The effect of medium chain saturated fatty acid ( monolaurin ) on levels of the cytokines on experimental animal in Entamoeba histolytica and Giardia lamblia infection

The aim of this study was to demonstrate the effect of medium chain saturated fatty acid (monolaurin) on experimentally infected Giardia lamblia and Entamoeba histolytica, and measurement of IFN-γ, TNFα, IL-4, IL-10, TGF-β levels of cytokines. To study the effect of monolaurin on the duodenal mucosa of the studied infected hamsters, a group of sixty golden Syrian hamsters were used, which were further divided into two subgroups: Subgroup I, in which hamsters were infected by oral administration of 10,000 G. lamblia cysts and Subgroup II, in which hamsters were infected by 10,000 E. histotytica cysts. Each subgroup was divided into (6) groups. Subgroup I included the group from (1 to 6), although subgroup II included from 7 to 12 groups. In G. lamblia infected subgroup I, best results were observed by the reduction in both vegetative and cystic forms, respectively shown in group (6) treated with combination of metronidazole and monolaurin post infection 94.68 and 96.55%, respectively. In the Subgroup II infected with E. histolytica, the high reduction in trophozoite and cystic forms in intestinal contents were in the group (12) which was treated with a combination of metronidazole and monolaurin post infection (90.12 and 92.56%, respectively). Cytokines levels IFN-γ, TNF-α, IL-4, IL-10 and TGF-β were measured in serum using sandwich enzyme-linked immunosorbent assay (ELISA). The best result was shown in the group (6) treated with a combination of metronidazole and mololaurin post infection 130,129, 35, 165 and 240 Pg/ml. Also histopathological examination gave the best healing in the groups (6) infected with G. lamblia than thos infected by E. histolytica.


INTRODUCTION
Entamoeba histolytica (E.histolytica) and Giardia lamblia (G.lamblia) are common causes of diarrhea and malabsorption in humans (Rauch et al., 1990).The infection may produce severe acute diarrhea in children less than five years of age with chronic infections resulting in weight loss and growth retardation (Fraser, 1994;Fraser et al., 1997;Newman et al., 2001).A parasitological assessment of drinking water in Egypt demonstrated a high prevalence of G. lamblia, an intestinal parasite of humans and various animals (Stauffer et al., 2006).E. histolytica is a protozoan causing amebic dysentery.Invasive amoebiasis is manifested by amebic colitis which *Corresponding author.E-mail: zein201244@yahoo.comcan be complicated by intestinal perforation and peritonitis or hepatic abscess which may be fatal (Petri et al., 1987).There are little data on the true prevalence and incidence of E. histolytica infection in Africa; however, Egypt has high rates of asymptomatic infection as detected by the stool examination (Stauffer et al., 2006).
Therapeutic strategy has included diverse pharmaceutical agents of traditional use such as metronidazole, quinacrine and furazolidone (Gardner and Hill, 2001;Harris et al., 2001).However, evidence points to an increasing frequency of cases refractory to treatment with these drugs (Mendelson, 1980;Brasseur and Favennec, 1995;Sarker et al., 2010).Of these, metronidazole and albendazole may be the most representatives of anti-E.histolytica and G. lamblia of traditionally and recently used.Lemee et al. (2000) pointed to an increased frequency of cases refractory to treatment with these drugs and emergence of the drug-resistance species.
Metronidazole has proved to acquire potential carcinogenicity and mutagenic effect in rats (Hill, 2000).
Cytokines may have a role in G. lamblia and E. histolytica infection.Cytokines or Interleukins are secreted by lymphocytes, monocytes or macrophages.They act on other cells of the immune system to regulate their function.Interleukins cause inflammatory response in parasitic diseases.(Beutler and Cerami, 1989).Tumor necrosis factors (TNF), a mediator of inflammatory response also plays an important role in parasitic diseases (Beutler and Cerami, 1986).Furthermore, this cytokine promotes the proliferation and recruitment of monocytes and neutrophils to inflammatory sites being the dominant cytokine for E. histolytica and G. lamblia parasite (Singer and Solaymani, 2010).Singer and Nash (2000) illustrated the importance of Tcells in the control of giardiasis.Neither Th1 nor Th2 cells were absolutely necessary for the clearance of Giardia infection.This suggests that in the absence of Th1 cells, Th2 cells are sufficient for clearance of the parasite, or that in the absence of Th2 cells, Th1 cells are sufficient.Alternatively, Th3 cells (mucosal T cells) may play the major role in E. histolytica and G. lamblia infection.However, in interferon-gamma deficient animals, parasite clearance was delayed when compared to controls, suggesting the Th1 response may be more substantial in controlling Giardia infections.T-cell cytokines may also induce the production and release of antigiardial defensins into the intestinal lumen (Singer and Nash, 2000).
The drugs of choice in amebiasis are nitroimidazoles (Freeman et al., 1997;Iamp et al., 1999).It is effective and available at low cost.Trophozites of E. histolytica are able to adapt to therapeutically relevant levels of the drug (Samarawickerma et al., 1997;Wassmann et al., 1999).Lauric acid is a naturally derived fatty acid belonging to the medium chain saturated fatty acids (MCSFAs) which is suggested to have antimicrobial and antiparasitic properties.MCSFAs are natural fats that are easily digested, quickly absorbed and readily utilized for energy production Fahmy et al. 107 in mammals.Due to their relative ease of absorption by the body, these compounds are ideal for individuals with digestive tract problems such as diarrhea and malabsorption of long chain fatty acids containing foods (Petschow et al., 1998).Lauric acid (dodecanoic acid, C: 12) is a medium chain saturated fatty acid which is reported to have anti-giardial effect with lethal dose 50 (LD 50 ) concentration comparable to that of metronidazole which is the drug of choice in treatment of giardiasis.Dodecanoic acid appears to induce in vitro trophozoite death by accumulating within the parasite cytoplasm resulting in rupture of cell membrane (Rayan et al., 2005).Lauric acid is transformed into a substance called "monolaurin" in the human body.Monolaurin is a glyceride ester derivative of lauric acid (Hegde, 2006).Also, it was reported that monolaurin can destroy various pathogenic bacteria and protozoa such as G. lamblia.This is why lauric acid is the fundamental building block of the most effective anti-pathogenic of all the medium chain saturated fatty acids (MCSFAs) (Rayan et al., 2005).
The present study was carried out to evaluate lauric acid, as a treatment of plant origin on G. lamblia or E. histolytica infection.This work also studies the histopathological changes occurring in the small intestine of infected animals and following treatment.

