Trichinellosis is a zoonotic disease affecting mainly temperate regions; many cases were reported all over the world including Egypt.
Heat shock proteins (HSPs) are widely distributed in nature. They perform important functions in the folding and unfolding or translocation of proteins, as well as in the assembly and disassembly of protein complexes. The heat shock response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stress. In non-stressed cells, HSPs are present in low concentration, while in stressed cells they accumulate at high levels (Zügel & Kaufmann, 1999). The parasites protect themselves against the host by activating various evasion mechanisms including HSP synthesis (Buchmeier & Heffron, 1990). Apparently, HSPs play specific functions in differentiation of parasites, in protection from the host killing mechanisms, including free radicals, and even in virulence (Devaney, 2006).
Heat shock protein 90 (HSP90) functions as a part of a multi-protein complex that promotes the folding and stability of client proteins. It is essential for survival of nematodes as confirmed in
This study aimed to evaluate the effect of HSP90 inhibitor, GA, on the adult worms and larvae of
Mice were infected with
Geldanamycin (C29H40N2O9, M.W. 560.64) was obtained from LC Laboratories, a division of PKC Pharmaceuticals, Inc. The drug was supplied as yellow microcrystalline powder. GA was dissolved in 0.1 % dimethyl sulfoxide (DMSO) to give a stock of 10 mM to be used in animals and
Twenty one mice were infected by 250
Similar parts of the diaphragm and thigh muscles were obtained and processed for histopathological examination by (H&E) and periodic acid-Schiff (PAS) staining. The PAS reaction was carried out as described elsewhere (Lechler
Adult worms were recovered (Wakelin & Lloyed, 1976). Briefly, mice were infected orally by 250
Muscle larvae were obtained from infected mice as described before (Dunn & Wright, 1985), and placed in PBS in a test tube. The larvae were then treated by GA exactly as described for adults.
Briefly, the parasites (larvae or adults) were transferred to 1-ml centrifuge tubes. The tubes were centrifuged at 7,000
Data were presented as mean ± standard deviation. The probability of significant differences among dual means of groups was determined by Student’s t-test. Differences were considered non-significant when (
No difference was found between control infected animals (25,428±1,397) and DMSO-treated infected animals (26.221 ±453) as regards the muscle larval count. In contrast, significant reduction of larval counts was observed in the treated groups compared to control animals. Moreover, there was significant difference between the groups treated with GA. The larval reduction rate was of 40.4 % in low dose (0.5 mg/kg), whereas that of the higher dose (1 mg/kg) was of 67.9 % (Table 1).
Muscle larval count in control and treated groups
Control (n=7) | Group 1 (n=7) | Group 2 (n=7) | |
---|---|---|---|
Mean ± SD | 25,428 ± 1,397 | 15,428 ± 975 | 8,142 ± 1,069 |
IE | --------- | 40.4 % | 67.9 % |
F. test | 14.526 | ||
0.001 | |||
Control & G1 | Control & G2 | G1 & G2 | |
0.001 | 0.001 | 0.001 |
Intensity effect (IE): IE (%) = [(N-n) /N] X 100
Where N is the average number of larvae in control group and n is the average number of larvae in treated groups.
Group 1 received low dose of GA (0.5 mg/kg), while group 2 received higher dose (1 mg/kg).
Muscle sections from mice receiving the solvent showed similar changes as in control animals. The striking changes observed in PAS or ordinary (H&E) staining was thinning of the capsule around
Photomicrographs of skeletal muscle sections 5 weeks p.i. showing: A,C encapsulated larvae from the control infected mice. The collagenous capsule (thick arrow) surrounds sections of
As regards the adult worms, the control group showed the characteristic structure of the cuticle being composed of two layers: upper striated and electron dense layer, and lower non-striated and electron light one. The cuticle is surrounded by the wavy two-layered epicuticle (Fig. 2A). In the treated groups the following changes were observed: a) haziness or blunting of the epicuticle which is the only change observed in lower dose of GA (Fig. 2B), b) loss of the electron dense layer of the cuticle (Fig. 2B), c) separation of the cuticle from the underlying hypodermis either in small areas forming blebs or extending through a large area (Fig. 2C), d) washed-out image of the cytoplasm of the hypodermis denoting cell damage (Fig. 2C, D).
Electron micrographs ofthebody wall of adult worms (A-D) and larvae (E,F):A, section in the body wall of an adult from control infected mouse (× 12,000). B, loss of the electron dense layer in the cuticle (arrow) is observed (× 25,000). C, separation of the cuticle from the underlying hypodermis (arrow) is noted (× 6000). D, there are blunting of the epicuticle and washed-out image of the cytoplasm (arrow) (× 10,000). E, section in the cuticle of a larva from the control group (× 20,000). F, disruption of the layers of the cuticle (arrows) is noted (× 25,000)
As regards the larvae, the cuticle in the control group was composed of two major regions separated by an electron dense layer. The outer surface of the cuticle was bounded by 4 electron dense layers (Fig. 2E). In the treated groups the changes were less striking consisting mainly of disruption of layers 3 and 4 (Fig. 2F).
The medical treatment of trichinellosis is problematic as there are “areas of uncertainty”. The classical medical treatment includes benzimidazole anthelmintics (mebendazole or albendazole) combined with steroids. Mebendazole is usually administered at a daily dose of 5 mg/kg but higher doses (up to 20 mg/kg/day to 25 mg/ kg/day) are recommended in some countries. Albendazole is used at 800 mg/day (15 mg/kg/day) administered in two doses. These drugs should be taken for 10 to 15 days(Bruschi & Dupouy-Camet 2014). The main problem in treatment is the poor susceptibility of migrating and encapsulated muscle larvae to anthelmintic drugs, making late treatment far from ideal (Gottstein
Heat shock proteins are best characterized for their role in cell survival during periods of stress, where their ability to bind denatured or misfolded proteins is essential for survival (Morimoto, 1998). HSPs also function during normal growth and division as chaper-ones for protein folding and transport. HSPs are essential for the survival of nematodes. For example, HSP70 has been found to be expressed in
Our study demonstrated the lethal effect of HSP90 inhibitor, geldanamycin, on the parasitic stages of
The body wall of a typical nematode consists of the cuticle, the hypodermis, and the somatic musculature. Integrity of nematode cuticle is essential for the nutritive and protective functions as well as to maintain shape. It provides protection from physical or immuno-logical injury, and plays a role in osmoregulation (Roberts & Janovy, 2013). The electron microscopy in our study demonstrated damage of the cuticle and hypodermis especially in adult worms. This may shed some light upon the mechanism of action of GA. In a similar way,
During the life cycle of
This study demonstrated the detrimental effects of GA against adults and larvae of
In conclusion, HSP90 seems to be essential for the survival of different stages of