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

Ibrahim Rabia1, Faten Nagy2 2, Eman Aly1, Amina Mohamed3 Fayza EL-Assal3 and Azaa El- Amir3

1 Parasitiology, 2 Immunology Departments, Theodor Bilharz Research Institute, (TBRI) Giza, Egypt  and  3
Faculty of  Science, Cairo Univeresity.

ibrahimshalash@yahoo.com

Abstract: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, Pentoxifyllin (PTX) and silymarin in combination with immunization. Parasitological parameters, dynamics of serum-specific  immunoglobulins (Igs)  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, 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 combined  with  either PTX  or  silymarin. In  conclusion,  in  this  study,  treatment  with  PZQ complemented  with immunization resulted in a significant reduction of parasitological parameters and a rise in specific Igs. The addition of  antifibrotic drugs PTX or silymarin to PZQ, potentiated an antipathological effect which minimized and ameliorated liver fibrosis. [Journal of American Science 2010; 6(5):208-216]. (ISSN: 1545-1003).

Key word: Schistosoma mansoni, Praziquantel, Pentoxifyllin, silymarin.

1- Introduction
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.,

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.

2- Material 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 at an institution responsible for animal ethics (TBRI) (Nessim et al., 2000).

Preparation  of  S.   mansoni   soluble   egg  antigen
(SEA):
Soluble egg antigen 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 postinfection (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 µg 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. Two groups were treated with PZQ combined with PTX or Silymarin. The sixth group- immunized, 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 and blood were collected,  the serum was separated by centrifugation at 3000 xg for 10 minutes and stored at -20  C until used..

Parasitological Parameters:
1-   Worm 	burden: 	Infected	 animals	 were perfused to recover hepatic and portomesenteric worms for subsequent counting (Duvall and DeWitt, 1967).
2-   Tissue  egg  load:  The  number  of  eggs  per gram tissue (liver and intestine) was studied according to the procedure described by Cheever (1968).
3-   Oogram 	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.

3- Results:
Parasitological parameters:
The  total  number  of  worms  and  the percent
reduction of worm burden showed no significant difference between infected control and the immunized infected control. On the other hand, the groups treated with PZQ alone or combined with PTX or silymarin showed	 highly	significant 	decrease	 (P<0.001)

compared to immunized infected control. It showed no significant or slight decrease (p<0.05) in groups treated with   PTX  or   silymarin   respectively compared  to immunized infected control. The mean ova count in intestine   and   liver   showed   significant   reduction (P<0.01) in immunized infected control compared to infected  control,  while  all  treated  groups  showed highly  significant  reduction  (P<0.001)  compared  to immunized  infected  control  (Table  1).  As  regards oogram   pattern,   there  was  no  significant  change between the infected control and immunized infected control. On the other hand, highly significant decrease was shown only in the groups treated with PZQ alone or  combined  with  PTX  or  silymarine  (P<  0.001) compared to immunized infected control (Table 2). 
Granuloma diameter:
Granuloma diameter showed slight decrease in immunized infected control compared to infected control (P<0.05), while in all treated groups, it showed highly significant decrease (P<0.001) except the group treated with PZQ alone which showed no significant change   compared   to   immunized   infected   control (Table  3).
Immunological Parameters:
Serum-specific immunoglobulin isotypes:
In  infected  control  group,  there  was  no  significant
change in IgG isotypes compared to normal control. However in immunized infected control there is significant increase in IgG1 (P< 0.01) and IgG4 (P<0.05) compared to the infected control. The level of IgG1  showed  no  significant  change  in  the  treated groups   except   in   the   groups   treated   with   PTX combined with PZQ or silymarin alone which showed slight decrease (P<0.05) compared to immunized infected control. On the other hand , there was highly significant  increase in IgG2 level in all treated groups

(P<0.001), while the increase in IgG4 level was shown only in the groups treated with PZQ alone or combined with PTX or silymarin (P<0.05, P<0.05 and P<0.01 respectively) compared to immunized infected control (Table (4).

