Sinistral, Dextral, and Mixed Coiling Patterns Observed in Heligmonellid Nematodes (Trichostrongyloidea).

Coiling patterns of heligmonellid nematodes were examined for 520, 208, and 33 individuals of Nippostrongylus brasiliensis, Orientostrongylus tenorai, and Sabanema sp., respectively, collected from murine rodents of Indonesia. Besides typical sinistral coiling, complete dextral coiling was found in 3.3% of N. brasiliensis and 12.1% of Sabanema sp. Mixed coiling with partial sinistral and dextral patterns was also observed in 38.8% of N. brasiliensis, 60.7% of Sabanema sp., and 3.4% of O. tenorai. In dextral coils, the left ventral area with large ridges was located inside as in sinistral coils, keeping the ability to cling to intestinal villi. The cuticular dilatation at left to left dorsal area was located caudally in sinistral coils but rostrally in dextral coils. Presence of mixed coiling indicates that the coiling patterns can change. As the transition of coiling pattern accompanies a change in direction of coil axis, it is surmised that the dextral coiling may be chosen when a worm leaves a villus to move to another villus.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Goblet cell mucins as the selective barrier for the intestinal helminths: T-cell-independent alteration of goblet cell mucins by immunologically 'damaged' Nippostrongylus brasiliensis worms and its significance on the challenge infection with homologous and heterologous parasites.

The aim of this study was to examine the role of T cells on the alteration of terminal sugars of goblet cell mucins in the small intestinal mucosa of parasitized rats and to clarify the biological significance of the altered mucins in the mucosal defence against intestinal helminths. For this purpose, Nippostrongylus brasiliensis adult worms obtained from donor rats at 7 ('normal' worms) or 13 days ('damaged' worms) post-infection were implanted intraduodenally into euthymic and hypothymic (rnu/rnu) rats. Expulsion of implanted normal worms and associated goblet cell changes were extremely delayed in hypothymic recipients compared with euthymic recipients. In contrast, intraduodenally implanted damaged worms were expelled by day 5 regardless of the strains. Around the time of expulsion of implanted damaged worms, euthymic recipients showed both goblet cell hyperplasia and alteration of mucins, whereas hypothymic rats showed only the latter. Dexamethasone treatment completely abolished goblet cell changes of both strains of recipients. To clarify the importance of the constitutional changes of goblet cell mucins in mucosal defence, euthymic rats were primed by implantation of damaged worms to induce goblet cell changes, and then 3 or 5 days later they were challenged by implantation with normal worms. The results show that when goblet cell changes were induced by priming with damaged worms, recipient rats could completely prevent the establishment of normal worms. When hypothymic rats were primed and challenged in the same manner, a similar but slightly less preventive effect was observed. Such a protective effect of altered mucins seems to be selective because priming of euthymic rats with damaged N. brasiliensis did not affect the establishment of Strongyloides venezuelensis. These results suggest that: (1) once N. brasiliensis adult worms are 'damaged' by the host's T-cell-dependent immune mechanisms, they can induce alteration of sugar residues of goblet cell mucins via host-mediated, T-cell-independent processes; (2) the expression of such altered mucins is highly effective not only in causing expulsion of established damaged worms but also in preventing establishment of normal worms; and (3) the preventive effect of altered mucins is selective against parasite species.

A freeze-fracture study of the cuticle of adult Nippostrongylus brasiliensis (Nematoda).

The surface of the cuticle of adult Nippostrongylus brasiliensis has been studied by means of the freeze-fracture technique and by transmission electron microscopy. Some of the surface coat appears to have been shed from the surface of the cuticle of adults fixed in situ in the intestine of its host and from the surface of individuals removed from the intestine and freeze-fractured. Freeze-fracturing the cuticle of individuals removed from the host has shown that this surface coat varies in thickness from 30 to 90 nm. The epicuticle is about 20 nm thick and cleaves readily to expose E- and P-faces. The P-face of the epicuticle possesses a small number of particles, similar to intra-membranous particles, whilst the E-face possesses a few, widely scattered depressions. Despite the presence of these particles the epicuticle is not considered to be a true membrane. Freeze-fracturing the remainder of the cuticle has confirmed its structure as described by conventional transmission electron microscopy. Clusters of particles on the P-face of the outer epidermal (hypodermal) membrane and corresponding depressions on the E-face of the membrane are though to be associated with points of attachment of the cuticle to the epidermis (hypodermis). No differences in appearance of the cuticle and its surface layers were observed in individuals taken from 7-, 10-, 13- and 15-day infections.

Microtopography of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae): free-living larval stages.

Microtopographic features of the various growth stages of the three free-living larval stages of the rat hookworm Nippostrongylus brasiliensis (Nematoda) were surveyed by scanning electron microscopy. These worms have a rounded anterior end and an elongated tail. Cuticular annulations were observed along the body, which also bore two ribbon-like lateral alae. Two rings of six lip-like lappets were observed around the triradiate oral opening in all larval stages. The cephalic space contained two lateral amphidial pits. The excretory pore in the third anterior part was observed in a ventral view of the larvae. No deirids were observed. The anus with a crescent-shape opening was located posteriorly. Phasmidial apertures, only observed in the third-stage larvae, opened on the lateral alae in the tail region.

Microtopography of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae): parasitic larval stages and adults.

