[Frontiers in Bioscience 3, a38-46, June 9, 1998]

	[Frontiers in Bioscience 3, a38-46, June 9, 1998] Reprints PubMed CAVEAT LECTOR
	*CHARACTERIZATION OF KINETICS OF ANTI-TRICHINELLA SPIRALIS* NEWBORN LARVAE IMMUNITY IN RATS Ching-Hua Wang** Biology Department, California State University, 5500 University Parkway, San Bernardino, CA 92407, USA Received 5/11/98 Accepted 5/24/98 5. DISCUSSION The experiments reported here systematically studied the kinetics of anti-newborn larvae immunity. Two approaches were employed in the experiments. First, the dynamics of the immune response were determined by comparing muscle larvae burdens of rats immunized with newborn larvae i.v. and then challenged with newborn larvae i.v. at various intervals after immunization. It was found that the onset of immune protection took place 1 week after immunization (table 1), which confirms the findings in mice by Bell et al. (22). The peak incidence of this immunity was at 4 weeks after newborn larvae immunization when 100% protection was established. The second approach involved immunizing rats with muscle larvae and then detecting anti-newborn larvae immunity directly in the peritoneal cavity of rats after challenging them with newborn larvae i.p. In addition, blood samples of these rats were also incubated with newborn larvae in vitro. It was found that 8 days after muscle larvae infection, newborn larvae recovery from the peritoneal cavity was considerably reduced (table 2). The cell adherence and larvicidal activity observed after i.p. injection of newborn larvae or after incubation of larvae with blood, however, were greatly increased compared to the controls (tables 2 and 3). These effects peaked on day 16 (table 2) for peritoneal cells and as early as on day 9 (table 3) for blood cells. The onset of anti-newborn larvae immunity detected here is 7 to 14 days earlier than the in vitro findings by Mackenzie et al. (19) and Philipp et al. (20), which suggests that the in vitro assay may not be sensitive enough to reflect an immunity which is expressed in vivo. The lag phase of immune response against newborn larvae is 6 days shorter in rats immunized with muscle larvae per os (tables 2 and 3) than in rats immunized with newborn larvae i.v. (table 1). Newborn larvae are first produced by adult T. spiralis worms at around day 6 post infection (36). When the anti-newborn larvae immune response is detectable on day 9 after muscle larvae infection, by this time, the host immune system has only been stimulated by newborn larvae for no more than 3 days. When rats are immunized i.v. with newborn larvae, however, they need at least 9-14 days to trigger the host immune system to respond (table 1). In addition, after muscle larvae immunization, the anti-newborn larvae response reaches maximum activity 11 to 18 days earlier (5 to 12 days after newborn larvae production, Tables 2 and 3) than in rats immunized with newborn larvae i.v. (26 days post inoculation, table 1). It is known that newborn larvae are continuously produced by adult worms in the small intestine of rats from day 6 onwards until the adult worms are rejected (36). During this period, due to the systemic larval dissemination and recirculation (37), newborn larvae antigens are extensively exposed to the host immune system so that the immune response is expected to be stronger. In contrast, when immunization is given by a single i.v. injection with newborn larvae, the exposure of the larval antigens to the host is significantly shortened (14). Hence, one is expected to observe a longer lag phase before the onset of the immune response and it requires a more extended period to mount the maximum anti-newborn larvae activity. Another possibility is that after muscle larvae infection the small intestine of rats is sequentially stimulated by muscle larvae, adult worms and newborn larvae and this may enhance the immune response against newborn larvae. When newborn larvae were incubated with the blood obtained from rats 14 days after muscle larvae infection, cell adherence to newborn larvae and the larvicidal effect seemed on the decline (table 3). The most likely reason is that by this time, newborn larvae that were bearing cells could no longer stand the laking treatment, i.e., they were lysed, and therefore, they were not counted. This phenomenon actually indicates a stronger larvicidal response of the host. The effects of different doses of immunization and challenge infection on the expression of anti-newborn larvae immunity were studied. The results demonstrate that a relatively weak anti-newborn larvae immunity is incident to the low immunization dose (500 muscle larvae) given (figure 1). When the immunization dose is high (5-6,000 muscle larvae per os), however, the effect of anti-newborn larvae immunity is not proportionally increased. On the contrary, it is decreased dramatically (figures 2, 3). The reduction of anti-newborn larvae immunity does not occur at an earlier stage (day 9) (table 4) but takes place at a later time (day 16) during the infection (figure 2). These results suggest that through low dose immunization, host immune system may not be sufficiently stimulated. High dose immunization, on the other hand, may induce a suppressive effect on host immunity. At an earlier stage of infection, e.g., 8 days after muscle larvae infection, newborn larvae that have been produced have not developed to muscle larvae. By day 16, however, many newborn larvae are maturing to muscle larvae in the striated muscle cells. Hence, it is likely that the maturing muscle larvae may be responsible for this suppressive effect. At the moment, it is not clear what the mechanism is for this suppressive effect. Compared to the low and high dose immunization, the most optimal immunization dose examined is 2,000 muscle larvae infection per os (figures 1, 2) or 20,000 newborn larvae injection i.v. (table 1), both of which induce the strongest immune response against newborn larvae. The results of challenge dose response on anti-newborn larvae immunity demonstrate that the higher the challenge dose is given, the lower the effects of cell adherence to newborn larvae and larval killing are observed (figure 3). These data suggest that first, the effectors of the immunity may be diluted by the large numbers of newborn larvae given in the challenge infection, and second, the effectors may not be reused. This means that once the antibodies and effector cells attach to the target larvae, they do not detach from these targets and reattack other larvae. In fact, the experimental observations proved this hypothesis. When placing the cell-coated larvae obtained from the peritoneal fluid of immune rats on a slide and pressing the slide with a cover slip, the outer layers of cells could be pressed away from the larvae. Nonetheless, the inner-layer cells never became loosened from the larvae (figure 4). Figure 4. Newborn lavae with cell adherence. (a) One with cells attached, one without. (b) A closer view of the cell adherence. Summarizing, the results described here demonstrate that anti-newborn larvae immune response is generated in rats 3-4 days after newborn larvae production during a primary T. spiralis infection. This response occurs within 2 weeks after i.v. injection of newborn larvae. Low dose (500 muscle larvae) immunization elicits a sufficient yet not strong anti-newborn larvae immunity. High dose (5-6,000 muscle larvae per os) immunization leads to a reduction of the immune effect against newborn larvae. The suppressive effect induced by high dose immunization with muscle larvae is not evident on day 9 of T. spiralis infection but on day 16. The most optimal immunization dose examined is 2,000 muscle larvae. Due to the fact that the immune effectors once attached to the first larva, do not re-deploy themselves to other unattended target larvae, high dose challenge infection reduces the effect of anti-newborn larvae immunity.

