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Abstract Schistosomiasis is a chronic water-borne helminthic disease with a major health and economic burden in tropical and sub-tropical areas, especially without access to safe drinking water and adequate sanitation. In Egypt, schistosomiasis is the most important endemic parasitic disease. S. mansoni is the causative agent for over 90% of all human schistosomiasis. In the definitive host, including man, adult male and female worms produce large numbers of eggs. Many eggs leave the body in feces, but some are trapped in tissues and induce a granulomatous immune response, leading to progressive tissue fibrosis and organ damage. miRNAs comprise a family of non-coding RNAs with approximately 21-25 nucleotides that can be detected in a wide range of body fluids and tissues. They downregulate gene expression at the post-transcriptional level and play an important role in controlling diverse biological functions including cell differentiation, development, proliferation and signal transduction. In schistosomiasis, it was found that the miRNA plays a variety of regulatory roles in the immunological responses occuring during the development of schistosoma. The present study aimed to assess the expression of host miRNA in serum and liver in mice experimentally infected with S. mansoni at different time intervals. In the present study blood and liver tissue samples were collected from control non infected mice (group I) and S. mansoni infected mice sacrificed at 4 , 8 and 12 weeks p.i. (groups II-IV) and from mice treated with PZQ eight weeks p.i. and sacrificed four weeks later (group V). At each time point, serum and liver tissue samples were processed for miRNAs extraction, reverse transcription and real time PCR analysis of miRNAs 223 & 146b expression. Liver sections were subjected to egg count estimation and histopathological examination. Parasitological assessment of hepatic egg count in the S. mansoni infected mice groups revealed no eggs in mice examined four weeks p.i. (group II) and a significantly higher mean Summary, Conclusions and Recommendations 84 egg count ∕ gm of liver tissue in group IV (12 weeks p.i.) compared to group III (eight weeks p.i.) with 142.4 % elevation. Treated mice (group V) showed the least mean egg counts with reduction percentages of 74.6 % compared to group III and 89.72 % compared to group IV. Examination of H&E stained liver sections revealed inflammation in the portal tracts as well as apoptosis in some hepatocytes four weeks p.i. On the other hand, large granulomas, more inflammation and appearance of fibroblasts and fibrocytes were observed eight weeks p.i. The changes become more obvious 12 weeks p.i with distorted hepatic architecture, chronic granulomatous lesions and fibrous transformation of granuloma around mature egg. It was observed that administration of praziquantel eight weeks p.i. improved the hepatic tissue pathology in S. mansoni infected mice. The hepatic granuloma size in the treated mice was significantly lower than that of the untreated mice (groups III and IV). Moreover, the treated group recorded the least inflammatory reaction. The decrease in granulomas size after treatment went hand in hand with diminished egg counts in the liver. Regarding miRNA-223, results of this study revealed that miRNA-223 expression in liver tissue in the early stage of infection (four weeks p.i.) was not significantly different from that of the uninfected mice. Significant down regulation was detected in mice examined 8 and 12 weeks p.i with more marked down-regulation in the latter. Moreover, the expression at 12 weeks was significantly lower than that observed at four weeks p.i. Expression is specifically associated with the hepatic egg count and the extent of hepatic pathological changes. It correlated negatively with tissue egg count. Serum miRNA 223 expression showed significant down regulation 8 and 12 weeks p.i, following the same pattern as tissue level. The levels of serum and tissue miRNA-223 were positively correlated and both were inversely related to the histopathological changes. Regarding liver expression of miRNA-146b, the present study showed nonsignificant difference between infected and control mice. However, expression increased gradually with progression of infection resulting in a significant higher level in mice examined in the later stage of infection (12 weeks p.i ) compared to mice examined earlier (4 weeks p.i.). Unlike miRNA- 223, expression of serum and tissue miRNA-146b correlated positively with egg count and pathological changes. Studying the expression Summary, Conclusions and Recommendations 85 profile of miRNA-146b in serum samples revealed a positive correlation with liver expression levels. The serum level was significantly elevated in the late stage (12 weeks p.i.) compared to the two earlier follow up periods (four & eight weeks p.i.). Regarding the effect of treatment, it was found that tissue and serum miRNA-223 expression levels nearly returned to normal level in infected mice within one month after PZQ treatment. Importantly, tissue miRNA 223 expression was significantly higher in the treated mice compared to the untreated groups examined 8 or 12 weeks p.i. (groups III and IV). PZQ treated mice also showed significant reduction in liver and serum miRNA-146b expression compared to the group with untreated advanced infection (group IV). Restoration of normal miRNAs expression in treated mice was accompanied by significantly lower hepatic egg counts, smaller hepatic granuloma size and diminished inflammatory cellular infiltration. Summary, Conclusions and Recommendations 86 Conclusions from the present study, it can be concluded that: - Down-regulation of miRNA-223 and up-regulation of miRNA-146b occurs in the liver tissue of mice experimentally infected with S. mansoni. Serum expression level of these two miRNAs follows the same pattern as the tissue expression level with significant positive correlation. - Expression levels of the studied miRNAs do not change until four weeks p.i. in mice. They may be useful as diagnostic markers of infection after the start of egg deposition. - Dysregulation of miRNAs expression correlates with liver egg count and becomes more obvious with the progression of infection and detection of more chronic granulomatous lesions, fibrous transformation and distorted hepatic architecture in liver sections. The expression level of these miRNAs can, thus, be used as a marker to reflect the extent of liver pathology. - The expression level miRNA146b in serum is sensitive to chemotherapy. Therefore, it could be potentially useful to monitor the therapeutic effects of PZQ in terms of improvement of sever hepatic histopathological damage. - Collectively, these findings provide new insights for further understanding of the mechanisms of host-parasite interaction in schistosomiasis mansoni. This may facilitate development of novel interventions for management of schistosomiasis associated morbidity. Recommendations - Further research is needed to investigate the molecular and functional role of miRNA-223 and miRNA-146b in the inhibition or activation of the immune system cells and HSC during the course of schistosomiasis Summary, Conclusions and Recommendations 87 - Further studies are needed to study the potential value of using a combination of miRNA-223 and miRNA-146b as well as other miRNAs as biomarkers to evaluate hepatopathology progression in patients with schistosomiasis. - It is also important to identify more potential miRNAs specific for schistosomiasis fibrosis. - Effective therapeutic methods for treating schistosomiasis-associated hepatic fibrosis are urgently needed as PZQ cannot completely reverse the progression of chronic liver fibrosis. - Considering the role of miRNAs in the hepatic immunopathology induced by schistosoma eggs, there is a need to explore the effectiveness of therapeutic strategies based on manipulating miRNA expression to prevent and/or treat the chronic complications of schistosomiasis. Safety issues of miRNA therapeutics in schistosomiasis require further studies. |