PARACTIN^® exhibit immuno-stimulant effect by increasing cellular immune response  
¹Juan L. Hancke, & ¹Rafael A. Burgos

PARACTIN^® has shown immunomodulatory activities, in reducing the production of inflammatory mediators at high concentrations (micro molar) and stimulating the immune response in mice and in vitro.  In previous studies, we have shown that high dosage of PARACTIN^® reduce the intensity of symptoms of uncomplicated respiratory tract infections suggesting immunosuppressant effects. We recently demonstrated that PARACTIN^® reduces the IFN-g and IL-2 in T-cells (Burgos et al., 2005) in high concentrations.  However, another study demonstrated that in healthy individuals using a daily dose of 25 mg of PARACTIN^® is able to diminish the incidence of common colds suggesting an immuno-stimulant effect.  In the present report we describe that PARACTIN^® in low concentrations is able to increase the cellular immunity, inducing IFN-gamma IL-2 production specifically increasing the Th-1 response cell.

Mice were treated intraperitoneally with a daily dose equal to 6.138 or 61.68 mg/kg of PARACTIN^® for six days. Then, the mice were inoculated with 2.5 mg of B. abortus protein or PBS as negative control.  7 days after immunization the animals were sacrificed and T-cells were isolated from the lymph nodes and treated with concanavaline-A (CON-A) (3.3 or 10 mg/ml), B. abortus protein (0, 1, 5 y 10 mg/ml) and/or PARACTIN^® (0.165, 1.65 or 16.5 nM).  The IFN-g, IL-2 and IL-4 production was measured using ELISA. 

Previous report of PARACTIN^® has shown immunostimulant and immunosuppressant effect at a micro molar range of concentration, however, the effects at lower concentrations (nanomolar range) has been until unknown. In this work, we demonstrated that T-cells murine cultured with PARACTIN^® at concentrations between 0.165 and 0.75 nM showed an increase of the cellular proliferation. PARACTIN^® showed a reduction in cellular proliferation in cells treated with 1.65 nM of PARACTIN^® and 3.3 mg/ml of con-A, however, 0.165 and 16.5 nM of andrographolide had not effect.  PARACTIN^® alone in presence of con-A did not induce changes in IFN-g, IL-2 and IL-4 production. 1.65 nM PARACTIN^® induced a significant increase in production of IFN-g and IL-2, while no changes in production of IL-4 were observed.

B. abortus is a parasite capable of evading the extra cellular defense mechanisms of the host. Therefore, favoring the Th-1 response is crucial to control the infection. T-cells from mice treated with 6.14 and 61.38 mg/Kg of PARACTIN^® and immunized with 2.5 mg of B. abortus protein then the cells were cultured in presence of B. abortus protein (0, 1, 5 y 10 mg/ml) for 24 hours and the cellular proliferation was measured. Treatment with 6.14 and 61.38 mg/Kg of PARACTIN^® significantly increase the cellular proliferation of T-cells and induces an increase of IFN-gproduction compared to control mice. The IL-2 production was increased only in mice treated with 61.38 mg/Kg of PARACTIN^®. The IL-4 production wasn’t modified.

The immunostimulatory effect of PARACTIN^® at low doses has shown an increase of antigen specific and nonspecific immune response in mice. PARACTIN^® can also be used for the prevention of common colds (a minimum of 15-25 mg of PARACTIN^® corresponding to a dose of 0.3mg/Kg/day of PARACTIN®) in healthy individuals, suggesting that the effect could be attributable to the presence of andrographolide in the extract.  In this sense, the increase in the cellular proliferation, IFN-g and IL-2 production in T-cells cultured with PARACTIN^® (between 1.65-16.5 nM) indicate that this labdane present an immunostimulatory effect in vitro. The most significant increases in the cytokine production was IFN-g, as was shown by ELISA and gene expression assay, indicating that PARACTIN^® increase the IFN-g by induction of the expression gene.  The IL-4 production was not modified in the concentrations used in this study.  The increase of cellular proliferation and induction only of IFN-g and IL-2, not IL-4, shown here suggest that PARACTIN^® at 1.65 nM could be a stimulant of immune system, specifically through of Th1 response.

B. abortus triggers antigen-presenting cells to release IL-12, which causes Th0 cells to differentiate into Th1 cells that secrete IFN-?. IFN-? finally causes switching of immunoglobulin to IgG2a in the mouse. In the present study we demonstrated that in T-cell from control and immunized with B. abortus mice, PARACTIN^® at 6.14 and 61.4 mg/ Kg stimulate a strong proliferate response. Furthermore, in T-cell from immunized B. abortus mice, PARACTIN^® at 61.4 mg/ Kg induces an increase of IFN-g and IL-2 production.

In summary, we showed clearly that PARACTIN^® has immunostimulatory effect at low concentration (1.65 nM), by increase of cellular proliferation and induction of Th-1 cytokine. This effect could explicate the immunostimulatory properties of PARACTIN^® observed in vivo.