cold fixation at 4°C, and short duration of fixation, up to 5 hours [18]. It was stated that no significant loss of nucleic acids was observed within the first 3 days of fixation-paraffin embedding [19]. Therefore, paraffin-embedded sections were processed for ISH examination in a period of not more than 3 days. Moreover, the scoring system used in this study did not rely on the quantitative measurement of staining intensity. Therefore, mRNA loss, which affects mainly the intensity of staining, was believed to affect the results of this study minimally. To evaluate the stringent conditions pursued for minimizing mRNA loss, the ISH immunostaining was compared between 6 randomly selected paraffin embedded sections and cryostat sections. Despite the bit lower intensity of staining, the mean percentage of the positively stained cells was not changed. Hence it was confirmed that no loss of mRNA took place which could affect ISH results. Each histopathological paraffin block of excisional biopsies of CRC patients and punch biopsies of control subjects and adenoma patients were sectioned into 4 um thick sections. Histopathological sections were made from both tumorous and non-tumorous tissues for each CRC (resection safe margins) and adenoma patient (punch biopsy 3-4 cm away from the polyp). Hematoxylin and Eosin slides were prepared and examined by a histopathologist for confirming the histopathological diagnosis, the grade of CRC, and the type and degree of dysplasia of adenoma tissue sections.
Extraction of cell wall antigens of S. gallolyticus and LPS of B. fragilis
The reference strain, S. gallolyticus subspecies gallolyticus CIP 105428 (Insitut de Louis Pasteur, France) was used. The extraction of cell wall antigens of S. gallolyticus was conducted by using the lysozyme method. Sufficient amount of the cultured reference bacteria in Columbia agar (Oxoid, UK) with 5% horse blood was obtained by making bacterial suspension in 30 mM (pH 8) Tris buffer (Fluka, Switzerland). The suspension was centrifuged at 3500 g for 10 minutes at 4°C. Pellet was washed 3 times in 30 mM Tris buffer (pH 8) by centrifugation at 3500 g for 5 minutes at 4°C. Pellet of washed bacteria was resuspended in a solution containing 4.75 ml of 30 mM Tris buffer (pH 8), 3 mM magnesium chloride (Sigma, USA), 25% sucrose (Fluka, Switzerland) and 0.25 ml of lysozyme (0.6 mg/ml) (Sigma, USA). The lysate solution was incubated for 2 hours at 37°C and was then centrifuged at 3000 g for 5 minutes at 4°C. Then, the supernatant was collected, which became the solution of cell wall antigens of the reference strain, S. gallolyticus [20]. The concentration of cell wall antigens was measured by Biurette method.
The extraction of B. fragilis LPS was obtained by the phenol-water extraction method, followed by the phenol-chloroform-petroleum ether extraction, as described by [21].
ELISA
For S. gallolyticus, 96-wells microtiter plate (Sterilin, UK) was coated with 40 μg/ml of cell wall antigen while, for B. fragilis, the microtiter plate was coated with 10 μg/ml of LPS extract. The concentration of coating antigens for the studied bacteria was determined after a series of standardization steps. The microtiter plates were incubated in a humid chamber for 2 hours at 37°C and were then washed and stored at -20°C until further use. It is noteworthy to mention that the cell wall antigens of S. gallolyticus had already been treated with 100 μl a well of 0.01 M sodium periodate (Sigma, USA) in PBS for two hours at room temperature to destroy the common polysaccharides antigen of group D. After 3 times washing, 50 μl sera of CRC (50), adenoma (14), control subjects (30), and healthy volunteers (30) were pipetted into microtiter plates and incubated for 2 hours at room temperature. For each run, two wells were dispensed with 50 μl of diluting buffer as a negative control and two wells were dispensed with known positive sera for either S. gallolyticus or B. fragilis. After 3 rounds of washing, 50 μl/well of 1:40,000 diluted horseradish peroxidase anti-human IgG conjugates (Sigma, USA) were dispensed and incubated for 2 hours at room temperature. Then, 50 μl/well of chromogen-substrate OPD.2HCL (Abbott, USA) were pipetted into wells and incubated in dark for 15 minutes at room temperature. Optical density was read by ELISA reader (Organic Technica, Spain) at 492 nm [22].
