الفهرس 
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Abstract
Toxoplasmosis is a cosmopolitan food and waterborne zoonotic infection. The disease is caused by Toxoplasma gondii which is a highly successful global pathogen due to its ability to infect almost any nucleated cell in any warm-blooded animal. It is estimated that one to two billion of the world’s population is infected. Epidemiological studies indicate that nearly 20–90% of adults have been exposed to the parasite. Almost 25–30% of the human population is infected with T. gondii. But, seroprevalence varies greatly between different countries from 10 to 80% and even within the same country. Three severe infection sequelae occur in humans: congenital infection, ocular toxoplasmosis and cerebral toxoplasmosis in HIV-infected and immunocompromised people.
The recommended chemotherapy of toxoplasmosis involving a combination of pyrimethamine and sulfadiazine plus folinic acid is mainly used in the therapy of HIV infected patients as well as in congenital and ocular toxoplasmosis. The therapy using these two antifolates is quite efficient but also toxic to human cells. These treatment regimens for infected patients have not essentially changed for years. The recommended drugs limit replication of T. gondii, however this may be associated with numerous and severe adverse effects. In addition, drugs don’t affect the tissue cysts of the parasite located predominantly in brain and muscles. Therefore, there is an urgent need to develop new drugs and establish a new “gold standard” treatment
Proteases have been validated as drug targets, so protease inhibitors are currently used as drugs to treat hypertension and HIV infection. Protease Inhibitor Cocktail (PIC) is a novel blend of five pan-protease inhibitors: AEBSF, aprotinin, E-64, leupeptin and bestatin. It has low toxicity and comprehensively protect proteins from degradation by various proteases. It is a cocktail of small molecules that inhibit the action of proteases.
Therefore, the present work aimed at studying the effect of PIC, which inhibits both cysteine and serine proteases, on in vitro cultured T. gondii tachyzoites. This was achieved by assessing its effect on the invasion of the host cells, the intracellular development and the secretory pathway of T. gondii tachyzoites.
To achieve these goals, the study was performed in vitro on HepG2 cell line. The cells were initially grown as monolayer in sterile T-25 plastic culture flasks at 37o C with an atmosphere containing 5% CO2. They were grown in DMEM containing 10% fetal bovine serum and the flasks of the cell line were divided into two groups: control group (I) and experimental group (II). The 18 flasks in the control group (I) were equally subdivided into: subgroup (Ia): non- infected, non- treated HepG2 cell culture, subgroup (Ib): non- infected, treated with PIC at a concentration of 1/40, HepG2 cell culture and subgroup (Ic): T. gondii tachyzoite Infected, non- treated HepG2 cell culture. The 12 flasks in the experimental group (II) were equally subdivided into two subgroups: subgroup (IIa): cell culture infected with pre-cultivation PIC treated T. gondii tachyzoites (cell culture infected with tachyzoites incubated with PIC at a concentration of 1/40 one hour prior to cultivation) and subgroup (IIb): cell culture infected with T. gondii tachyzoites then treated with PIC (24 hour-infected cell culture then treated with PIC at a concentration of 1/40).
Counting and viability of the tachyzoites in the culture supernatant were used to assess the effect of PIC on the ability of tachyzoites to invade, intracellularly multiply and re-emerge from the cultured cells. They were done on the third, fourth and fifth days post infection. In addition, assessment of the morphological changes was done using light microscopic and TEM study. These studies were done on the infected control subgroup and experimental subgroups.
To assess the effect of PIC on the secretory pathway of Toxoplasma tachyzoites, electrophoretic study was performed after two, three and five hours of incubation with the PIC. It was done to detect the effect of PIC on the total protein composition of the excretory/secretory antigens (ESAs) using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE allows the identification of the electrophoretic pattern of proteins and estimation of their molecular weights and intensity in each band using a standard wide range protein marker.
Regarding the assessment of invasion and multiplication of T.gondii tachyzoites in different studied subgroups, the results showed that subgroup (IIa) (infected with pre-cultivation treated tachyzoites) had the highest reduction of tachyzoite count and viability. This reduction was statistically significant compared to infected control subgroup (Ic). Subgroup (IIb) (infected then treated) showed a slight reduction of tachyzoite count and viability which were not statistically significant compared to infected control subgroup (Ic). Moreover, there were statistically significant reduction of tachyzoite count and viability in the subgroup (IIa) (infected with pre-cultivation treated tachyzoites) compared to subgroup (IIb) (infected then treated). On the other hand, a comparison between the tachyzoite count and viability in the supernatant fluid of within the same subgroup on the third, fourth and fifth day post infection was done. The results showed no statistically significant change of tachyzoite count and viability in all studied subgroups between the three days.
