Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats. Part II.

Toll-like receptors in the gastrointestinal tract can influence intestinal homeostasis and play a role in the repair and restitution of intestinal epithelium following tissue damage. In our previous study a statistically significant increase in the level of TLR4 and TLR2 gene expression was observed in rats in early stages of hymenolepidosis. Moreover, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLRs within intestinal epithelial cells) increased over the infection period. In this paper, we determined changes in the expression of TLR2 and TLR4 and the number of anaerobic intestinal commensal bacteria in Hymenolepis diminuta infected rats. In the isolated jejunum of infected rats at 16 days post infection (dpi), the expression of TLR4 and TLR2 was significantly higher than uninfected rats. In the colon, a statistically significantly increased expression of TLR2 was observed from 16 to 40 dpi, and TLR4 from 16 to 60 dpi. The jejunum and colon of infected rats contained Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Enterococcus, Streptococcus, Staphylococcus, Bacillus, Lactobacillus) and Candida. The total number of intestinal bacteria was higher in H. diminuta infected rats, but the observed microbiota had only minor effects on the expression of TLR2 and TLR4. Toll-like receptors play a role in maintaining epithelial barrier function in response to enteric pathogens and parasites. In our study, the alteration of TLR2 and TLR4 expression in the infected rats indicates the potential role of the innate immune system in the pathomechanism of this infection.

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats.

Toll receptors play a critical role in the rapid activation of innate immune responses to a variety of pathogens. In mammals, Toll-like receptors (TLR) have been found in both immune related cells and other cells. At present little is known about the participation of TLR in host defense mechanisms during parasitic infections. The aim of this study was to determine the expression of TLR2 and TLR4 genes in rat intestines during experimental hymenolepidosis. There is difference in expression of TLR2 and TLR4 genes in the colon and jejunum in uninfected rats: in the colon, mRNA of the examined TLR is present in much higher amounts than the jejunum, while the protein of the TLR also had a segmented specific distribution. In the jejunum isolated rats infected with Hymeolepis diminuta 6 and 8 days post infection (dpi), mRNA for TLR4 and TLR2 were significantly more strongly expressed in comparison with the uninfected controls. In the colon, a statistically significantly increased expression of TLR4 gene was observed only at 6 dpi, and at 8 dpi for the TLR2 gene. Moreover, we observed that during inflammation, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLR within intestinal epithelial cells), increased together with the duration of the infection period.

Glutathione-S-transferase activity in mouse muscle during experimental trichinellosis.

The role of glutathione-S-transferase (GST, EC 2.5.1.18) in biochemical host defence in experimental trichinellosis was evaluated. The activity of GST in mouse skeletal muscles was measured during the muscular phase of trichinellosis, starting from the 3rd week post infection (w.p.i.) to the 11th w.p.i. Activity was determined spectrophotometrically by monitoring the formation of thioether (S-2,4-dinitrophenylglutathione) from the reduced form of glutathione and 1-chloro-2,4-dinitrobenzene used as a substrate and as an example of xenobiotics. The changes in the activity of GST were as follows: an increase in activity starts in the 4th w.p.i., peaks (up to 310% of the normal value) in the 6th w.p.i., decreases in the 8th week and a final, weak rise was observed in week 11. The statistically significant changes in GST activity in this phase of experimental trichinellosis suggest that this enzyme participates in the biochemical defence of the host against Trichinella infection.

Biochemical investigations of the dynamics of proteinase activity at different stages of trichinellosis in mice.

In the literature available hitherto there are many reports on enzymatic changes in tissues and a correlated rise in enzymatic activity in blood serum during experimental and human trichinellosis. In this study we were characterised proteinase activity in crude extracts from muscles of mice infected with Trichinella spiralis and T. pseudospiralis and the dynamics of their changes in different stages of disease. The activity of proteinase in muscle of mice infected with T. spiralis showed an increase in the 1st-5th week post infection, and then a slight decrease. The biggest proteinase activity was observed in 5th-6th week post infection. In the muscle of mice infected with T. pseudospiralis the increase of proteinase activity was observed in 1st-4th week post infection. In the 4th week the activity reached its maximum and in the 5th-10th week post infection there was a decrease of the activity in comparison with the control. As we could see, the dynamics of the changes of proteinase activity in mice is similar in the case of the disease with other biochemical and immunological indices observed in trichinellosis and with the increase of regeneration and transformation processes observed in histopathological studies.

Dynamics of rat muscle mitochondria uncoupling in trichinellosis.

Parallel oxygraphic measurements of respiratory control index (RCI) and spectroscopic measurements of the activity of two mitochondrial enzymes from rat muscle, mtATPase and succinate dehydrogenase were performed. In Trichinella spiralis infection, in comparison with Trichinella pseudospiralis infection, a delayed stimulation of mtATPase in 3rd week p.i. was observed and the degree of stimulation was weaker. A second peak of mtATPase stimulation appeared at the beginning of the encystation phase. The two-phase phenomenon was accompanied by a sharp decrease of RCI to 39% of normal values and almost 2-fold increase in the ratio of Mg(2+)-stimulated to Mg(2+)-unstimulated mtATPase. By the end of investigations (7th week) the level of SDH normalized, but mtATPase was still elevated. The results, as in the case of T. pseudospiralis infection, support the testing of mtATPase activity to follow bioenergetic changes in tissue parasite infections. In comparison with oxygraphic measurements ATPase testing needs a much smaller amount of biological material and ATP.

[Use of restriction enzyme analysis in parasitology]. / Zastosowanie analizy restrykcyjnej dla celów parazytologii.

Application of analysis of nucleic acid using restriction enzymes in parasitology is discussed. The main advantage of the new technique is the possibility of direct and detailed studies at the level of DNA. At present, the "genetic probe" becomes more and more commonly applied to identification of both parasites and their transmitters. It appears that the technique of restriction analysis is of great significance for solving taxonomic problems in parasitology.