Serum cytokines level:
The  profile  of  Th-1  related  cytokine  IFN-γ  showed
significant increase in infected control (P < 0.001) compared to normal control. On the other hand it showed slightly significant decrease in immunized infected control compared to infected control (P<0.05). In treated groups, the groups treated with PZQ alone or combined with PTX or silymarin showed significant increase (P < 0.05) compared to immunized infected control. On the other hand, significant decrease in IFN- γ level was observed in groups treated with silymarin or PTX alone (P<0.01 – P< 0.001 respectively) compared to immunized infected control. The Th-2- related cytokines IL-4 showed highly significant increase in  the infected control compared to normal control (P< 0.001). At the same time, it showed significant decrease in the immunized infected control (P<0.01) compared to infected control. Also, it showed no  significant  change  in  all  treated  groups  except groups treated with PTX or PZQ alone which showed slight decrease (P<0.05) compared to immunized infected control. On the other hand, the Treg-related cytokine   IL-10   level   showed   significant   highly increase in infected control (P<0.001) compared to normal control and slight increase in immunized infected control (P<0.05) compared to infected control. In  the treated  groups,  it  showed  slightly significant increase in the groups treated with PZQ, silymarin or PTX alone (P<0.05) compared to immunized infected control (Table 5).

Table  1: Worm  burden and tissue load in mice immunized with SEA (10 μg X3) 6 wks before  infection and treated with different  types of drugs then sacrificed  12 wks post infection.

Animal group	Mean no. of worms + SEM	% reduction	Mean no. of ova count + SEM / g tissue	
			Intestine	% reduction	Liver	% reduction	
Infected control	31.6 ± 0.33		17234 ± 1291		3011 ± 374		
Immunized infected control	29.5 ± 0.17	6.65%	7450 ± 114 **	56.8%	1420 ± 210 **	53%	
Treated groups							
PTX	27.1 ± 0.29	14.2%	#4158 ± 234	75.9%	# #988 ± 277	67.3%	
PTX + PZQ	# # #1.3 ± 0.30 	95.9%	# # #612 ± 133	96.4%	# # #158 ± 33 	94.8%	
Sily	# 21.6 ± 0.21	31.6%	# #3889 ± 303	77.4%	# # #812  ± 90 	73.1%	
Sily + PZQ	# # #1.1 ± 0.21	96.5%	# # #500 ± 125	97.1%	# # #99  ± 11 	97.1%	
PZQ	# # #1.2 ± 0.3	96.2%	# # #638 ± 131	96.3%	# # #112  ± 10 	96.3%	

# # # P<0.001, # # *** P < 0.001 , **  P < 0.01, *  P <0.05 relative  to infected control. P<0.01,  # P<0.05  compared to  immunized infected  control.

Table  2: Oogram  pattern in mice immunized with SEA (10 μg x3). 6 wks before  infection  and treated with different  types of   drugs then sacrificed  12 wks post infection.
Animal Group	Oogram pattern ( % ova)	
	Immature	Mature	Dead	
Infected control	67.2 ± 5	33.1 ± 2.6	1.7 ± 0.3	
Immunized infected control	62.3 ± 2.4	31.8 ± 3.5	21.6 ± 0.7*	
Treated groups				
PTX	# 47.9 ± 3.9	29.9 ± 3.1	22.2 ±1.1 	
PTX + PZQ	# # # 4.9 ± 5.4	# # # 3.3 ± 1.4	# # # 91.8 ± 7.4 	
Sily	# # 22.1 ± 3.8	40.8 ± 3	37.1 ± 2 	
Sily + PZQ	# # # 9.5 ± 1.2	# # #8.8 ± 1.1	# # 81.7 ± 4.1 	
PZQ	# # # 2.0 ± 0.3	# # #1.7 ± 0.2	# # 96.3 ±4.9 	

*** P < 0.001 ,  **  P < 0.01,  *  P <0.05   relative   to  infected  control.
# # # P<0.001, # # P<0.01, # P<0.05  compared to  immunized infected  control.