MATERIALS AND METHODS
G. lamblia and E. histolytica cysts were obtained from diarrheic patients attending parasitology laboratory in outpatient clinic of Theodore Bilharz Reasearch Institute (TBRI).Each hamster was infected orally by 10,000 of either G. lamblia or E. histolytica cysts.The excreta of hamsters were examined daily to evaluate the time of maximal cyst excretion.A group of sixty golden Syrian hamsters were used, which were further subdivided into two subgroups: Subgroup I: in which hamsters were infected by oral administration of 10,000 G. lamblia cysts and Subgroup II: in which hamsters were infected by 10,000 E. histotytica cysts through an esophageal tube.Each subgroup divided into (6) group: Subgroup I included the group from (1 to 6) although subgroup II included 7 to 12 groups.Group (1) constituted infected animals added as control (5 animals), group (2) infected with Giardia cysts (5 animals) and treated with metronidazole in a dose of 120 μg/kg receiving twice daily for 5 successive days.Group (3) infected with Giardia cysts (5 animals) received dose of 500 mg/kg monolaurin for 7 consecutive days pre infection, group (4) infected with Giardia cysts (5 animals) received 500 mg/kg monolaurin for 7 consecutive days post infection.Group (5) infected with Giardia cysts (5 animals) received dose (2/3) 300 mg/kg monolaurin for 7 days consecutive days pre infection, then treated with metronidazole 1/3 dose twice daily for 5 successive days.Group (6) infected with Giardia cysts (5 animals) received a dose of 300 mg/kg monolaurin for 7 consecutive days post infection in a combination treated with metronidazole 1/3 dose twice daily for 5 successive days.Group (7) infected with E. histolytica cysts.Group (8) infected with E. histolytica cysts and treated with metronidazole120 μg/kg twice daily for 5 successive days.Group (9) infected with E. histolytica cysts (5 animals) and treated dose 500 mg/kg monolaurin for 7 consecutive days pre infection.Group (10) infected with E. histolytica cysts (5 animals) three consecutive days post infection.Group (11) infected with E. histolytica cysts (5 animals) received a dose of 300 mg/kg monolaurin for 7 consecutive days pre infection, then treated with metronidazole 1/3 dose twice daily for 5 successive days and group (12) infected with E. histolytica cysts (5 animals) received a dose of 300 mg/kg monolaurin for 7 consecutive days post infection in combination with metronidazole 1/3 dose twice daily for 5 successive days.The number of trophozoites were investigated in the duodenal part of intestine of hamsters.

