دور أشباح بكتيريا Salmonella Typhimurium في التعديل المناعي لسرطان القولون والمستقيم المستحدث في ذكور الجرذان البيض

The role of Salmonella Typhimurium ghost bacteria in the immunomodulation of induced colorectal cancer in male albino rats

Abstract

The current study aimed to investigate the immunostimulatory role of Salmonella Typhimurium ghosts (STGs) vaccine to reduce the effect of Azoxymethan (AOM)-induced colorectal cancer in male albino rats (Rattus rattus), which were used as a model to achieve the experiments of the current study, which were conducted in the laboratories of Biology Department belong to both the College of Education for Pure Sciences and the College of Science / University of Kerbala, and in the animal house of the College of Pharmacy / University of Kerbala, and in the training center of the Iraqi Biotechnology Company in Al-Qadisiyah Governorate for the period from the beginning of February 2023 to the beginning of November 2023.
The study included the use of (41) male albino rats, and included two axes. The first axis represented the initial evaluation to the locally prepared vaccine safety and its immunogenicity by studying the immune response of the vaccinated rats and comparing it with the unvaccinated rats, as well as testing the complete vaccination program. The second axis included investigating the immunomodulatory activity of the prepared vaccine on colorectal cancer induced rats before, during and after treatment with the complete vaccination program with (STGs) by measuring the expression level of some immune parameters represented by Toll like receptor- 4 (TLR-4), Programmed cell death ligand- 1 (PDL-1), and Tumor necrosis factor- alpha (TNF-α), in addition to studying some changes in histological and blood parameters.
S. Typhimurium ghosts were prepared from the activated bacterial isolate provided by the Al-Amin Center for Advanced Biotechnology and Research / Najaf, using the Sponge Like Reduced Protocol (SLRP) as a vaccine and to evaluate its efficiency in stimulating or modifying the immune response of laboratory rats (6 rats with an average weight of 150 grams were allocated for the experiment). The agglutination ability test was performed for S. Typhimurium cells and STGs using standard antisera. The results showed that agglutination appeared clearly by the presence of granular aggregation on the glass slide when using standard antibodies with the prepared S. Typhimurium cells and STGs, while the agglutination ability was absent in the negative control group. The scanning electron microscope imaging results also revealed that these ghost cells which containing micropores are characterized by having intact and high-quality outer shells.
The full vaccination program (42 days) was tested and the experiment included the use of (15) rats with an average weight of 150 grams, divided into 3 groups, each group (5) rats included (the control group, the oral vaccination group, and the subcutaneous injection group). The STGs vaccine was used with Freund’s adjuvant (1:1) at a dose of 100 μl/rat. The serum was collected from each animal before each vaccine dose and after the end of the vaccination program (14, 28, and 42 days). Then the ability of the antibodies present in the serum to interact with the live S. Typhimurium antigen was measured using the agglutination ability test. The positive agglutination result appeared on the glass slide by the presence of milky-colored granular fog that included the subcutaneous vaccination group, which gave a clear clump (within one minute) for the antibody reaction with the antigen after treatment with the STGs vaccine compared to the tests of the oral vaccination group.
When studying the effect of the prepared STGs vaccine on some histological, blood and immunological parameters for colorectal induced rats , the experiment included the use of (20) male white rats with weights ranging between (75-50) grams and ages of (5-4) weeks, they were divided into four groups with (5) rats for each group, the first group was considered the negative control group, and the second group was the positive control group that was injected with (AOM) at a rate of (15 mg/kg of animal weight) once a week for two consecutive weeks, the third group was the group of animals that were injected with a single dose subcutaneously with equal volumes (1:1) of STGs + Freund’s adjuvant at a dose of (100 μl /rat) after (20) weeks from the date of the second injection of the carcinogenic (AOM) substance, then the full vaccination program continued (14, 28 and 42 days) ,while the fourth group represented the group of animals treated with carcinogenic (AOM) substance, which they were subjected to interference with live bacteria by subcutaneous injection with (1) ml of the bacterial suspension of live S. Typhimurium bacteria at a concentration of (1×108 cells/ml) once after the completion of the complete vaccination program. Blood samples were collected during the experiment and tissues were collected after sacrificing the animals at the end of the experiment to evaluate the required standards.
Gross changes were observed in the colon tissue of the positive control group, represented by the loss of its normal appearance, in addition to the appearance of multiple masses of polyps. In the histopathological sections, severe infiltration of inflammatory cells between the crypts was observed, with marked hyperplasia in the glandular foci of the mucosal layer compared to the negative control group. As for the third group, histopathological changes in the colon mucosa and significant hyperplasia in the lymphoid tissue with infiltration of inflammatory cells and damage to the crypts of the mucosa layer were observed. The cross-section of the liver tissue of the fourth group showed simple sinusoidal congestion, degeneration of some hepatocytes, and irregularity of the sinuses accompanied by infiltration of some inflammatory cells in the portal area compared to the positive control group. The cross-section of the spleen tissue of the fourth group also showed small clusters of white pulp lymphocytes scattered across the red pulp, accompanied by slight infiltration of inflammatory cells compared to the positive control group.
The immune response measurements in the blood showed a significant increase (P<0.05) in the total number of white blood cells among the four study groups, as it reached its highest value in the positive control group with a mean and standard error of (17.52 ± 0.28 cells/mm3 blood), while the granulocytes showed a significant increase (P<0.05) in the rate and standard error of (7.9 ± 1.92cells/mm3 blood) in the fourth group when compared with the experimental groups, in addition to a significant increase (P<0.05) in the rate and standard error of monocyte cells in the same group when compared with the experimental groups, while the red blood cells showed a non-significant increase (P>0.05) in all four experimental groups and recorded a probability value between the groups of (0.28).
When estimating the expression levels of the immune parameters (TLR-4, PDL-1 and TNF-α), the results showed that there was no statistical significance at a significance level (P>0.05) between the four groups at most times regarding the study to the effect of the vaccine on the expression levels of the immune parameter TLR-4 in the serum of the rat groups, except that the averages in the groups (induced – vaccinated) tended to rise over time compared to the negative control, but it was not sufficient to achieve statistical significance. The results showed that the expression level of PDL-1 increased according to the vaccination serial time, and there were significant differences between the groups at all times at a significance level of P<0.05, the third group showed high levels during the entire vaccination program compared to the other groups, which also indicates a clear significant increase (P<0.05) compared to the negative control group, which indicated the presence of non-significant differences (P>0.05), while the results recorded a clear variation in the levels of TNF-α between the different groups over the vaccination period, the negative control group did not show a significant difference in the immune response (P>0.05), while the positive control group showed a clear statistical significance at the significance level (0.05) in the inflammatory response, the most prominent being the third and fourth groups that showed a statistically significant effect on the immune response.
We conclude from the above that subcutaneous vaccination using S. Typhimurium ghost vaccine (STGs) in rats can generate a strong cellular immune response to reduce bacterial colonization in infected rats, and also provides complete protection against live strain bacteria, thus these chemically induced bacterial ghosts can be used as a promising vaccine. We also conclude that the induction of colorectal cancer in male white rats by sub-peritoneally injecting them with AOM can lead to changes in some histopathological, blood and immune parameters when injected subcutaneously with S. Typhimurium ghost vaccine, and it had an immunostimulatory effect with colorectal cancer.