Table 3: Hepatic  granuloma diameter and % reduction in mice immunized with SEA (10 μg x3). 6 wks before infection and treated with different  types of   drugs then sacrificed  12 wks post infection

Animal Group	Hepatic granuloma diameter(a)
Mean μm ± SEM	% Reduction (b)	
Infected control	299.8  ±  12.5		
Immunized infected control	254  ±  14.3*	15.3%	
Treated groups			
PTX	# # 139.9  ±  20.1	53.3%	
PTX + PZQ	# # # 103.4  ±  17.5	65.4%	
Sily	# # 164.4  ±  13.2	45.2%	
Sily + PZQ	# # # 128.6  ±  20.7	57.1%	
PZQ	225.1  ±  19.3	24.9%	

 (a) 	The mean  granuloma diameter per  group  was calculated  from the mean  values   (10-15  granulomas
per  mouse).
(b) 	Percentage reduction was calculated  relative to infected control.
*** P < 0.001 ,  **  P < 0.01,  *  P <0.05   relative  to infected control.
# # # P<0.001, # # P<0.01, # P<0.05 compared to immunized infected control.

Table  4: Serum  anti-SEA  IgG subclasses levels in mice infected   with SEA (10 μg x3). 6 wks before infection and treated with different  types of   drugs then sacrificed  12 wks post infection

Animal Group	X` O.D ± SEM
Ig G1	X` O.D ± SEM
Ig G2  	X` O.D ± SEM
Ig G4  	
Normal control	0.202 ± 0.178	0.294 ± 0.21	0.321 ± 0.02	
Infected control	0.462 ± 0.103	0.606 ± 0.207	0.99 ± 0.126	
Immunized infected control	0.954  ± 0.34 **	0.521 ± 0.383	1.12 ± 0.135*	
Treated groups				
PTX	0.815 ± 0.251	# # #0.939 ± 0.178	1.08 ± 0.204	
PTX + PZQ	# 0.783 ± 0.187	# # # 0.998 ±0.231	# 1.58 ± 0.179 	
Sily	# 0.791 ± 0.245	# # #1.049 ± 0.159	1.21 ± 0.321 	
Sily + PZQ	0.833 ± 0.213	# # #1.180 ± 0.256	## 1.69 ± 0.155	
PZQ	0.802 ± 0.421	# # #0.897 ± 0.534	# 1.43 ± 0.254 	

*** P < 0.001 ,  **  P < 0.01,  *  P <0.05   relative  to infected control.
# # # P<0.001, # # P<0.01, # P<0.05  compared to immunized infected control.

Table 5: Serum cytokine level in mice immunized with SEA (10 μg x3) 6 wks before infection and treated with different  types of   drugs the  sacrificed  12 wks post infection

Animal Group	IFN – γ
Pg/ml ± SEM	IL – 4
Pg/ml ± SEM	IL – 10
Pg/ml ± SEM 	
Normal control	235 ± 26.5	15.6 ± 0.57	90 ± 14.5	
Infected control	611 ± 34  	69.6 ± 12    	510 ± 29.1 	
Immunized infected control	420 ± 22.4 *	35.4 ± 9.77 **	625 ± 16.5*	
Treated groups				
PTX	# # #140 ± 51	#  28.8 ± 5.1	#733 ± 21.9	
PTX + PZQ	# 587 ± 94	40.2 ± 11.7	655 ± 40 	
Sily	# # 221 ± 59			# 25.5 ± 14.4	# 719 ± 46.5	
Sily + PZQ	# 613 ± 72.2	45.1 ± 14.2	685 ± 50.3	
PZQ	# 633 ± 53.4	39.8 ± 13.3	# 723 ± 45.5	
P<0.001  relative   to  normal   control
*** P < 0.001 ,  **  P < 0.01,  *  P <0.05   relative  to infected control.
# # # P<0.001,  # # P<0.01,  # P<0.05  compared to immunized infected control.