Drugs
*Metronidazole (flagyle) was supplied by Rhone Opulence Rorer Company, as suspension.The dose given to each hamster was 120 μg/kg twice daily for 5 successive days.The dose for hamsters was calculated according to the chart for drug doses in experimental animals Paget and Barnes (1964).*Monolaurin (lauric acid) was supplied by Manufactured for Ecological formulas CONCORD, CA 9456 (Rayan et al., 2005).The dose was 500 mg/kg given for 7days, pre and post infection.

Parasitological study
In the group infected with G. lamblia and E. histolytica one week following infection, stool analysis was performed for all (12) groups to verify infection.Analysis was repeated every other day till end of second week post-infection.Treatment to all groups were given one week following infection.One week later stool analysis was done by direct examination of fresh stool for trophozoites and merthiolate iodine formaldhyde concentration (MIFC) technique (Blagg et al., 1955).Hamsters infected with G. lamblia and E. histolytica were sacrificed ten days after treatment and small bowel was removed and the duodenal contents were analyzed and the number of vegetative forms was counted.In hamsters infected with E. histolytica, large intestine was excised and colonic contents were examined.In the group treated with monolaurin pre-infection, hamsters were administered monolaurin daily for one week pre infection and the hamsters were sacrificed after three weeks post infection.

Histopathological examination
After sacrifice of the animals, part of the small intestine were fixed by formaline then pieces of tissues were processed for paraffin embedding stained with hematoxylin-eosin and masson trichrome stain (Bancroft and Stevens, 1975).

Serum cytokine and chemokine measurement
Levels of the cytokines IFN-γ, TNF-α, IL-4, IL-10, TGF-β (R & D systems Inc., Minneapolis) were measured in serum using sandwich enzyme-linked immunosorbent assay (ELISA) (eBioscience).The results were expressed as pg/ml, based on standard curves (Baqai, 1996).In brief, ELISA plates were coated with 50 µl (Iµg/ml) of capture antibody (IFN-γ, TNF-α, IL-4, IL-10, TGF-β) (Beckton Dickenson & Co.) and allowed to incubate at 4°C overnight.Plates were washed six times with Phosphate buffered saline (PBS)/Tween 20.Excess protein binding sites were blocked with 200 µl of skimmed milk.The wash step was repeated and 50 µl of serum samples were added and incubated 1 h in water bath at 37°C.The washing step was repeated and the biotin labeled anti-(IFN-γ, TNF-α, IL-4, IL-10, TGF-β) monoclonal detector antibody (lµg/ml) (Beckton & Dickenson & Co) was added to each well and the plates incubated at room temperature for an hour.After a further washing step 100 µl of avidin-alkaline phosphatase was added to each well and the plates incubated at room temperature for 30 min.The washing step was repeated and 100 µl of pnitrophenyl phosphate (pNpp) (Sigma Aldnch) (lmg/ml) in 0.2 M tris buffer was added to each well to detect bound antibody.The reaction was visualized by the addition of 100 µl/well of p-nitrophenyl phosphate (pNpp) (Sigma) substrate solution for 30 min in the dark at room temperature.The reaction was stopped by adding 50 µl/well of 8 N H2SO4 and plates were read at 405 nm using ELISA microplate reader (Bio Rad).

Ethical considerations
The experimental animal studies were conducted in accordance with international valid guidelines and they were maintained under convenient conditions at the Schistosom Biological Supply Progrm (SBSP) animal house of Theodor Bilharz Research Institute (TBRI), Cairo, Egypt.

Statistical analysis
The statistical package for social sciences (SPSS) for Windows (version 11) computer program was used for statistical analysis.Means of different groups were compared using unpaired 2-tailed students t-test.Data were considered significant if "p" values were less than 0.05.

