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Intraepithelial infiltration of eosinophils and their contribution to the elimination of adult intestinal nematode, Strongyloides venezuelensis in mice.

Eosinophils were examined for the capacity of attacking Strongyloides venezuelensis adult worms in the intestinal mucosa by using interleukin (IL)-5 transgenic mice. In IL-5 transgenic mice, most of the subcutaneously inoculated infective larvae were killed during migration, and only a few worms could reach the small intestine. When the same number of adult worms were surgically implanted in the small intestine of IL-5 transgenic and control mice, fecal egg output as well as the number of adult worms recovered from the intestine was significantly lower in IL-5 transgenic mice. In the intestinal mucosa of IL-5 transgenic mice, large number of eosinophils was present in the lamina propria even before adult worm implantation. The number of eosinophils increased significantly as early as 24 h after implantation and tripled by day 3, whereas mucosal eosinophilia remained low in wild-type mice. Most notably, eosinophils infiltrated into the intestinal epithelium and surrounded adult worms in IL-5 transgenic mice, which was never seen in wild-type control mice. However, IL-5 transgenic mice required the same period as normal mice to completely expel implanted adult worms. The amount of specific IgA as well as total IgA in the stool was high in IL-5 transgenic mice before adult worm implantation, and dropped rapidly after adult worm implantation. The present study suggests that eosinophils are capable of attacking adult nematodes in the intestinal epithelia, probably in conjunction with secretory IgA, although they are not enough for the complete worm expulsion.

Animals↗

Analysis of T cell populations and IL-3 mRNA expression in mesenteric lymph node cells and intestinal intraepithelial lymphocytes in Strongyloides ratti-infected mice.

T cell populations and IL-3 mRNA expression were analysed in mesenteric lymph node cells and intestinal intraepithelial lymphocytes (IEL) in Strongyloides ratti-infected mice. On days 7 and 12 post-infection, 2.6 times as many mesenteric lymph node cells were present in S. ratti-infected mice compared with uninfected mice. Although the percentages of CD3+, CD4+ and CD8+ cells decreased during infection, the absolute numbers of these cell types increased on day 7 due to an overall increase in the mesenteric lymph node cell number. The CD4/CD8 ratio in IEL was increased on day 5, whereas no significant change in the CD4/CD8 ratio was observed in the mesenteric lymph node cells. Expression of IL-3 mRNA, which is an important cytokine for the induction of murine mucosal mastocytosis and S. ratti-expulsion, was examined in mesenteric lymph nodes and IEL of uninfected and infected mice. IL-3 mRNA was detected in mesenteric lymph nodes of S. ratti-infected mice but not detected in the lymph nodes of uninfected mice. IL-3 mRNA was detected in IEL from both infected and uninfected mice with an 20-fold increase in expression in IEL of infected mice. Overall, IL-3 mRNA levels were higher in IEL than in mesenteric lymph nodes following S. ratti-infection. Expression of IL-4, IL-10, stem cell factor (SCF or c-kit ligand) and IFN-gamma mRNA was also examined in these two tissues. IL-10 mRNA was not detected in any tissue examined and IFN-gamma mRNA levels were unaltered as a result of an S. ratti-infection. Elevated expression of mRNA for SCF (5-fold) and IL-4 (20-fold) was observed in the mesenteric lymph nodes of infected mice. In contrast, SCF mRNA levels were similar in IEL of uninfected and infected animals and only a modest increase in IL-4 mRNA was observed in IEL of infected mice.

Animals↗

The effect of method of infection on the pathway of juvenile Strongyloides ratti in the host.

Subcutaneous injection of the larvae is the almost universally adopted means of initiating experimental infections of skin-invading roundworms but, so far, the possibility that this procedure introduces artefacts of one kind or another has not been critically studied. Experiments described in this paper were used to compare the effect of (a) injection and (b) skin application, of a small, precisely counted ('exact') dose of larvae. Results with two strains of S. ratti showed that the same proportion of the dose developed to adults in the intestines of rats irrespective of the method. With the same exact dose technique it has been shown that milk-borne infection of the pups of lactating rats is not an artefact produced by injection. Large doses (mean 4000) of larvae of the homogonic strain of S. ratti carrying a radioactive label of 75Se were tracked in their migration to the mammary gland following injection or skin application at two different sites on the right-hand side of nursing mother rats. The broad conclusion of earlier work in this laboratory using injection, that larvae move by a local route and not a systemic one, was supported by the results. The detailed distribution of the label and of unlabelled worms of the heterogonic strain in families was, however, different for the two methods, indicating that subtle variations in pathway can be brought about by the use of injection. If migration involves the lymphatic system, then the interpretation of immunological experiments in terms of lymphatic anatomy must take account of such procedural effects. The extent to which these results contribute to theories of migration in Strongyloides ratti is discussed.

Anesthesia↗

The effect of fatty acids on the developmental direction of Strongyloides ratti first-stage larvae.