4- Discussion:
Schistosomal infection is cryptic as it frequently	 goes	 undetected		 and	developed significant 	  pathology	before 		chemotherapy administration	 (Wilson	  et 	al.,	   2006).	The combination of protection using SEA fractions and treatment was recommended in several studies as it provided many complementary goals, a reduction of egg- induced pathology, minimal parenchymal changes and the eradication of worms. Therefore, the assessment of the effect of treatment of immunized infected mice is important by studying several  criteria  related  to the parasitic  intensity, stages and distribution through the tissues of the host   for   the   evaluation   of  the   magnitude   of infection and efficacy of the treatment (Abdel- Ghaffar et al., 2005).
The   present   study   revealed    that 	the immunization  schedule used	did  not  cause any significant change in worm burden but slightl significant  reduction  in  tissue  egg  load  which agreed with Botros et al. (1996). The treatment of PZQ alone or combined with PTX or silymarin in immunized infected animals caused almost similar high percentage of eradication of worms and tissue egg  load  which  also  agree  with  the  work  of Suleiman et al. (2004). The death of the worms due to the treatment with antischistosomal drugs was attributed to metabolic disorders, mechanical destruction and muscular contraction of the treated worms (Doenhoff et al., 2002). At the same time, percent reduction in the egg count in both immunized infected and treated groups was found to be higher in the intestinal tissue than in hepatic tissue. This variation was attributed to excretion of some ova from the intestine prior to digestion and

to hepatic shift of worms after treatment (Abdel- Ghaffar and Qurtam, 2001, Abdel –Ghaffar, 2004). On the other hand, the treatment with PZQ alone or combined with PTX or silymarin caused decrease in immature egg stages and number of mature eggs with high increase in number of dead eggs which agree with the findings of Botros et al.( 1996). The parasitological improvement is due to antiparasitic drug (PZQ) which  causes  direct or indirect toxic effect 	in	combination	 with	 the 	effect 	of immunization with SEA which lead to reduction in tissue egg load. This may be attributed to marked decrease in the worm number or fecundity due to hindering  the  process  of  oviposition  (Guirguis,
2003).
The manifestations  of schistosomiasis  are mainly attributed to granulomatous inflammation around parasite eggs (Abath et al., 2006). The formation  of  granulomas depends predominantly on CD4+  T cell specific for egg antigen and represents a delayed – type hypersensitivity (Stadecker, et al., 2004 ; Pearce, 2005; Garcia et al., 2008). At the same time, hepatic stellate cells (HSCs) comprise 10-15% of all hepatic cells and they are recruited to areas of hepatic injury and become activated (Cassiman et al., 2002). They adopted a myofibroblast–like phenotype, secreting extracellular matrix components (Iredale, 2003; Mann et al., 2009).
In this work, although all treated groups revealed significant diminution of granuloma diameter, but the groups treated with PTX or silymarin alone or combined to PZQ revealed more diminution in granuloma diameter. This is because PTX reduces transdifferentiation of HSC to myofibroblasts and inhibits HSC proliferation that

leads to inhibition of extracellular matrix synthesis, beside  the  effect  of PZQ which  reduces  CD4 T cells and increase CD8 cells  (Raetsch et al., 2002; El-Ahwany et al., 2006; El-Lakkany and Nosseir,
2007). At the same time, the groups treated with silymarin or PTX alone revealed lower pattern than the other treated groups and this many be due to the effect of previous immunization of the infected animals before treatment. This effect is considered complementary to the immunization effect  which modulate the immune response by limiting the immunopathological reactions against schistosome eggs    trapped	in	the   liver.    In	this   study, immunization before infection increased the levels of  production  of  IgG1  and  IgG4  .		All  treated groups had increased levels of IgG2 , but slight increase in the level of  IgG4 was observed in the groups treated with PZQ alone or combined with PTX or silymarin. This increase in the production of immunoglobulins have an important role in the improvement of the pathology and the reduction in the ova count and worm burden (Soren et al., 2009; El-Ahwany et al., 2006).
Cytokines which act on lymphocytes are of special interest because of their role in regulating cells of the immune response (Kim et al., 1997). During schistosomal infection, both Th1 and Th2 responses directed against egg antigen and produce IFN-   , IL-4, IL-5 and IL-13 (Hoffman et al., 2002; Sadler et al.,2003; Stadecker et al., 2004). In this study, the diminished production of Th1-cytokine IFN-   and  Th2-cytokine  IL-4  in  the immunized group may be implicated in the down modulation of the granulomatous response due to immunization (Chensue et al., 1992). Groups treated with PTX or silymarin  alone  showed  significant  decrease  in IFN-   and IL-4 . On the other hand, groups treated with   PZQ   alone   or   combined   with   PTX   or silymarin showed increase in Treg cell cytokine IL-
10. Recent studies suggest that Treg cells play a pivotal role in suppressing Th1 cell development as well  as  limiting  the magnitude of Th2 response directed against egg antigen by a process dependent upon IL-10 (Hori et al., 2003; Wynn, 2004; Stadecker et al., 2004). The increasing level of IL-
10 is probably implicated in the down regulation of
granuloma formation as it reduces the intrahepatic inflammatory response and hence it has an antifibrotic effect (Nelson et al., 2003; Thompson et al., 1998). These results indicate the importance of   the   effect   of   PTX   as   it 	has   a   potent antifibrogenic role. Also using silymarin as an antioxidant drug can  inhibit HSC activation  and slow down the progression of liver fibrosis (Afdhal and Nunes, 2004;   Jhy-wen et al., 2009).   Recent studies recommended using PTX in early stage of