Parasitological parameters
Table 1 showed that the reduction in trophozoite forms of G. lamblia in intestinal contents was 67.0% in group (3) treated with monolaurin pre-infection and 87.34% in the group (4) treated with monolaurin post-infection compared to infected control group (1).The reduction in the cysts of G. lamblia treated with monolaurin pre and post infection (group 3 and 4) was 73.96 and 91.15%, respectively (Table 2).Tables 1 and 2 showed that there was a reduction in trophozoite and cystic forms of G. lamblia in intestine (92.15 and 93.23, respectively), when treated with metronidazole (group 2).The best results in a percentage reduction rate in both vegetative and cystic forms, respectively was shown in the group (6) treated with a combination of metronidazole and monolaurin post infection 94.68 and 96.55%, respectively (Tables 1 and  2).In Tables 3 and 4, in the group infected with E. histolytica, the high reduction in trophozoite and cystic forms in intestinal contents were in the in group ( 12) treated with a combination of metronidazole, and monolaurin post infection were 93.08 and 92.56%, respectively.The reduction in trophozoite forms of E. histolytic in the groups (9 and 10) treated with monolaurin pre and post infection were 75.32 and 88.15%, respectively (Table 3).The difference for both treated groups was statistically significant from respective untreated control hamsters at (P < 0.001).The reduction in number of the cysts of E. histolytic pre and post infection (group 9 and 10) were 76.66 and 89.59%, respectively (Table 4).When treated with metronidazole in group (8) there were reduction in trophozoite and cysts forms of E. histolytic in intestinal contents were 89.14 and 90.03%, respectively (Tables 3 and 4).

Serum cytokine and chemokine measurement
Levels of the cytokines IFN-γ, TNF-α, IL-4, IL-10, TGF-β (R&D systems Inc., Minneapolis) were measured in serum using sandwich ELISA.There is a significant reduction observed in all groups, the greatest reduction in different cytokines was observed in group 3 and 6 while there is no significant reduction in the level of TGF-β (Table 5).The Levels of the cytokines IFN-, TNF-α, IL-4, IL-10, TGF-β were measured in serum using sandwich ELISA.
In sub-groups infected with E. histolytica.There is a significant reduction observed in all groups, the greatest reduction in different cytokines was observed in group 9 and 12 while there is no significant reduction in the level of TGF-β (Table 6).

Histopathological examination
In the control infected non treated hamsters, atrophic degeneration of the intestinal villi was observed (Figure 1).Again, the group given monolaurin post-infection revealed partial villi atrophy (Figure 2), but exhibited complete healing in the group treated post-infection (Figure 3 and 4).While the group given combination of metronidazole and monolaurin post-infection with E. histolytic showed degeneration and healing in intestinal villi (Figure 5).