Specimens of the rat hookworm, Nippostrongylus brasiliensis (Nematoda) were recovered from lungs (third- and fourth-stage larvae) and intestine (fourth-stage larvae and adults). The following features were studied in the different stages by scanning electron microscopy: cephalic structures, especially sense organs, synlophe, cervical region, and caudal part. The main differences between the third and fourth stages concerned the lip-like structures around the oral aperture, the appearance of the cephalic space with the presence of a cephalic cap in fourth-stage larvae, the pattern of longitudinal ridges, and sexual differentiation. Pore-like papillae, not seen in third-stage larvae, developed in later stages. Deirids were observed only in adults, and phasmids were poorly discerned. Some of these morphological features, such as the cephalic sense organ apertures and cuticle pores and micropores, can be observed only by scanning electron microscopy. The possible functions of these different structures and their relationship with the behavior of the worms during their life cycle are discussed.

Pathology of pulmonary parasitic migration: morphological and bronchoalveolar cellular responses following Nippostrongylus brasiliensis infection in rats.

Nippostrongylus brasiliensis has an obligatory migratory phase through the lungs during its development in rats. This migration is associated with marked tissue damage and pronounced cellular reaction. Given that cells from the lower respiratory tract, especially alveolar macrophages, can adhere to and kill larvae of N. brasiliensis in vitro, we studied the time course of morphological changes associated with parasitic migration. Compared to a primary infection, a secondary infection resulted in significant changes in the pulmonary tissue characterized by an early acute inflammation leading to granulomatous reaction in the parenchyma and a leucocytosis in the bronchoalveolar lavage fluids with an anamnestic increase in absolute numbers of neutrophils, alveolar macrophages, eosinophils, and lymphocytes. Scanning electron microscopy showed that inflammatory cells, especially alveolar macrophages, granulocytes, lymphocytes, erythrocytes, and platelets, adhered to the larvae following secondary infection and this adhesion was associated with disruption of cuticular surface in some larvae. Secondary infection also resulted in retention of larvae in granulomatous lesions in the lungs even up to 21 days postinfection. There was mast cell and type II pneumocyte hyperplasia and these cells appeared to be activated. Thus, the histopathological changes in lungs correlated with the bronchoalveolar cellular responses and further document the inflammatory and immunological reactions during the migration of N. brasiliensis larvae.

Two- and three-dimensional locomotion of the nematode Nippostrongylus brasiliensis.

Locomotion of adult Nippostrongylus brasiliensis has been studied in saline, in 0.6% agar, in sodium alginate of different viscosities and amongst sand grains in these media. In saline the nematode formed two-dimensional waves but there was little forward progression. Amongst sand grains in saline the nematode moved forwards by thrusting against sand grains, but thigmokinetic behaviour later resulted in quiescence. In 0.6% agar and in alginates of weak viscosity the nematode produced two-dimensional waves and sometimes a three-dimensional helical wave which resulted in forward movement. The formation of three-dimensional waves and the distance travelled increased with increasing viscosity up to 4% sodium alginate and also amongst sand gains in these media. In 8% sodium alginate the nematode became coiled like a spring but remained almost stationary. The three-dimensional wave is formed with torsion and obtains thrust from the viscous medium. In the intestine of the host thrust will be obtained from the mucus and villi of the intestinal mucosa. The ability of this nematode to move by two- and three-dimensional undulatory propulsion is probably related to its complex ridged cuticle. Attention is drawn to the role that increased viscosity of mucus may play in entrapping nematodes during their immune rejection.

Nippostrongylus brasiliensis and Ascaridia galli: characterization of peroxisomes.

A method is described for the isolation of peroxisomes from mitochondria-enriched fractions obtained from both species of nematodes. The distributions of these organelles are characterized after density gradient centrifugation in sucrose or Percoll by urate oxidase and catalase activities. The possession of peroxisomes may be part of an important defence mechanism in parasites.

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages.

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.

The use of plasma etching to reveal the internal structure of Nippostrongylus brasiliensis (Nematoda).

Plasma etching has been used to strip away the cortical layers of the cuticle of adult Nippostrongylus brasiliensis to reveal the struts, with their supporting fibres, which are found in the fluid-filled middle layer of the cuticle, and the basal fibre layers. The etched specimens were studied by means of scanning electron microscopy. The results support earlier work, obtained by transmission electron microscopy, on the cuticle of this nematode. Plasma etching has been shown to have potential in studying the structure of nematodes.

Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages.

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.

Ultrastructural observations on the in vitro interaction between rat eosinophils and some parasitic helminths (Schistosoma mansoni, Trichinella spiralis and Nippostrongylus brasiliensis).

Rat eosinophils form an intimate association with the surfaces of parasitic helminths, in vitro, in the presence of immune serum. The parasite presents a non-phagocytosable surface to the cell. The initial response of the eosinophil is degranulation which leads to the formation of large cytoplasmic vacuoles. Peroxidase, an enzyme localized in the matrix of the crystalloid secretion granules, is discharged into these vacuoles as a consequence of degranulation. The vacuoles eventually become connected to the adherent basal plasma membrane of the eosinophil, and peroxidase is secreted directly onto the surface of the parasite. There is no morphological evidence to suggest that this particular secretion affects the integrity of the parasite surface.

Nippostrongylus brasiliensis: further properties of antibody-damaged worms and induction of comparable damage by maintaining worms in vitro.

When adult Nippostrongylus brasiliensis were maintained in vitro they became damaged. Using the criteria of ultrastructural morphology, acetylcholinesterase isoenzyme pattern and the behaviour of the worms after transfer to a normal rat, this damage appeared to be similar to that produced by the in vivo action of antibodies. Antibodies were shown to be responsible for the anterior migration of adult worms which occurs during primary infections in mature rats and in the prolonged infections seen in lactating and immature rats. Antibody damaged worms and worms unaffected by antibodies were equally able to stimulate the immune response required for worm expulsion. Apparently antibody damage is not required for the initiation of the second immune component necessary for expulsion of this parasite.