The effect of fatty acids was studied on the developmental direction of Strongyloides ratti first-stage larvae (L1). The proportion of third-stage infective larvae increased markedly when L1 were cultured in faeces with added fatty acids such as palmitic (C16), stearic (C18), oleic (C18:1) and linoleic (C18:2) acids. Unsaturated fatty acids were more effective than saturated ones. Moreover, the proportion of infective larvae increased with quantity of linoleic acid but the triacylglycerols of any fatty acid had no effect. These results suggest that these free fatty acids cause physiological changes that determine the developmental course of L1 of S. ratti in nature.

Animals↗

Dissociation of the protective immune response in the mouse to Strongyloides ratti.

The generation of protective immunity by various stages in the life-cycle of Strongyloides ratti and the phases against which resistance is directed has been examined in murine strongyloidiasis. Mice were exposed to natural, complete infections, were treated with thiabendazole (which largely resembles the natural infection), were treated with cambendazole (which restricts infection to the larval stage), or infected directly by oral transfer of adult worms. Mice that were infected with infective larvae alone did not become resistant to infective larvae or the complete infection but were resistant to adult worms implanted directly into the gut. Mice exposed to adult worms alone were resistant to natural infections and adults worms implanted directly but were not resistant to infective larvae. On the other hand, mice that had received prior natural infections showed evidence of resistance to infective larvae, adult worms, and natural, complete infections. It is concluded that there is immunological cross-reactivity between infective larvae and adult worms but that under certain circumstances the infective larvae are able to evade the host's protective immune response.

Animals↗

Studies of the effect of contralateral versus ipselateral challenge of rats primed on one side with Strongyloides ratti.

The initial pathway of skin penetrating larvae of Strongyloides ratti inside the host is not systemic and could well involve local components of the lymphatic system. The experiments described were an attempt to detect an effect on immunity depending on whether larvae of a challenge infection were committed to a pathway through heavily primed or lightly primed lymph nodes. Female rats were immunised by subcutaneous injection into the right forearm of 10,000 [corrected] heat killed, or 1000 live, third stage larvae of S. ratti. Animals given living parasites were placed on a diet containing 0.1 or 0.2% thiabendazole 48h or 36h after infection. Challenge infections of less than 100 larvae ('exact' doses) were applied on day 21 to either the right or left flank and, similarly, to controls that had received the anthelmintic but not the priming infection. Heat killed parasites elicited no response at all. Priming with live larvae stimulated a significant immunity (16% and 37% depression in 2 experiments), but there was no difference between rats whose challenge was on the same side as the priming dose and those which had the contralateral treatment. The significance of these results to theories of pathfinding in general, and to practical immunology, is discussed.

Analysis of Variance↗

The ultrastructure of the peri-vulval region of Strongyloides cebus.

Ultrastructural studies were undertaken on a discrete peri-vulval area of cuticular modification in the parasitic female of Strongyloides cebus. Cilium-like arrangements of microtubules were observed which suggested that this region was sensory in nature. The possible function of this is discussed.

Animals↗

Analysis of polyunsaturated fatty acid composition of Strongyloides ratti in relation to development.

The effect of linolenic acid (C18:3 omega 3) on the development of Strongyloides ratti first-stage larvae (L1) in culture was studied. The fatty acid composition of S. ratti free-living generations was analyzed by gas chromatography. L1 had abundant linoleic acid (C18:2 omega 6) but its proportion decreased with development. On the contrary, eicosapentaenoic acid (C20:5 omega 3) and C20:4 omega 3 were prominent in the filariform larva (L3). Because C20:5 omega 3 is generally synthesized from C18:3 omega 3 via C20:4 omega 3, the high ratio of C20:5 omega 3/C18:3 omega 3 of L3 in all the free-living generations suggested that polyunsaturated fatty acid metabolism, particularly the omega-3 series, and eicosanoids produced had important roles in the development of S. ratti L1.

Animals↗

On the biological and biochemical nature of cloned populations of Strongyloides ratti.

Seven geographically varied isolates of Strongyloides ratti were cloned. Each clone was examined for the degree of homogonic development of the free-living generation and by enzyme electrophoresis. Considerable variation was found between the clones in the degree of development that was homogonic. In contrast, the clones were indistinguishable by enzyme electrophoresis.

Animals↗

Protease resistant interleukin-3 stimulating components in excretory and secretory products from adult worms of Strongyloides ratti.

Excretory and secretory (ES) products collected from adult worms of Strongyloides ratti stimulated interleukin-3 (IL-3) production with mesenteric lymph node cells from infected C57BL/6 mice, but not with normal mesenteric lymph node cells. The IL-3 stimulating components were not major IgG binding antigens. Activity of the IL-3 stimulating components was stable by treatment with protease, although reduced by heating in boiling water.

Animals↗

Characterization of the surface polypeptides of Strongyloides ratti: a comparison of homogonic and heterogonic strains.

Surface iodination, extraction and SDS-PAGE analysis techniques were employed to characterize and compare the surface polypeptides of two strains of Strongyloides ratti. Third stage infective larvae and parasitic adults of homogonic and heterogonic strains were studied using a variety of surface labelling procedures and detergents for the extraction of labelled molecules. Profiles obtained from SDS-PAGE analysis demonstrated that homogonic and heterogonic strains of S. ratti have identical surface antigens.