DISCUSSION
Millions of people are annually infected with E. histolytica and G. lamblia, making the diseases a major cause of morbidity worldwide (Teles et al., 2011).Metronidazole is known to be the drug of choice for treatment of trichomoniasis and giardiasis.However, some adverse reaction appears to be related to the high dosage and duration of treatment, resistance to the drug had led to the search for other suitable treatment (Martinez and Caumes, 2000).In human giardiasis, therapeutic failure that was recorded recently is up to 5 to 20% of cases (Fallah et al., 2007) occurs due to low compliance of drug therapy, frequent re-infection or emergence of parasite resistance to metronidazole and/or The Levels of the cytokines IFN-, TNF-α, IL-4, IL-10, TGF-β were measured in serum using sandwich ELISA.nitromidazole (Lemee et al., 2000).In this study regarding the effect of metronidazole on G. lamblia infection, it was found that there was a high significant difference between control and all treated G. lamblia infected groups.Metronidazole administration resulted in a percentage reduction rate of 92.15 and 93.23% in both vegetative and cystic forms, respectively (Tables 1 and 2) group (2), compared to 67 and 73.96% when monolaurin was given preinfection in group (3).The best results were in a percentage reduction rate in both vegetative and cystic forms obtained in group (6) which was treated with a combination of metronidazole and monolaurin post infection 94.68 and 96.55%, respectively.These results are in agreement with Amer et al. (2007) who made a trial to increase the effectiveness of metronidazole on G. lamblia.The study revealed a highest cure rate in vegetative forms (99.32%) and cystic forms (98.6%) in hamsters infected with Giardia and treated by a combination of metronidazole and lactospore.The authors added that, when metronidazole or lactospore were given separately, the reduction rates of trophozoites  were 92.22 and 63.4%, respectively and of cystic forms were 93.8 and 79.5%, respectively.Another study reported 96.4 to 99.14% cure from Giardia cysts and 86.5 to 78.1% reduction in the vegetative forms in hamsters treated with metronidazole.Moreover, it was found that a cure rate over 90% in cystic forms Giardia when treated with metronidazole in other reports (Fawzy et al., 2003).
Monolaurin, the monoglyceride of lauric acid is the most powerful antiviral, antibacterial and antifungal fatty acid found in coconut oil.It has the greatest overall antimicrobial effect.It also had antiprasitic effect on blastocysts in vitro (Hassan et al., 2010).Monolaurin which is a natural compound derived from coconut was evaluated for its antigiardial effects on giardia as stated by Rayan et al. (2005).
In the present study in subgroup infected by G. lamblia, the best results in a percentage reduction rate in both vegetative and cystic forms was respectively observed in group (6) which was treated with combination of metronidazole and monolaurin post infection 94.68 and 96.55%, respectively.In the group (4) receiving monolaurin post infection, the number of trophozoites and cysts forms of G. lamblia in intestinal contents were 87.34 and 91.15%, respectively compared to infected control group.This agree with Helmy (2010) who evaluated the effect of monolaurin, against G. lamblia in infected hamsters which the highest percentages of reduction in the number of Giardia cysts and trophozoites were in the group that received combined treatment (98.83 to 96.95%) followed by the group that received the metronidazole as treatment (93.77 to 95.5%) and the lowest percentage of reduction in the group that received the lauric acid as treatment (82.03 to 78.76%).
Similarly, in study of Hassan et al. (2010) reported the administration of monolaurin (lauric acid) was effective in the group infected with blastocystis.Dodecanoic acid (monolaurin ML) at 500 and 700 µg/ml induced highly significant reduction of concentration of blastocystis cells in culture after 2 h incubation (p < 0.01).Higher concentration (1000 µg/ml) caused rapid death of the parasite with viable cells were detected after 30 min incubation.These results agree with those found by Rayan et al. (2005) who confirmed that the dodecanoic acid has an anti-giardia effect with an LD 50 concentration comparable to that of metronidazole.
Our result agrees with Fahmy et al. (2008) whose results showed that combined treatment of metronidazole together with arthemer and rosemary gave best results in highest percent reduction of cyst count in stool analysis and vegetative forms in small intestine caused by G. lamblia.Mixed treatment of metronidazole plus artemisia and rosemary showed complete regeneration of intestinal cells and sings of recovery was noticed.El-Shennawy et al. (2009) studied the effect of pomegranate on the intestinal G. lamblia and concluded that the highest trophozoite reduction 98.7% was obtained in the group receiving metronidazole with pomegtanate (leaves).By histology, healing of mucosal ulcerations, preserved villi and reduced chronic inflammatory infiltrate of the lamina propria were detected with combined therapy.
Role of cytokines in G. lamblia infection is not clear.G. lamblia normally does not penetrate the epithelial barrier, therefore the spontaneous elimination of the parasite depends largely on immune mechanism.Cytokines or other inflammatory mediators may play a role in G. lamblia infection.IL4 appears to have some relationship in patients with G. lamblia infection as reported previously (Baqai, 1996).
Chronic diarrhoea and malabsorption produces mucosal inflammation associated with T cell activation and cytokine release (Farthing, 1993).Cytokines were not altered after infection of colonic cell with G. lamblia (Jung et al., 1995).As G. lamblia is a non invasive parasite, TNF alpha did not appear to have any role in giardiasis.This is in contrast with patients suffering from amoebiasis where TNF alpha was found because E. histolytica is an invasive parasite (Wang et al., 1992).TNF appears to act synergistically with other cytokines (Neta et al., 1988) and may be of therapeutic benefit in G. lamblia infection (Belosevic and Daniels, 1992).
In this study, the Levels of the cytokines IFN-γ, TNF-α, IL-4, IL-10, TGF-β were measured in serum using sandwich ELISA.The best result was shown in group (6) which was treated with a combination of metronidazole and mololaurin post infection 130,129, 35, 165 and 240 Pg/ml in the subgroup I, infected with G. lamblia.In the present study, there is no significant reduction observed in all groups, the greatest reduction in different cytokines was observed in group 3, 6, 9 and 12, while there is no significant reduction in the level of TGF-β.
Our results agreed with Huma and Rakhshanda (2000) whose results indicate that IL-4 being an inflammatory regulator appears to have some relationship with it, probably because G. lamblia is a non invasive parasite giardiasis, while TNF alpha was not detected in patients.In our study, histopathological examination revealed complete healing of intestinal mucosa and regeneration and healing in intestinal villi after treatment with combination of metronidazole and monolaurin postinfection in group infected with G. lamblia, while partial healing of the lining epithelium of the intestine was noticed after treatment with monolaurin post infection or metronidazole treatment.This agrees with Helmy (2010) who showed that histopathological examination and electron microscopic examination revealed complete healing of intestinal mucosa after the combined treatment, while partial healing of the lining epithelium of the intestine was noticed after metronidazole or lauric acid treatment.Amoebiasis is a significant cause of morbidity worldwide and is the third leading cause of death from parasitic diseases.Although, metronidazole is the drug of choice for treatment of amoebiasis and has been used in clinical practice for many years, inappropriate usage could lead to drug resistance (Bansal et al., 2006).Drug resistance in E. histolytica is uncommon but differences in drug susceptibility between different isolates have been reported and resistance of metronidazole may be develop in future (Bansal et al., 2006).
In subgroup II, infected with E. histolytica, results showed that the high reduction in trophozoite and cystic forms in intestinal contents in group (12) treated with combination of metronidazole and monolaurin post infection were 93.08 and 92.56%, respectively.Then, the group (10) treated with monolaurin post-infection gave the best result in percent of reduction of cyst count in stool analysis and vegetative form 88.15 and 89.59% when infected with E. histolytica.
In our study, in the reduction in cytokines when infected with E. histolytica, the best result was observed in group (12) which was treated with combination of metronidazole and mololaurin post infection; the level of different cytokines was 150,135, 30, 150 and 234 Pg/ml (IFN-γ, IL-4, IL-6, IL-10).In the group (8) which treated with metronidazole, the reduction in cytokines levels show in group (2) which was 170,150,43.1,.
Histopathological examination revealed complete healing in the group treated with combination of metronidazole and monolaurin post-infection.While the Fahmy et al. 113 group given the treatment post-infection with E. histolytic showed degeneration and healing in intestinal villi.Our work showed promising results with monolaurin, which can be used as a complementary food product in combination with metronidazole in treatment for G. lamblia or E. histolytica infection.

