Effect of Treatment with Antifibrotic Drugs in Combination with PZQ in Immunized Schistosoma mansoni Infected Murine Model

The main problem in schistosomal hepatic morbidity is fibrosis and extensive scarring induced by living eggs. In this study, we tried to study the effect of treatment using antihelminthic drug (PZQ) and/or antifibrotic drugs (PTX and silymarin) in combination with immunization. The parasitological parameters, the dynamics of serum-specific immunoglobulins and splenic cytokines associated with changes in granuloma diameter were assessed. Naïve mice were immunized intravenously with 10 ug of SEA in three doses at 2 days intervals 6 weeks before infection. Animals were infected by tail immersion with 100 cercariae and divided into several groups. Three groups were treated with PZQ, PTX or silymarin administered alone. Another two groups were treated with PZQ combined with PTX or silymarin. All treated animals and respective controls were sacrificed 12 weeks post infection. Immunization did not affect worm reduction , but slight decrease in granuloma diameter, increase in immunoglobulins and cytokines was observed . Reduction in worm burden was associated with reduction in ova count and changes in oogram pattern which were mainly due to PZQ treatment. Increasing reduction in granuloma diameter, elevation of immunogloulins and cytokines levels were observed in the groups treated with PZQ alone or cmbined with PTX or silymarin. In conclusion, in this study, treatment with PZQ complemented with immunization resulted in significant reduction of parasitological parameters and rise of specific Igs. Addition of antifibrotic drugs PTX or silymarin to PZQ, potentiated an antipathology effect which minimized and ameliorated liver fibrosis by inhibition of HSC activation and accentuation of the effect of suppressor Treg cells. [Journal of American Science 2010; 6(5):208-216]. (ISSN: 1545-1003). Key word: Schistosoma mansoni, Praziquantel, Pentoxifyllin, silymarin. 1Introduction Schistosomal pathology is a direct consequence of the immunological response to ovideposition in host tissue especially liver. Liver injury is typically associated with infiltration of inflammatory cells leading to fibrosis (Friedman, 2003). Liver fibrosis results from chronic damage of the liver and activation of hepatic stellate cells (HSC) which leads to excess production of extracellular matrix (ECM) components (Friedman and Arthur, 2002 ; Friedman, 2003 ; Bartley et al., 2006). Various investigators have focused on the protective immunization against schistoromiasis using several soluble egg antigen (SEA) fractions which were identified and tested in experimental models with the induction of variable levels of protection against infection (Tendler et al., 1996). Immunization of mice stimulates specific immunity which causes reduction in worm burden, intestinal egg load and liver pathology (Romeih et al., 2008 ; Garcia et al., 2008). Until recently, non of immunizing fractions was able to induce more than 67% protection, but the existence of at least partially protective immunity would make a logical complement to drug therapy (Bergquist et al., 2008 ; Maher et al., 2003 , Zovain et al., 2001). Praziquental (PZQ) is the drug of choice for all species of Schistosoma as an effective antischistosomal drug (Utzinger and Keiser, 2004). Although treatment with this drug is effective, but frequent schistosome reinfection occurs after treatment due to relative resistance to schistosomicide drugs (Silva et al., 2003). At the same time, it is stated that it is preferable to develop combination of PZQ and anti-fibrotic drugs in the treatment of murine schistosomiasis which could minimize liver fibrosis simultaneously with worm elimination (Mahmoud et al., 2002 ; Doenhoff et al., 2002). Pentoxyfilline (PTX) has been identified as an antifibrotic drug which can interfere on a large spectrum of cytokines with proinflammatory action and causes inhibition of ECM synthesis (Bienvenu et al., 1995 ; Reis et al., 2001). Antioxidants such as silymarin have received attention as potential antifibrotics which inhibit HSC activation and protect hepatocytes from undergoing apoptosis (Leiber et al., The journal of American Science 2010 6(5) Ibrahim et. al. Effect of Treatment with [email protected]. [email protected]. 