infection and in a long-term treatment. Also, it can be used as an adjuvant therapeutic tool when combined with PZQ in treatment of schistosomiasis (El-Lakkany and Nosseir, 2007). 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.

Acknowledgment
We acknowledge Prof. Dr. Zeinab Shaker professor
of immunology , TBRI, for her time spent in reviewing this work.

Corresponding auther
Dr. Ibrahim Rabia Bauiomy Aly
Associate professor
Immunoparasitology Dep.
Theodor Bilharz Research Institute, (TBRI) Giza, Egypt.
e- mail :- ibrahimshalash@yahoo.com

5- References
1-		Abath,   F.G.;   Morais,   C.N.;  Montenegro, C.E., Wynn, T.A. and Montenegro, S.M. (2006): Immunopathogenic mechanisms in schistosomasis: what can be learnt from human studies? Trends Parasitol., 22:  85-91.
2-  Abdel-Ghaffar, O. (2004): Assessment of the efficacy of Ro 16-2308 against the Egyptian strain of Schistosoma mansoni in mico: Parasitological, hematological and biochemical criteria. Egypt. J. Zool., 42: 173-203.
3- Abdel-Ghaffar, O. and Qurtam, A.A. (2001): Parastiological, hematological and biochemical assessment of the efficacy of Ro 15-5458 against the Egyptian strain of Schistosoma marsoni in mice. J. Egypt. Ger. Soc. Zool., 36;
571-603.
4-  Abdel-Ghaffar,  O.;  Rawi,  S.M.  and  Ishaq, A.I. (2005): Evaluation of the curative efficacy of	RO	15-8843	against	mansonian schistosomiasis in albino mice. J. Egypt. Ger. Soc. Zool. , 47 : 15-22.
5- Afdhal, N.H and Nunes, D. (2004): Evaluation of liver fibrosis: a concise review. Am. J. Gastroenterol. 99: 1160-1174.
6-		Bartley,   P.B.;  Ramm,   G.A;  Jones.  M.K.; Ruddel,   R.G.;  Li,  Y. and  McManus,   D.P. (2006):   A   contributory  role   for   activated hepatic   stellate   cells   in   the   dynamics   of

