الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Schistosomiasis is a parasitic disease caused by trematode flukes of genus Schistosoma (S.), which affects hundreds of millions worldwide. It is one of the neglected tropical diseases, causing significant morbidity and can result in permanent damage to various organs with major effects on childhood development and adult productivity. Schistosomes alternate generations between definitive hosts (mammals), in which sexual reproduction takes place, and intermediate hosts (snails), in which asexual multiplication takes place.
Despite the adult schistosomes are faced with humoral and cellular immune responses of the human host, they can live in the blood for many years due to several unusual parasite adaptations which occur soon after infection. This unusual ability directs the control attempts either toward the very early schistosomula stage or to the schistosomal tegument as it was proved to be a crucial target for many antischistosomal drugs. It performs vital functions and plays a critical role in modulation of the host response and parasite survival. Praziquantel (PZQ) is the only drug available for the treatment of schistosomiasis mansoni since the 1970s, induces extensive tegumental and subtegumental alterations that result in its unparalleled potency.
The lipid composition of the developing and adult schistosomes tegument showed high sphingomyelin (SM) content (about 20%), representing an important structural component in the membrane leaflets. The cellular sphingolipid homeostasis is maintained by the control of its synthesis, breakdown and inter-organellar transport of its metabolites. The SM molecules form tight network of hydrogen bonds with surrounding water molecules allowing nutrients, but not host antibodies to access at the host-parasite interface.
Mg+2-dependent neutral sphingomyelinase (Mg2+-nSMase) is an integral membrane protein in mammals. It hydrolyses SM into ceramide and phosphorylcholine. This enzyme was identified in Schistosoma tegument. Much is known about the activators of Mg2+-nSMase and excessive SM hydrolysis; that destroy the intramolecular hydrogen bond, increasing the surface membrane fluidity and permeability. Contrarily, the role of Mg2+-nSMase inhibitors on the developing schistosomes has not been evaluated yet.
Coenzyme Q10 (CoQ10), is a natural fat-soluble, vitamin-like, ubiquitously existing benzoquinone derivative. The antioxidant function of CoQ10 is attributed to its active reduced form ubiquinol. Recent data pointed out a novel role for ubiquinol as an efficient inhibitor of Mg2+-nSMase. (213)
The chemical instability of ubiquinol upon exposure to air, ultraviolet light or high temperature hampers its adequate oral delivery. The large molecular weight, poor aqueous solubility and high lipophilicity also contribute to its low absorption. To solve such a problem, recent approaches; via nano-formulations, were invented to improve its stability and bioavailability. Niosomes are lipid based nanosized drug delivery system which is composed of spherical lipid bilayers composed of cholesterol and nonionic surfactants. They are biodegradable, biocompatible, and non-immunogenic vesicles, possessing a potential role to overcome the challenge of poor water solubility by increasing the absorption across the gastrointestinal tract and acting as a depot, releasing the drug in a controlled manner and restricting its effects to target cells, so enhance the bioavailability of such a compound.
These gave us the challenge to investigate the efficacy of this drug in its plain and niosomes-encapsulated forms on lung schistosomula and adult S. mansoni stages in experimentally infected mice regarding their potential tegumental alterations with subsequent survival attrition. Furthermore, the antioxidant and anti-inflammatory properties of the drug encourage us to demonstrate the associated histopathological changes of the affected tissues.
This study was conducted on 60 male Swiss strain albino mice equally divided into two main groups; group I (30 mice) was designed to study the efficacy of ubiquinol and ubiquinol-encapsulated niosomes (U-N) against lung-stage schistosomula. Mice were infected with 3000 cercariae each. It was further subdivided into five equal subgroups (SGs). Subgroup (SG) Ia: Infected, non-treated (non-treated control), Subgroup Ib: Infected, PZQ –treated (therapeutic control); which received 200 mg PZQ/kg/day/mouse, Subgroup Ic: Infected, niosomes–treated; that received 0.3 ml niosomes/day/mouse, Subgroup Id: Infected, ubiquinol–treated; which received 300 mg ubiquinol/kg/day/mouse and Subgroup Ie: Infected, U-N–treated; that received 0.3 mL U-N suspension/day/mouse (equivalent to 300 mg ubiquinol/kg/day). The treatment started on the first day of experimental infection and continued for five successive days to coincide with the development of lung schistosomula. Animals were sacrificed seven days PI which is the proper time to isolate lung schistosomula. group II (30 mice) was designed to study the efficacy of ubiquinol and U-N against adult stage. It was equally subdivided into five subgroups, corresponding to those of group I with the following difference: Mice were infected with 100 ± 10 cercariae each, PZQ was given in a dose of 100 mg /kg/day/mouse and all treatment regimens started on the 30th day PI before the onset of significant oviposition and continued for 15 days. Animals were sacrificed on the 47th day PI.
The efficacy of the different treatment regimens was assessed by determination of the parasite burdens (lung schistosomula and adult worms) recovered from all studied subgroups. Prior to fixation in 2.5% glutaraldehyde, half of the schistosomula and adult worms recovered from infected non-treated subgroups (SGs Ia and IIa) and U-N treated subgroups (SGs Ie and IIe) (couple worms were excluded) was fixed in 4% paraformaldehyde for indirect immunofluorescence (IF) assay. Couple worms recovered from infected, non-treated subgroup (SG IIa) and U-N-treated subgroup (SG IIe) were allowed to depair separately for each subgroup for few minutes before their fixation in 2.5% glutaraldehyde and were processed for TEM examination. The body perimeters for lung schistosomula and adult worms were measured for all subgroups. Scanning electron microscopy (SEM) for 2.5% glutaraldehyde fixed lung schistosomula and adult worms recovered from infected, non-treated subgroups (SG Ia and IIa) and U-N treated subgroups (SG Ie and IIe) was demonstrated. Transmission electron microscopy (TEM) for the testes and ovaries of the depaired adult worms was performed. Both hepatic and intestinal tissue-egg counting were performed along with calculating the fecundity of the female worms and recording the oogram changes for all the animals of group II. The histopathological changes of pulmonary and hepatic tissue were demonstrated in group I and II, respectively with determination of the hepatic granulomas number and size in group II.
