Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production.

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.

Increased rat alveolar macrophage expression of functional iNOS induced by a Dirofilaria immitis immunoglobulin superfamily protein.

Dirofilaria immitis is a worldwide filarial nematode causing heartworm disease in dogs and cats. Several mosquito species, which are able to feed both on humans and animals, can transmit this parasite. Inflammatory progression of host tissues induced by parasites are mediated by several molecules, including nitric oxide (NO), which usually exerts deleterious effects on parasites and occasionally on the host. We analyze the in vitro effect of total D. immitis adult worm somatic antigens on naïve rat alveolar macrophage NO production and further separation of parasite proteins to define specific D. immitis somatic molecules influencing host cell NO secretion. Additionally, we address the possible influence of Wolbachia spp. on the in vitro production of NO by macrophages. Our results demonstrate that D. immitis adult worm soluble antigens are able to specifically induce NO production from host macrophages. Furthermore, we demonstrated that this effect is due to nematode antigens rather than to defined components (LPS and metabolic molecules) derived from its endosymbiont, Wolbachia spp. In addition, we were able to isolate and identify one of the parasite specific components from the DiSo extract, denominated DiID35.3 and putatively belonging to the Immunoglobulin Superfamily Protein (ISP) group, triggering NO release from macrophages in a dose-dependent and specific manner.

Antigens from Ascaris suum trigger in vitro macrophage NO production.

SUMMARY We investigated the in vitro effect of total excretory/secretory and somatic antigens from Ascaris suum adults (ESA and SA) and larvae 3 (ESL3 and SL3), and of 10 purified protein fractions from ESA components on rat alveolar macrophage nitric oxide (NO) production. Our results showed that in vitro incubation of macrophages with SA and SL3 antigens of A. suum did not result in NO release from cells, whereas incubation with ESA or ESL3 antigens resulted in the stimulation of NO production by these cells, both in a specific (inhibited by L-NAME and L-canavanine) and dose-dependent manner. In addition, we could demonstrate that a purified ESA fraction consisting of three Coomassie-stained bands of approximately 37, 44 and 46 kDa is involved in the in vitro triggering of NO production by host cells. These three bands were subjected to MALDI-peptide mass fingerprint, showing similarities with phosphoglycerate kinase, elongation factor Tu and enolase molecules, respectively. Future studies will focus on the characterization of these parasite-derived molecules.

Lung-migrating digenean parasites: in vitro influence on nitric oxide production from normal rat pulmonary macrophages.

Nitric oxide (NO) is one of the most versatile players in the immune system. Most parasites induce inflammation in the host associated with NO production. Here, we compare the in vitro effect of Schistosoma bovis somatic (SbS) and excretory-secretory (SbES) antigens, and excretory-secretory Paragonimus mexicanus adult worm (PmES) molecules on rat alveolar macrophages NO production measured by the Griess method and by RT-PCR. Additionally, we address the divergence of the NO stimulatory/inhibitory effects of these two parasites. Polymyxin B was used to assess possible LPS contamination. In vitro incubation of rat alveolar macrophages with PmES (10 microg/ml) and SbS (50 microg/ml), but not with SbES extracts, resulted in NO production and an increase in iNOS cell mRNA. This production was specific and inhibited by L-NAME and L-canavanine. Different effects were observed when cells were incubated with P. mexicanus and S. bovis antigens.

Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.