Schistosoma japonicum egg- induced fibrosis. Int. J. Parasitol., 36 (9): 993-1001.
7- Bergquist,   R.;  Utzinger,  J.  and  Mc  Manus, D.P. (2008): Trick or treat: the role of vaccines in integrated schistosomiasis control. PLoS. Negl. Trop. Dis., 2 : 244-252.
8-	Bienvenu,  J.;  Doche,  C.;  Gutouski,  M.C.; Lenoble,  M.;  Lepape,  A. and  Perdrix, J.P. (1995): 	Production	 of	proinflammatory cytokines and cytokines involved in the TH1/ TH2 balance is modulated   by pentoxifylline. J. Cardiovasc. Pharmacol., 25 : 580-84.
9- Boros, D.V. and Warren, K.S.(1970): Delayed hypersensitivity type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from schistosoma mansoni eggs. J. Exp. Med. 132: 488-507.
10- Botros, S.; Doughty, B.; Shaker,  Z.; Akl, M.; Sharmy,   R.;  Diab,  T.  and  Hassanein,   H. (1996):  Efficacy of antipathology vaccine in murine schistosomiasis administered with and without	 chemotherapy.	 Int.	J. Immunopharmacol., 12: 707-718.
11- Bradford, M.M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-
54.
12-  Carter, C.E.  and  Colley,  D.G.  (1978):  An elecrophoretic 	analysis	 of	Schistosoma mansoni soluble egg antigen. J. Parasitol., 64:
385-390.
13- Cassiman,   D.;  Lib  brecht,   L.;  Desnet,  V.; Denef, C. and  Roskams,  T. (2002): Hepatic Stelalte cell/myofibroblast subpopulations in fibrotic human and rat livers. J. Hepatol., 36 :
200-209.
14-Cheever,  A.W. (1968): Conditions affecting accuracy of KOH digestion techniques for counting S. mansoni eggs in tissues. Bull. WHO, 39: 329-331.
15-Chensue  S.W., Terebuch, P.D., Warmington, K.S., Hershey,  S.D.; Evanoff, H.L.; Kunkel, L. (1992): Role of IL-4 and IFN- γ in Schistosoma			 mansoni	  egg-induced hypersensitivity		   granuloma		formation. Orchestration,		relative 	contribution,	 and relationship	 to		macrophage	 function.	     J. Immunol, 148: 900-906.
16- Doenhoff, M.J. ; Kusel, J.R.; Coles, G.C. and Cloll,  D.  (2002):  Resistance  of Schistosoma mansoni to praziquantel: is there a problem? Trans R. Soc. Trop. Med. Hyg., 96: 465- 469.
17- Duvall, R.H. and  De Witt,  W.B. (1967): An improved  perfusion  technique for  recovering

adult  Schistosomes from  laboratory animals. Am. J. Trop. Med. Hyg. , 16: 483-486.
18-El-Ahwany,   E.G.;  Nosseir,  M.  M.  and  Ali.
I.R.  (2006): Immunonsdulation of pulmonary
and hepatic granulomatous response in mice immunized	 with	 purified	lung-stage schistosomulae antigen. J. Egypt . Soc. Parasitol., 36: 335-350.
19- El-Lakkany, N. and Nosseir, M. (2007): Pharcodynamics of pentoxifylline and /or praziquantel 	in	murine 	schistosomiasis mansoni. APMJS. 115: 184-194.
20- Engvall,  E. and Perlman, P. (1970): Enzyme linked	 immunosorbent 	assay	(ELISA). Quantitative  assay of  immunoglobulin  G.  J. Immunochem., 8: 871-874.
21-   Friedman   SL,   Arthur   MJD.    (2002)   : Reversing	 hepatic 	fibrosis.	Science 	and Medicine , 8 : 194 – 205.
22- Friedman SL (2003): Liver fibrosis –from bench to bedside. J. Hepatol. 38: 238 : 538-53.
23- Garcia,  T.C.;  Fonseca,  C.T.;  Pacifico, L.G., Duraes   Fdo,   V.;  Marinho,  F.A.;   Penido, M.L.;  Caliari,  M.V.;  de Melo, A.L.; Pinto, H.A., Barsante, M.M.;  Cunha-Neto, E. and Oliveia,  S.C.  (2008):  Peptides containing T cell epitopes drived from Sm 14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in nice. Acta. Trop. , 106  : 182-7.
24- Garcia,  T.C.;  Fonseca,  C.T.; Pacifico, L.G.; Duraes   Fdo,   V.;  Marinho,  F.A.;   Penido, M.L.,  Caliari,  M.V.,  de Melo, A.L.;  Pinto, H.A., Barsante, M.M.; Cunha  – Neto, E. and Oliveira,  S.C. (2008): Peptides containing T cell  epitopes,  derived  from  Sm  14,  but  not from   paramyosin,   induce   a   Th1   type   of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice. Acta. Trop. 106(3):
162-7.
25-		Guirguis,	 F.R. 	(2003):	Efficacy	 of praziquantel   and   Ro   15    -   5458,   a   q- acridanonchydrazone		derivative,	 against Schistosoma  haematobium.  Arzeim,  Forsch, Drug, Res., 53 (1): 57-61.
26-Hoffmann,  K.F.;   Wynn,   T.A.  and   Dunne, D.W. 	(2002): 	Cytokine 	mediated	 host responses	 during	schistosome	infections; walking the fine line between immunological control and immunopathology. Adv. Parasitol.
52: 265-307.
27-  Hori,   S.;   Nomura,    T.   and   Sakaguchi,S. (2003): 	Control 	of	regulatory	 T	cell