Animals↗

The migration and attrition of Strongyloides ratti in naive and sensitized rats.

Compressed organ autoradiography has been utilized to study the migration of the homogonic strain of Strongyloides ratti from the site of skin penetration to the gut. This transit is characterized by rapid disappearance of parasites from the cutaneous site of infection, followed by vascular dispersal throughout all organs investigated. For the first time, parasite migration in both naive and previously sensitized hosts is compared; the principal stations of migration are the same in the two groups although there is a quantitative difference in accumulation of parasites with time in these stations. Parasite attrition occurs in both groups of animals; however, in naive rats it is not manifest until day 20-25 post challenge, whereas in sensitized rats immune elimination occurs as early as 48 h post challenge.

Animals↗

Larval somatic antigens effective in inducing an IgG response and protection against Strongyloides papillosus infection.

A serum IgG response to Strongyloides papillosus infection was analysed in rabbits. IgG production was induced against 30-200 kDa larval somatic antigens and 21-160 kDa adult excretory/secretory (ES) antigens at 4 weeks postinfection. No immunoreactivity was detected between sera of infected animals and any proteins of larval ES and adult somatic antigens. Protection against larval challenge infection was investigated in rabbits immunized by primary infection and by intradermal inoculations of larval somatic and adult ES antigens. Primarily infected animals had 90% fewer or less worms from day 2 onwards after challenge, and lower faecal egg outputs after challenge, when compared with those in susceptible animals. Immunization with larval somatic antigens induced effective protection, showing 52.6-81.5% reductions in worm recovery from day 5 onwards after challenge, and lower faecal egg outputs after challenge. Systemic immunization with adult ES antigens failed to protect the animals against challenge. The possibility that resistance to S. papillosus reinfection is performed on the killing of migrating larvae in the early stages of infection is discussed. Systemic immune responses to larval somatic antigens might play an important role in the resistance.

Animals↗

Comparison of the trapping ability of Arthrobotrys robusta and Monacrosporium gephyropagum on infective larvae of Strongyloides papillosus.

In an in vitro trial, the trapping ability of Arthrobotrys robusta and Monacrosporium gephyropagum against Strongyloides papillosus infective larvae on corn meal agar plates, was evaluated after seven days of interaction at 25 degrees C. Monacrosporium gephyropagum trapped 93.1% of the larvae whereas A. robusta trapped only 32.3%. Variability in the capture of S. papillosus infective larvae by both trapping fungi is discussed.

Animals↗

Attempts to establish infections with Strongyloides stercoralis in mice and other laboratory animals.

Infection of a dog with Strongyloides stercoralis filariform larvae resulted in a persistent infection. Patent infections were not seen in rabbits, guinea-pigs, rats and 11 inbred strains and one outbred strain of mice. Manipulation of factors known to influence S. ratti infections in mice, such as age and sex of the host and the route of larval presentation, did not facilitate the appearance of rhabditiform larvae in the stools. Administration of immunosuppressive doses of corticosterioids to rabbits, guniea-pigs and C57Bl/6 mice did not permit complete development. Similarly, th course of infection was not altered in T cell-deficient hypothymic (nu/nu) mice. The fate of filariform larvae applied to the skin of mice was ascertained; filariform larvae were observed to migrate from the skin via the lungs to the muscles within several days of infection. Although S. Stercoralis does not develop to maturity in the small intestine of mice, this system does allow in vivo studies of the actions of anthelmintics against filariform larvae as well as a number of aspects of the immune response to this parasite.

Age Factors↗

The occurrence of Strongyloides ratti in the tissues of mice after percutaneous infection.

The migration of infective larvae of Strongyloides ratti has been examined in C57Bl/6 mice after percutaneous infection of the anterior abdominal wall. Lateral migration of larvae through the skin and subcutaneous tissues was not seen. Large numbers of larvae were recovered from the muscles between 2 and 24 hours after infection and larvae were seen in the cerebrospinal fluid 24 and 48 hours after infection. Insignificant numbers of larvae were seen in the blood, serosal cavities, liver, spleen, kidneys, brain or nasopharynx. Larvae arrived in the lungs between 24 and 72 hours after infection and worms were first noted in the small intestines at 48 hours. It is concluded that larvae migrate preferentially to the muscles and CSF before passing to the lungs, but the exact mode of travel is uncertain.

Abdominal Muscles↗

Strongyloides ratti in virgin female rats: studies of oestrous cycle effects and general variability.

There were no differences in mean intestinal worm burdens 8 days after subcutaneous injection of 4000 infective larvae of Strongyloides ratti into rats in dioestrus, pro-oestrus, oestrus and metoestrus. Thus, changes in the hormonal environment of the migrating larvae dependent on the oestrous cycle did not alter the worms' destination or affect their potential for development. In particular, the results are prima facie evidence that prolactin is not, on its own, responsible for the re-orientation of larvae in the tissues of nursing mothers. Other sources of variability in experimental S. ratti infections are analysed and the 'exact dose' technique offered as a corrective for some procedural errors.

Animals↗