[Frontiers in Bioscience 3, a38-46, June 9, 1998]
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CHARACTERIZATION OF KINETICS OF ANTI-TRICHINELLA SPIRALIS NEWBORN LARVAE IMMUNITY IN RATS

Ching-Hua Wang

Biology Department, California State University, 5500 University Parkway, San Bernardino, CA 92407, USA

Received 5/11/98 Accepted 5/24/98

5. DISCUSSION

The experiments reported here systematically studied the kinetics of anti-newborn larvae immunity. Two approaches were employed in the experiments. First, the dynamics of the immune response were determined by comparing muscle larvae burdens of rats immunized with newborn larvae i.v. and then challenged with newborn larvae i.v. at various intervals after immunization. It was found that the onset of immune protection took place 1 week after immunization (table 1), which confirms the findings in mice by Bell et al. (22). The peak incidence of this immunity was at 4 weeks after newborn larvae immunization when 100% protection was established.

The second approach involved immunizing rats with muscle larvae and then detecting anti-newborn larvae immunity directly in the peritoneal cavity of rats after challenging them with newborn larvae i.p. In addition, blood samples of these rats were also incubated with newborn larvae in vitro. It was found that 8 days after muscle larvae infection, newborn larvae recovery from the peritoneal cavity was considerably reduced (table 2). The cell adherence and larvicidal activity observed after i.p. injection of newborn larvae or after incubation of larvae with blood, however, were greatly increased compared to the controls (tables 2 and 3). These effects peaked on day 16 (table 2) for peritoneal cells and as early as on day 9 (table 3) for blood cells. The onset of anti-newborn larvae immunity detected here is 7 to 14 days earlier than the in vitro findings by Mackenzie et al. (19) and Philipp et al. (20), which suggests that the in vitro assay may not be sensitive enough to reflect an immunity which is expressed in vivo.

