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.

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.

Toxocara canis antigens stimulate the production of nitric oxide and prostaglandin E2 by rat alveolar macrophages.

The effect of four Toxocara canis antigens on nitric oxide (NO) and prostaglandin E2 (PGE2) synthesis was studied in vitro using rat alveolar macrophages. Somatic and excretory/secretory T. canis antigens prepared from adult worms and LII larvae were incubated with rat alveolar macrophages obtained by bronchoalveolar lavage at concentrations of 0.1-50 microg/ml. Both excretory/secretory adult antigen (ESA) and somatic LII antigen (SLII) stimulate the release of nitrites by alveolar macrophages. This effect was specific (inhibited by L-NAME and L-canavanine) and dose-dependent; 30 microg and 10 microg being the most effective concentrations of ESA and SLII, respectively. Western blot and reverse transcriptase-polymerase chain reaction analyses revealed that ESA antigen stimulates the production of NO at transcriptional level. T. canis ESA also stimulated macrophages to produce PGE2 at transcriptional level. The addition of L-canavanine decreased the release of PGE2 significantly, which suggests that NO mediates the production of this prostaglandin. These results indicate that T. canis can stimulate the release of vasodilatory mediators by macrophages of the host.

Cytoplasmic signalling pathways in alveolar macrophages involved in the production of nitric oxide after stimulation with excretory/secretory antigens of Toxocara canis.

We studied the cytoplasmic signalling pathways involved in the generation of nitric oxide (NO) after stimulation with adult excretory/secretory antigens (ESA) of Toxocara canis. The pathways of phospholipase A2 (PLA2) and phospholipase C (PLC) were considered as potentially involved in the synthesis of nitric oxide. We used inhibitors of these pathways at different levels. Several concentrations of lithium chloride, verapamil, TMB-8 and staurosporine were used to inhibit the PLC pathway. Inhibition of the PLA2 pathway was attempted with mepacrine, diethylcarbamazine or meloxicam. Lithium chloride, verapamil and TMB-8 reduced the production of NO induced by ESA in a concentration-dependent manner. Regarding the PLA2 pathway, a range of concentrations of mepacrine greatly reduced the production of NO induced by ESA. Meloxicam inhibition was always higher than 50%. Diethylcarbamazine showed a dose-dependent effect on the production of NO induced by the ESA. Our results suggest that both the PLC and the PLA2 pathways play an essential role in activating the production of macrophage NO triggered by the ESA of T. canis. This could indicate that NO production in our experimental conditions is due to both an increase of intracellular calcium and to the participation of the arachidonic acid cascade. The implications of these activations on the host-parasite relationship are discussed and compared with LPS-stimulated macrophages.

In vivo inhibition of inducible nitric oxide synthase decreases lung injury induced by Toxocara canis in experimentally infected rats.

The direct effect of nitric oxide (NO) on the viability of Toxocara canis larvae was studied. We observed that the nitric oxide donors, SIN-1 and SNOG, exert no cytotoxic effect on the in vitro viability of T. canis larvae. In addition, we developed a model in rats to elucidate the role of NO during T. canis infection. We evaluated different indicators in four experimental groups: morphological parameters, the total number cells and cell types recovered, nitrite and protein concentration, lactate dehydrogenase and alkaline phosphatase enzymatic activity in the bronchoalveolar lavage fluid, lung index and detection of anti-T. canis specific antibodies. We observed significant differences between non-infected and infected groups. The infected animals treated with the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine were less damaged than infected, non-treated animals. Our results suggest that the in vivo inhibition of the synthesis of NO triggered by iNOS diminishes the deleterious effects of the parasite upon the host, especially the vascular alterations in the lungs. We could show that in vivo production of NO induced by infection with T. canis results in direct host damage. Thus, this induction may constitute an evasion/adaptation mechanism of the parasite.

Desarrollo mental en niños con infección chagásica crónica / Mental development in children with chronic chagasic infection

1.- Se practicó en la zona rural de Los Naranjos (Estado Carabobo) en épocas no lluviosas, la evaluación mental y de la organización intersensorial en un grupo de 31 niños chagásicos asintomáticos diagnosticados por dos pruebas (fijación de complemento y xenodiagnóstico) y en otro grupo control de 26 niños de la misma zona, aparentemente sanos y diagnosticados como negativos a dicha infección. Todos los diagnósticos se habían hecho por lo menos 2 o más años antes de esta investigación y pertenecían a un amplio estudio realizado en esta zona por el Prof. Pifano (fueron examinadas 1.000 personas de 5 a 45 años de edad). En todos se aplicó una batería de 4 tipos de test (Wechsler, Goodenough, Ayres y Bender). 2.- Aplicando el test de Student a los resultados de todos los tests empleados, no se demostró diferencia estadísticamente significante entre los dos grupos en estudio. Por tanto, de acuerdo a estos hallazgos y a otros señalamientos detallados en el texto original, no hay evidencia (sometida a estudios más amplios) que la infección chagásica crónicos asintomáticos, no tiene casi agresividad para las áreas, psico-intelectuales estudiadas; conclusión que por ahora, alivia, en razón del extremo volumen epidemiológico chagásico de nuestro país. 3.- Después de una minuciosa búsqueda bibliográfica, este estudio también revela la aplicación por primera vez en un medio esencialmente rural venezolano, de una batería de tests bastante confiable internacionalmente, y sus resultados reafirman por medios psicotécnicos, las conocidas deficientes condiciones socio-intelectuales de este ambiente antes dicho..