development by the transcription factor Foxp
3. Science 299: 1057-1061
28- Iredale, J. (2003): Cirhosis: new research providesa basis for rational and targeted treatments. Br. Med. J. 327 : 143-147.
29- Jhy-wen  wu;  Lie-chwen  Liu  and  Tung-Hu Tsai (2009) : Drug- drug interactions of silymarin	on	the 	perspective 	of pharmacokinetics. J. Ethnpharm. 185-193.
30- Kim, J.J., Aggvoo, V.; Bagarazzi,  M.L.; CHattergoon, M.A.; Dang, K.; Wang, B.; Boyer,  J.  D and  Weiner,  D.B. (1997):   In vivo   Engineering   of   a   cellular   immune response	 by	co-administration	 of	IL-12 expression vector with a DNA immunogn. J. Immunol.  158: 816-826.
31- Lieber,  C.S.; Leo, M.A. and Cao, Q. (2003): Silymarin retards the progression of alcohol- induced in baboons. J. Clin Gastroenterol., 37:
336-339.
32- Maher,  K.M.; El- Shennawy, A.M.; Ibrahim, R.B.; Aly, E. and Mahmoud, S. (2003) : Protective and antipathology potential of immunization with S. mansoni Schistosomula- secretory / excretory product.  New Egypt. J. Med. 28 : 173- 182.
33- Mahmoud, M.A.; El-Shafei,  M.A.; Mtta,  R. and  Hany,  A.A. (2002):  Parasitological and immunological changes in murine hepatic schistosomiasis before and after praziquantel treatment J. Egypt. Soc. Parasitol., 32: 551 –
560.
34-		Mann, 	J. 	and 	Mann,	D.A.	(2009): Transcriptional  regulation  of hepatic  stellate cells. Advanced drug delivery reviews 61: 497-
512.
35- Nelson, D.R. ; Tu, 2.; and soldevila-Pico,  C., (2003): long-term interleulin 10 therapy in chronic hepatitis C patients has a proviral and anti-inflammatory effect. Hepatology, 38: 859-
868.
36- Nessim, N.G.; Hassan,  S.I.; William , S. and El-Baz,   H.   (2000):   Effect   of   the   broad spectrum anthelmintic drug flubendazole upon Schistosoma mansoni experimentally infected mice. Arzneimitted-Forrschung/Drug Res.  50:
1129-1133.
37- Norden, A.P. and Strand, M. (1984): Schistosoma mansoni, S. haematobium and S. Japonicum:   identification	 of   genus-   and species-specific antigenic egg glycoproteins. Exp Paasitol, 58: 333-44.
38- Pearce  , E.  (2005):  Priming of the immune
response	 by	schistosome	egg.	 Parasite
Immunol. ,27 : 265-270.

