The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells

Background: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats.Methods: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0.Results: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-β) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.Conclusions: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite

The role of nitric oxide (NO) in parasitic infections

Nitric oxide (NO) has been shown to play a crucial role in various physiological and pathological conditions. NO plays a role in the immunoregulation and it is implicated in the host non-specific defence in a variety of infections. Abundant evidence indicates that NO contributes to the host defence function of macrophages. High levels of NO are mediated by up-regulated expression of the iNOS gene in response to the activating signals, in particular to the secretion of pro-inflammatory cytokines (IFN-y, TNF-a, IL-1, IL-2) by Thl cells. In this review, the role of NO during a number of parasitic infections has been summarized. Up to now, enhanced levels of NO production and expression of iNOS gene have been described in such infective diseases as malaria, toxoplasmosis, leishmaniosis, trypanosomosis and schistosomosis. During these infections, the preferential production of pro-inflammatory cytokines predisposes to the increased synthesis of NO, which mediates host protection through either direct parasite killing or by limiting parasite growth. The evidence presented in this review supports the conclusion that NO plays an important role in the majority of parasitic infections

Application of restriction enzyme analysis as an aid in parasitology

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 indentification of both parasites and their transmitters. It appears that the technique of restriotion analysis is of great significance for solving taxonomic problems in parasitology

Effect in vitro of albendazole on the kinetics of cytosolic glutathione transferase from the rat liver

Introduction. Since the idea of multifunctional mode of action of anthelmintics is considered and in experimental trichinellosis in vivo albendazole seems to act as an allosteric activator of cytosolic GST from mice muscles, in this study a termosensitivity after in vitro incubation with albendazole of purified commercial cytosolic glutathione transferase (GST) from the rat liver was investigated. Methods. Two extremal temperatures: −80°C and +30°C were used to destroy the dimer in quaternary structure of this enzyme. Results. In control preparations both extremal temperatures destroy this structure, so the Michaelis−Menten kinetic curves of substrate saturation show the typical hyperbolic shape. After a long (15 h) freezing at −80°C or heating (up to 14 h at +30°C) the kinetics of substrate saturation of GST after incubation with albendazole show the sigmoidal or "double sigmoidal" shape, pointing out the quaternary GST structure as a complex of "frozen subunits". Drug inhibits about 6−times the total activity of GST after incubation at +30°C. We conclude that albendazole in vitro influences the structure of cytosolic GST from the rat liver and inhibits its activity, but, in opposite to in vivo study in mouse muscles infected with Trichinella spiralis larvae, does not act as an activator of this enzyme

Glutathione-S-transferase activity in mouse muscle during experital 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 1 st- 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

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 1 st- 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

The intriguing influence of albendazole on glutathione reductase activity in the muscles from Trichinella spiralis infected mice.

A clear stimulation of the activity of glutathione reductase (RG, EC 1.6.4.2), one of the major enzymes participating in glutathione metabolism, was observed in the skeletal muscles from Trichinella spiralis infected mice. The maximal, almost twofold increase in RG activity was observed in week 5 post-infection. Administration of albendazole, an anthelmintic widely used in trichinellosis treatment, resulted in an additional, about 40% stimulation of RG activity (small, but statistically significant) on week 2 post-infection. In weeks 6 and 7 postinfection, when no stimulation of RG activity in infected mice caused by drug was observed, the drug modified the structure of that allosteric enzyme, affecting the kinetics of its substrate saturation in this way that the shape of the substrate saturation curve changed from „double sigmoidal”, typical of this enzyme kinetics in the muscles from infected mice, to hyperbolic, typical of this enzyme kinetics in the control, uninfected animals