209 2003). In this work, parasitological parameters and the dynamics of serum-specific immnoglobulins and splenic cytokines associated with changes in hepatic pathogenesis and granuloma diameter, were assessed in an attempt to study the effect of treatment with PZQ alone and in combination with PTX or silymarin in immunized infected mice model. 2Material and Methods: Animals: C57 BL/6 mice (6-8 weeks old), (18-20g) were bred and maintained at Schistosome Biology Supply Center (SBSC) of Theodor Bilharz Research Institute (TBRI) and kept under standard housing conditions. The animal experiments have been carried out according to the internationally valid guidelines in an institution responsible for animal ethics (TBRI) (Nessim et al., 2000). Preparation of S. mansoni soluble egg antigen (SEA): SEA was prepared (Boros and Warren, 1970, Carter and Colley, 1978) and purified from host antigen by affinity chromatography using cyanogen bromide activated sepharose-4B beads (Nordon & Strand, 1984). SEA was sterilized by filtration and protein content was estimated using Bio-Rad kit (Bradford, 1976). Drug and doses: a) Praziquental (PZQ) (Distocide ®, Epico Pharma Cairo, Egypt) was orally administered 7 wks p.i. at a dose of 500 mg/kg body weight for 2 consecutive days. It was freshly prepared before use as a 2% suspension in Cremophor-El (Sigma chemicals Co. St. Louis, Missouri). b) Pentoxifylline (PTX) (Trental ®, Aventis Pharma, Cairo, Egypt), was orally administered 4 wks post infection (PI) 5 days/wk at dose of 400 mg/Kg body weight. The treatment was continued until the date of sacrifice. c) Silymarin: (SEDICO Pharmasetuical-co) was orally administered starting from the day of infection at dose of 140 mg / kg three times / week until the day of sacrifice. Experimental design: 140 mice were immunized with SEA (10 ug X 3). Six weeks later, they were infected by tail immersion with 100 cercariae of an Egyptian strain of S. mansoni supplied from SBSP, TBRI and were divided into 6 groups. Three groups were treated with PZQ, PTX or silymarin as described before. Another two groups were treated with PZQ combined with PTX or Silymarin. The sixth groupimmunized, infected untreated mice were used as immunized infected control. Infected not immunized, untreated animals were used as infected control. Clean uninfected, untreated animals were used as normal control. All animals were sacrificed 12 weeks post infection. Parasitological Parameters: 1Worm burden: Infected animals were perfused to recover hepatic and portomesenteric worms for subsequent counting (Duvall and DeWitt, 1967). 2Tissue egg load: The number of eggs per gram tissue (liver and intestine) was studied according to the procedure described by Cheever (1968). 3Oogram pattern: The percentages of immature, mature and dead eggs in the small intestines were computed from a total of 100 eggs per intestinal segment and classified according to categories previously defined by Pellegrino et al. (1962). Immunological Study:Determination of anti-SEA immunoglobulin subclasses IgG1, IgG2 and IgG4 were measured using indirect ELISA, based on the method of Engvall and Perlman (1971). ELISA microtiter plates were coated with 100 ul / well of 30 ug/ml of SEA. Sera were diluted 1:20 and anti-mouse IgG subclasses (Binding site, Birmingham, UK) were used at a dilution of 1:500. Absorbance at 492 nm was measured. Cytokine assay: Serum IFN-γ, IL-4 and IL-10 levels were measured by a sandwich ELISA technique. Briefly, plates were coated with capture antibodies and 100 ul of serum samples or recombinant cytokines were added. Following addition of the biotinylated detection antibody and streptavidin-alkaline phosphatase conjugate, the reaction was developed with paranitrophenyl phosphate (Sigma) and absorbance was measured at 405 nm. Granuloma measurement: Hepatic granuloma diameter was measured according to Von Lichtenberg (1962). The percent reduction in granuloma diameter relative to infected control was calculated as follows: % reduction of granuloma diameter = mean diameter of controls mean diameter of test groups / mean diameter of control group x 100. Statistical analysis: The data were presented as mean ± standard error of the mean (X ± SE). The means of the different groups were compared globally using the analysis of variance ANOVA. Data were considered significant if p values were less than 0.05.