Immune Response on Mice Infected with Schistosoma mansoni and Treated with Myrrh

MATERIALS AND METHODS

Infected animals: Swiss albino infected (100 cercariae of S. mansoni) mice were purchased from Theodore Bilhars institute, Cairo Egypt.

Mice treatment with praziquantel and myrrh: Praziquantel was suspended in dist. water then mice were treated orally using stomach tub with 250 mg kg^-1 body weight for 3 alternative days starting from the 42th day of infection.

Myrrh (commercially, Merazide, Pharco Co. Alex. Egypt) were suspended in corn oil and mice were treated for 3 successive days with a dose of 10 mg kg^-1 body weight using stomach tub after 6 week of infection. After 3 weeks of the last dose of treatment, mice from all groups were sacrificed and blood samples were collected.

Determination of antischistosomal antibodies: Polystyrene flat bottom microtiter plate was coated with 50 μL mL^-1 of 25 μg mL^-1 from schistosome antigenic preparation (SWAP) at room temperature over night. After blocking, pooled sera from treated and non treated mice diluted in PBS-T20 were added (50 μL mL^-1) and incubated at 37°C for 2 h. After washing with PBS-T20, 50 μL mL^-1 of goat anti-mouse IgG Alkaline phosphatase conjugate (Sigma) diluted 1:500 in 0.2% (w/v) non-fat dry milk in PBS-T20 was added and incubated at 37°C for 1 h. After washing, 1 mg mL^-1 of P-nitrophenyl phosphate (Sigma) was used as a substrate and the absorbance was read at 405 nm using micro plate auto reader (Bio-Tek Instruments).

Cytokine assay: A polystyrene flat bottom microtiter plate was coated with 50 μl mL^-1 of treated and non treated sera diluted (1:100) in carbonate/bicarbonate buffer, (pH 9.6) overnight. After blocking, rat anti-mouse IFN - γ mAb and goat anti-mouse IL-2 mAb (Sigma) diluted 1:1000 in PBS, PH 7.2 were added at 37°C for 1 h. After washing, 50 μL mL^-1 of goat anti-rat IgG alkaline phosphatase conjugate (for IFN-γ) and rabbit anti goat IgG alkaline phosphatase conjugate for IL-2 diluted (Sigma) 1:600 in PBS-T 20 were added at room temperature for 2 h. One milligram per milliliter of P-nitro phenyle phosphate was used as Substrate and the absorbance was read at 405 nm using microtiter plate auto reader (Bio-Tek Instruments).

Statistical analysis: Student t-test was performed using the statistical program package, instate software, version 2.03 (Graphpad, USA) and on IBM PCIAT compatible computer. The degree of significance, p<0.05 significant, p<0.01 highly significant and p<0.001 extremely significant.

RESULTS

Serum levels of anti-schistosomal antibodies: From Table 1, the level of anti-schistosomal antibodies was decreased significantly (p<0.0001) in mice infected with 100 cercariae of S. mansoni and treated with PZQ as well as mice infected and treated with myrrh (p<0.0001).

Table 1:	Antischistoaomal antibody titer in mice infected with S. mansoni and treated with myrrh and praziquantel compared with the infected non-treated control group
p, probability; t, student t-test

Table 2:	Serum level of gamma interferon and interleukin-2 in mice infected with S. mansoni and treated with myrrh and praziquantel compared with the infected non-treated control group
p, probability; t, student t-test

This result this result was confirmed by the result that, there no difference between PZQ and myrrh (p<0.05). The serum level of anti-schistosomal antibodies was decreased also after combined treatment with PZQ and myrrh (p<0.05).

Serum levels of IFN-γ: Table 2 shows that, the level of IFN-γ was decreased nonsignificantly after treatment with myrrh while decreased significantly after combined treatment with PZQ and myrrh (p = 0.05). No difference in the mean level was observed after treatment with PZQ (p>0.05).

Serum levels of IL-2: Unlike IFN-γ, the level of IL-2 was decreased in mice infected with S. mansoni and treated with PZQ (p<0.05) and myrrh (p<0.01) and combined treatment with PZQ and myrrh (p = 0.05).

DISCUSSION

Schistosomiasis is a widespread helminthic disease and infects one in 30 people (Elliott, 1996). A significant advance in the control of schistosomiasis chemotherapy was the introduction of praziquantel (Shekhar, 1991) but resistance to praziquantel is an emerging problem (Ismael et al., 1994, 1996; Stelma et al., 1995). Several possibilities were discussed that might explain the low cure rates of praziquantel, first it act in synergy with the immune system (Gryseeles et al., 1994), second in mice drug resistant schistosomiasis is drug specific (Fallon and Doenhoff, 1994), third praziquantel is less active against the juvenile stages of Schistosoma mansoni than in adult (Xiao et al., 1985; Sabah et al., 1986b) and fourth, it has been suggested that cure rates depend on infection intensities with higher cure rates in lower infection intensities (Andrews, 1981). In the present study, mice infected with S. mansoni were treated with PZQ in a dose of 250 mg/kg/day for three alternative days or with myrrh in a dose of 10 mg/kg/day for three days. Anti-schistosomal antibodies were used to evaluate the reduction of the infection and for the humoral immune response. The most important finding was that antischistososmal antibodies were decreased significantly after treatment with myrrh and PZQ separatly (p<0.0001). Myrrh was found to an putative herbal antischistosomal drug. Sheir et al. (2001), found that treatment with myrrh in a dose of 10 mg kg^-1 of body weight for three days can induce cure rates of 91.7%. The respective roles of Th1 and Th2 cells in the immune response to S. mansoni infection has been the focus of intensive research (Kaplan et al., 1998). Previous evidence suggested a role for both subsets, since the early stage of granuloma formation has the characteristic of Th1 (IFN-γ and IL-2) cell response (Henderson et al., 1992; Cook et al., 1993). The results of our study revealed that, treatment of mice infected with S. mansoni with myrrh can improve the levels of both gamma interferons and interleukin-2. Otherwise treatment with PZQ can improve interleukin -2 only. From these result one can conclude that myrrh can act via in synergy with IFN-γ and interleukin-2 (Th1 response), this can explain why myrrh give higher cure rates than PZQ as discussed by Sheir et al. (2001). Also we selected these cytokines because, at the time of dissecting their levels were dominant (Boros et al., 1989) to give a clear difference after treatment. In the murine model of S. mansoni infection, the parasite ova settle in the liver, leading to the recruitment of various inflammatory cell types, primarily esinophils and to a lesser extent lymphocytes and macrophages to form granuloma which its function is to protect liver from the dissemination of toxic egg products (Weinstock et al., 1992). In conclusion, myrrh is a new effective and save anti-schistosomal drug that can eliminate schistosomal infection compared with the already known praziquantel. The mechanism of action of may be take place via the improvement of cellular immune response.