The assessment of the morphological changes by light microscopic examination revealed that the tachyzoites from the culture supernatant of cell culture infected with pre-cultivation treated tachyzoites (subgroup IIa) showed swelling with loss of characteristic crescentic morphology. While T. gondii tachyzoites from culture supernatant of infected then treated cell line (subgroup IIb) showed vacuolated cytoplasm and irregularities of surface outline. The transmission electron microscope (TEM) examination showed that the cell line infected with pre-cultivation treated tachyzoites (subgroup IIa) showed a scanty number of tachyzoites inside the parasitophorous vacuoles. Cross section of multiple tachyzoites showed ill-defined cytoplasmic membrane with decreased number of host mitochondria closely associated with the parasitophorous vacuoles. Whereas the longitudinal section of tachyzoites showed destructed rhoptries and apical complex with multiple coalesced vacuoles. They also showed swelling and loss of characteristic crescentic tachyzoite morphology with an apparently normal structure of the nucleus and deformed apical complex, rhoptries, increased dense granules and widening of the space surrounding the tachyzoite with destruction and almost absence of the tubulovesicular network. In cell line infected then treated with the PIC (subgroup IIb), parasitophorous vacuoles showed numerous tachyzoites inside it with apparently normal apical complex and dense granules. Some of the tachyzoites formed parasite masses, others developed cytoplasmic protrusions. Some tachyzoites also lost the integrity of the cytoplasmic membrane and the nuclear membrane with leakage of their contents. Longitudinal and cross section of the tachyzoites showed multiple separate vacuoles. Moreover, the parasitophorous vacuoles retained the tubulovesicular network but with disintegrated granular appearance. No host mitochondria or endoplasmic reticulum surrounding the parasitophorous vacuoles could be detected but tachyzoite mitochondria could be seen.
Assessment of the effect of PIC on the secretory pathway of T.gondii tachyzoites by SDS-PAGE analysis showed that the molecular weights of antigens/ protein bands for all samples either non-treated or treated at all durations were in the range from 164.3 to 12.9 KDa. There were five common bands in all durations in the non-treated and PIC treated samples. Their arrangement and molecular weights were (2nd)137.9-134.6, (4th)64.7-63.4, (5th)60.7-59.6, (7th)51.7-48.8 and (9th)14.4-12.9 KDa. It is worth to mention that after three hours of incubation, samples either treated with PIC or not, showed three unique bands. They were the (1st)164.3-161.2, (3rd)122.8-121.5 and (8th)32.1-31.7 KDa. They were not recorded in the non-treated or treated samples after two hours and five hours of incubation. After five hours, the molecular weights of the separated bands were more or less identical to that recorded after two hours of incubation either in non-treated or treated samples.
Regarding the intensity of the separated bands, it was observed that two of the common bands (4th) and (5th) with molecular weights 64.7-63.4 and 60.7-59.6 KDa respectively, were of high intensity in comparison to other bands in all studied groups. Moreover, the 5th band with molecular 60.7-59.6 KDa in the treated sample after three hours of incubation had the highest intensity among all other bands. This band could be formed of the two bands 5th and 6th. But, due to the high intensity of the protein content within this range, the bands overlapped and appeared as one band.
from the previous results, it was evident that the inhibitory action of the PIC was effective when applied to tachyzoites before host cell invasion. Pre-treatment of T.gondii tachyzoites with PIC resulted in failure of the invasion of most of the tachyzoites and decrease the intracellular multiplication and viability of the tachyzoites that succeeded in the initial invasion process. This effect was irreversible till the complete lysis of cell monolayer. Evident deformed morphology was seen by both light and TEM with absence of the tubulovesicular network. On the contrary, the effect on already established infection was not significant compared to the infected control and pre-treated subgroups regarding counting and viability. However, in this subgroup, morphological changes were evident. Although the parasite number inside the PV was not affected, large irregular-shaped parasites were detected. This means that the parasites were defective. The analysis of ESAs was done to recognize the effect on the secretory pathway. Although, the protein contents of ESAs from PIC treated samples were higher in comparison with that in the non-treated samples, this can’t exclude the effect on secretory protein maturation which indirectly affects the parasite invasion. Finally, it can be concluded that the efficiency of the PIC in preventing invasion and intracellular multiplication was proven. In addition, it was proven to be cost effective compared to using individual protease inhibitors separately. It also had the benefit of combined therapy as it lowered the concentration of each protease inhibitor used in the cocktail. Further studies may be needed to establish the exact mechanism by which the PIC exerts its effect on the T.gondii tachyzoites behaviour and its secretory pathway.