The lag phase of immune response against newborn larvae is 6 days shorter in rats immunized with muscle larvae per os (tables 2 and 3) than in rats immunized with newborn larvae i.v. (table 1). Newborn larvae are first produced by adult T. spiralis worms at around day 6 post infection (36). When the anti-newborn larvae immune response is detectable on day 9 after muscle larvae infection, by this time, the host immune system has only been stimulated by newborn larvae for no more than 3 days. When rats are immunized i.v. with newborn larvae, however, they need at least 9-14 days to trigger the host immune system to respond (table 1). In addition, after muscle larvae immunization, the anti-newborn larvae response reaches maximum activity 11 to 18 days earlier (5 to 12 days after newborn larvae production, Tables 2 and 3) than in rats immunized with newborn larvae i.v. (26 days post inoculation, table 1).

It is known that newborn larvae are continuously produced by adult worms in the small intestine of rats from day 6 onwards until the adult worms are rejected (36). During this period, due to the systemic larval dissemination and recirculation (37), newborn larvae antigens are extensively exposed to the host immune system so that the immune response is expected to be stronger. In contrast, when immunization is given by a single i.v. injection with newborn larvae, the exposure of the larval antigens to the host is significantly shortened (14). Hence, one is expected to observe a longer lag phase before the onset of the immune response and it requires a more extended period to mount the maximum anti-newborn larvae activity. Another possibility is that after muscle larvae infection the small intestine of rats is sequentially stimulated by muscle larvae, adult worms and newborn larvae and this may enhance the immune response against newborn larvae.

When newborn larvae were incubated with the blood obtained from rats 14 days after muscle larvae infection, cell adherence to newborn larvae and the larvicidal effect seemed on the decline (table 3). The most likely reason is that by this time, newborn larvae that were bearing cells could no longer stand the laking treatment, i.e., they were lysed, and therefore, they were not counted.

This phenomenon actually indicates a stronger larvicidal response of the host.

The effects of different doses of immunization and challenge infection on the expression of anti-newborn larvae immunity were studied. The results demonstrate that a relatively weak anti-newborn larvae immunity is incident to the low immunization dose (500 muscle larvae) given (figure 1). When the immunization dose is high (5-6,000 muscle larvae per os), however, the effect of anti-newborn larvae immunity is not proportionally increased. On the contrary, it is decreased dramatically (figures 2, 3). The reduction of anti-newborn larvae immunity does not occur at an earlier stage (day 9) (table 4) but takes place at a later time (day 16) during the infection (figure 2). These results suggest that through low dose immunization, host immune system may not be sufficiently stimulated. High dose immunization, on the other hand, may induce a suppressive effect on host immunity. At an earlier stage of infection, e.g., 8 days after muscle larvae infection, newborn larvae that have been produced have not developed to muscle larvae. By day 16, however, many newborn larvae are maturing to muscle larvae in the striated muscle cells. Hence, it is likely that the maturing muscle larvae may be responsible for this suppressive effect. At the moment, it is not clear what the mechanism is for this suppressive effect. Compared to the low and high dose immunization, the most optimal immunization dose examined is 2,000 muscle larvae infection per os (figures 1, 2) or 20,000 newborn larvae injection i.v. (table 1), both of which induce the strongest immune response against newborn larvae.

The results of challenge dose response on anti-newborn larvae immunity demonstrate that the higher the challenge dose is given, the lower the effects of cell adherence to newborn larvae and larval killing are observed (figure 3). These data suggest that first, the effectors of the immunity may be diluted by the large numbers of newborn larvae given in the challenge infection, and second, the effectors may not be reused. This means that once the antibodies and effector cells attach to the target larvae, they do not detach from these targets and reattack other larvae. In fact, the experimental observations proved this hypothesis. When placing the cell-coated larvae obtained from the peritoneal fluid of immune rats on a slide and pressing the slide with a cover slip, the outer layers of cells could be pressed away from the larvae. Nonetheless, the inner-layer cells never became loosened from the larvae (figure 4).

Figure 4. Newborn lavae with cell adherence. (a) One with cells attached, one without. (b) A closer view of the cell adherence.

Summarizing, the results described here demonstrate that anti-newborn larvae immune response is generated in rats 3-4 days after newborn larvae production during a primary T. spiralis infection. This response occurs within 2 weeks after i.v. injection of newborn larvae. Low dose (500 muscle larvae) immunization elicits a sufficient yet not strong anti-newborn larvae immunity. High dose (5-6,000 muscle larvae per os) immunization leads to a reduction of the immune effect against newborn larvae. The suppressive effect induced by high dose immunization with muscle larvae is not evident on day 9 of T. spiralis infection but on day 16. The most optimal immunization dose examined is 2,000 muscle larvae. Due to the fact that the immune effectors once attached to the first larva, do not re-deploy themselves to other unattended target larvae, high dose challenge infection reduces the effect of anti-newborn larvae immunity.