Calculation of the cutoff value for S. gallolyticus seropositivity
The cut off value is considered as the upper limit above which all of readings are considered positive. ELISA readings of control subjects (n = 30) were used to calculate the cutoff value according to the following formula [23]:
[23] (2.462): taken from the table of student's t-test under the P = 0.01 for the 29 degrees of freedom.
The cutoff value was used to demarcate between the S. gallolyticus- seropositive and seronegative subjects in the participants of this study other than control group, namely CRC, adenoma, and HV groups
In situ hybridization assay
For each run of ISH, one negative control tissue section (diluting buffer instead of probes), one positive tissue section (already tested as strongly positive), and one endogenous positive probe control were used. Biotinylated long DNA probe for human NF-κB mRNA and human IL-8 mRNA were used (Maximbio, USA). The used procedure was according to "DNA probe Hybridization/Detection System - in situ Kit" (Maximbio, USA).
Slides were baked overnight at 70°C and deparaffinized in xylene (Merck, Germany) and descending grades of ethanol (Merck, Germany) starting from 100%. Freshly diluted 1× proteinase K (Sigma, USA) solution was applied for 15 min at 37°C. The working solutions used for probes were 10% v/v for NF-κB and 7% v/v for IL-8 mRNA. Ten μl of the working cDNA probe was added onto each slide. Slides were incubated in a humid chamber overnight at 37°C. Next day after washing slides, RNase A (Maximbio, USA) was added for 30 min to abolish any unbound RNA. Slides were then washed three times with a pre-warmed protein block for 3 minutes at 37°C. One to two drops of alkaline phosphatase-streptavidin conjugate (Abbott, USA) were added onto tissue sections for 1.5 hour at 37°C. Then, 1-2 drops of NBT/BCIP (Abbott, USA) substrate were placed on tissue sections at 37°C until color was developed. Dark blue colored precipitate was seen in positive cells. Slides were counterstained by nuclear fast red, dehydrated by graded alcohols, and mounted with a permanent-mounting DPX medium.
Staining analysis
The scoring systems for in situ hybridization staining of NF-κB and IL-8 mRNA calculate the percentage of the glandular mucosal (adeno) cells, which were stained with nuclear blue/black color, out of total cells in 5 high power fields. The scores for NF-κB mRNA staining are; negative for less than 5% staining, low for 5-25% staining, intermediate for 26-50% staining, and positive for more than 50% staining [24]. The scores for IL-8 mRNA are; score 1 for 1-10% staining, score 2 for 11-50% staining, and score 3 for 51-100% staining [25]. In addition, stromal cells rather than glandular mucosal cells were observed too. The percentage of IL-8- or NF-κB- positively stained stromal cells were calculated out of total cells in 5 high power fields. However, no specific scoring system was found for stromal cells of human colonic tissues. Therefore, the ISH staining percentage of stromal cells was used for comparisons without using a predetermined scoring system.
Statistical analysis
SPSS software version 12 for Windows (SPSS Inc., USA) and Excel XP (Microsoft, USA) were used. To validate the scoring systems used in this study, two sets of statistical analyses were used. The first set, Chi square for independence and Mann-Whitney tests were used for the scoring systems of NF-κB and IL-8 respectively. The second set, a univariate student t test was used for direct comparisons of the mean percentages of the positively stained cells. On the other hand for stromal cells, only student t test for mean percentages of the positively stained cells was used. Pearson's correlation coefficient was used to correlate the expression of IL-8 mRNA and NF-κB mRNA with S. gallolyticus IgG antibodies in both CRC and adenoma patients. P values less than 0.05 were considered significant.