Regarding the efficacy of ubiquinol (SG Id), on the lung schistosomula burden, the therapeutic activity was limited with percentage reduction of 22.9% in comparison to their control (SG Ia). The encapsulation of the ubiquinol into niosomes (SG Ie) enhanced its efficacy with a statistically significant reduction of 39.12%. Similarly, a corresponding statistically significant reduction in the mean total, female and couple worms load with percentage reductions of 50.81%, 49.56% and 68.3%, respectively were recorded in SG IIe. This drug regimen had a potential worm de-pairing potency as percentage reduction in the couple worm burden was much higher than that of the total and female ones. On the other hand, there was a statistically significant reduction by 14.5% in the mean perimeters of lung schistosomula in the U-N–treated SG Ie. Similarly, female worms were significantly affected after treatment with U-N (SG IIe), with reduction of their mean perimeters by 25.94%. While a non-significant reduction of the male worm perimeters was recorded.
SEM of lung schistosomula in U-N-treated SG Ie revealed thickening, swelling of the surface, excessive longitudinal corrugations and disruption of the tegumental ridges and troughs with irregularly distributed spines. Notably, in U-N treated SG IIe, the male schistosomes showed excessive widening of the gynaecophoric canal, evident tegumental corrugations and collapsed, spineless, eroded or completely sloughed dorsal tubercles. Tegumental focal erosions and attached host cells and leucocytes were observed. The female worms showed extensive tegumental swelling, microsrosions, loss of sensory papillae with formation of long irregular disorganized splits with honey combed appearance. Rough and bushy spines with drastic organization of the posterior end were also observed.
TEM of the male testicular tissue in U-N-treated SG IIe revealed ultrastructural changes in the form of degenerations in the spermatocytes, Sertoli cells, follicular wall, smooth muscle layer, spermatozoa and spermatids. Furthermore, the ovarian oocytes of female schistosomes showed signs of immaturity and degeneration.
Under the fluorescence microscope, treatment with U-N yielded up schistosomula with diminished immunofluorescence reactivity (fluorescence intensity of 2+). Schistosomula possessing an intensity of 3+ were also observed. Notably, 60% of the adult worms, treated with U-N expressed fluorescence intensity of 4+ and the remaining revealed an intensity of 1+.
Oral treatment with PZQ, ubiquinol and U-N against adult schistosomes (SGs IIb, IId and IIe) revealed statistically significant reductions in the mean hepatic and intestinal tissue-egg counts in comparison to their corresponding controls. The comparison between PZQ-treated (SGs IIb) and U-N-treated (SG IIe), demonstrated a statistically non-significant difference indicating a relatively equivalent potency of both drugs regarding this demonstrative parameter. U-N regimen (SG IIe) induced significant reduction in the female worm fecundity with a percentage reduction of 43.0% with statistically non-significant difference between ubiquinol in its two forms (SG IId and SG IIe). Oogram pattern changes with statistically significant increase in the mean of mature and dead eggs on the expense of immature eggs were observed in mice treated with either PZQ (SG IIb) or U-N (SG IIe). Meanwhile, ubiquinol treatment (SG IId) induced a statistically significant increase in the mean of dead eggs only. This increase was statistically non-significant, when compared to U-N treatment (SG IIe).
Lung sections of ubiquinol-treated SG Id and U-N-treated SG Ie, showed small foci of minimal to mild interstitial inflammation adjacent to schistosomula.
The reductions in the mean number of hepatic granulomas induced by the different therapeutic regimens (PZQ, ubiquinol and U-N) were statistically significant in comparison to their control with percentage reductions of 49.20%, 33.91% and 36.66%, respectively. However, these findings were statistically non-significant when compared to each others. Similarly, the mean hepatic granulomas size was significantly reduced under the same drug regimens, when compared to SG IIa. Notably, U-N-treated SG IIe recorded the highest significant reduction among the different studied subgroups (38.2%). Treatment with ubiquinol in its two forms caused amelioration of the hepatic histopathological changes and revealed scanty, small granulomas surrounding degenerated ova with minimal hepatic fibrosis (5-10%).
The results of the present study showed comparative advantage of PZQ over U-N regarding its effectiveness on diminution the parasite burden, while they elicited a relatively equivalent potency considering tissue-egg loads and hepatic granuloma numbers. Furthermore, ubiquinol in its free and niosomes-incorporated forms through the assumed anti-inflammatory and anti-oxidant properties induced marked reduction of granulomas size and obvious amelioration of the associated hepatic pathology and fibrosis, significantly over PZQ.
These findings together with the drug safety profile and the recorded impact on maturation of worms’ reproductive organs suggest that U-N could be a promising novel antischistosomal drug and motivate us to investigate its efficacy when administrated along the time of S. mansoni development either alone or in combination with PZQ. The different mechanisms of action postulated for these compounds can make their combination very propitious.