39-Pellegrino, J.; Oliveria, C.A.; Faria, J. and Cunha,  A.S. (1962): New approach to the screening	 of	drugs	in	experimental schistosoma  mansoni  in  mice. Am. J. Trop. Med. Hyg., 11: 201-215.
40- Raetsch,  C.; Jia, J.D.; Boigk, G.; Bauer,  M.; Hahn,  E.G.;  Riecken,  E.O.  and  Schuppan, D.	(2002): 	Pentoxifylline 	downregulates profibrogenic cytokines and procollagen I expression  in  rat  secondary  biliary  fibrosis. Gut. 50: 241-247.
41- Raetsch,  C.; Jia, J.D.; Boigk, G.; Bauer,  M.; Hahn,  E.G.;  Riecken,  E.O.  and  Schuppan, D.	(2002): 	Pentoxifylline 	downregulates profibrogenic cytokines and procollagen I expression  in  rat  secondary  biliary  fibrosis Gut. 50: 241-247.
42-  Reis,   L.F.,   Ventura,  T.G.;   Souza,   S.O.; Arana-Pino,	 A.; 	Pelajo-Machado,	 M.; Pereira, M. J.; Lenzi, H.L.; Conceicao, M.J. and Takiya, C.M. (2001): Quantitave and qualitative interferences of pentoxifllyne on hepatic Schistosoma mansoni granulomas: effects on extracellular matrix and eosinophil population. Mem Inst oswaldo Cruz; Rio de Janeiro, 96 : 107-112.
41- Romeih, M.H.; Hassan , H.M.; Shousha, T.S. and Saber, M.A.(2008): Immunization against Egyptian Schistosoma mansoni infection by multivalent DNA vaccine: Acta. Biochim. Biophys. Sin. (Shanghai). 40 : 327-38.
42- Sadler,  C.H.; Rutitzky,  L.I.; Stadecker, M.J. and  Wilson, R.A. (2003): IL-10 is crucial for the  transition  from  acute to chronic  disease state 	during	infection	 of	mice 	with Schistosoma  mansoni.  Eur.  J.  Immunol.  33:
880-888.
43-  Silva,  L.M.;  Menezes,  R.M.;  De  Oliveira, S.A.	and 	Andrade, 	Z.A 	(2003): Chemotherapeutic effects on  larval  stages of Schistosoma mansoni during infection and reinfection  of  mice.  Rev.  Soc.  Bras.  Med. Trop. 36: 335-341.
44- Stadecker, M.J.; Ashai, H.; Finger, E.; Hernandez,   H.J.;    Rutitzky    and   Sun.   J. (2004): 	The 	immunobiology	of	Th1 polarizaiton in high-pathology Schistosoniasis. Immunol. Rev., 201 : 168-179.
45- Soren,  K.;  Monard, J.;  Johnsen,  M.V. and Lindberg, R. (2009): Persistent immune responses in late infection and after treatment in experimental Schistosoma bovis infections in goats. Res Vet Sci.  86 : 472-8.
46- Suleiman,  M.I.;  Akarim, E.I.;  Ibrahi, K.E., Saad,   A.M.;   Mohammed,   A.E.;   Ahmed, B.M.	 and 	Sulaiman, 	S.M.	 (2004):

Antischistosomal effects of praziquantel, its alkaline hydrolysis and sun decomposed products	on	experimentally	 S.	mansoni infected albino mice. (A) Efficacy assesament based   on   clinicopathological   findings.   J. Egypt. Soc. Parasitol. 34 : 131- 42.
47-  Tendler,   M.;   Brito,   C.A.;   Vilar,   M.M.; Serra-Freire,  N.'   Diago,   C.M.;   Almedin, M.S.;  Delbem,  A.C.;  da Silva, J.F.;  Savino, W.;  Garratt, R.C.;  Katz,  N. and  Simpson, A.S.  (1996):  A  Schistosoma  mansoni  fatty acid- binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine. Proc. Nath. Acad. Sci. USA 93: 269-273.
48-Thompson, K.; Maltby,  J. and Fallowfied,  J. (1998): Interleukin-10 expression and function in experimental murine livr inflammation and fibrosis. Hepatology, 28: 1597- 1606.
49-		Utzinger,	 J. 	and 	Keiser,	J. 	(2004): Schistosomiasis		and	soil-transmitted helminthiasis: common drugs for treatment and control. Expert. Opin. Pharmacother., 5 : 263-
285.
50-  Von  Lichtenberg  (1962):  Host  response  to eggs of S. manosni  I. Granuloma formation in the unsensitized laboratory mouse. Am .J. Pathol., 41: 711-31.
51-  Wynn,   T.A.;  Thompson,   R.W.;   Cheever, A.W. and Mentink Kane, M.M. (2004): Immunopathogenesis	 of	schistosomiasis. Immunol. Rev. 156-167.
52-Zouain,  C.S.; Gustavson,  S.; Oliveira,  S.C.; A zevedo,V.   Alves,   J.B.   and    Goes,   A.M. (2001):  The  role  of  IL-10  and  IgG1 in  the protection and granulomatous response in Schistosoma mansoni P24-immunized mice. Vaccine. 19: 1218-1224.

3/25/2010