Conclusion
In G. lamblia infected subgroup I, best results were observed by the reduction in both vegetative and cystic forms, respectively shown in group (6) treated with combination of metronidazole and monolaurin post infection 94.68 and 96.55%, respectively.In the subgroup II infected with E. histolytica, the high reduction in trophozoite and cystic forms in intestinal contents were in group ( 12) which was treated with a combination of metronidazole and monolaurin post infection (90.12 and 92.56%, respectively).Cytokines levels IFN-γ, TNF-α, IL-4, IL-10, TGF-β were measured in serum using sandwich ELISA.The best result was shown in the group (6) treated with a combination of metronidazole and monolaurin post infection 130,129, 35, 165 and 240 Pg/ml.Also histopathological examination gave best healing in groups ( 6) infected with G. lamblia than those infected by E. histolytica.

Figure 5 .
Figure 5. Treated by combination of metronidazole and monolaurin for week post infection with E. histotytica showing complet healing in intestine.

Table 1 .
Effect of monolaurin on vegetative forms (trophozoite) in the small intestine infected with Giardia lamblia.
Date were as mean ± SE (mean ± standard devination).*Significant difference compared to the infected control group (P > 0.001).

Table 2 .
Effect of monolaurin on the number of cysts excreted in stool infected with Giardia lamblia.

Table 3 .
Effect of monolaurin on vegetative forms (Trophozoite) in the small intestine infected with E. histolytic.

Table 4 .
Effect of monolaurin on the number of cysts excreted in stool infected with E. histolytic.

Table 5 .
Effect of monolaurin on cytokines infected with Giardia lamblia.

Table 6 .
Effect of monolaurin on cytokines infection with E. histolytica.