Parasite Expulsion Research Articles

Effect of inducible costimulator blockade on the pathological and protective immune responses induced by the gastrointestinal helminth Trichinella spiralis.

Infections with gastrointestinal helminths elicit potent Th2 responses, which ultimately result in their expulsion. However, during expulsion of Trichinella spiralis this Th2 response also induces a severe enteropathy characterized by villus atrophy and crypt hyperplasia. Inducible costimulator (ICOS), a homologue of CD28, interacts with B7-related protein 1, and has been shown to be important in T-B cell interactions and antibody class switching. Significantly, ICOS appears to be involved in the induction of both Th1 and Th2 responses, but may be of heightened importance in Th2 responses. Here we employed a blocking antibody against ICOS to investigate the contribution of ICOS costimulation to the development of the protective and pathological immune responses induced during infection with T. spiralis. We show that, although blocking ICOS resulted in a decrease in TNF-alpha and the Th2 cytokines IL-4 and IL-5 and serum levels of total IgE, it did not affect the expulsion of the adult parasites. Surprisingly, levels of IL-9, IL-13 and IL-10 were elevated and protection against the larval muscle stage of the parasite was enhanced. Importantly, these findings may relate to the fact that ICOS blockade significantly ameliorated the enteropathy that usually accompanies expulsion of the adult parasite.

Mouse mast cell protease-1 is required for the enteropathy induced by gastrointestinal helminth infection in the mouse

Background & Aims: The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. Mast cell hyperplasia is a hallmark of infection with gastrointestinal nematodes, in particular Trichinella spiralis. Although the precise mechanism by which mast cells induce expulsion of these parasites has not been elucidated, it has been proposed that mast cell mediators, including cytokines and granule chymases, act to create an environment inhospitable to the parasite, part of this being the induction of intestinal inflammation. Therefore, the aims of this study were to dissect the role of mast cells and mast cell proteases in the induction of parasite-induced enteropathy. Methods: Mast cell-deficient W/Wv and mouse mast cell protease-1 (mMCP-1)-deficient mice were infected with T. spiralis, and parasite expulsion, enteropathy, and Th2 responses were determined. Results: Expulsion of the parasite was delayed in both strains of mice compared with wild-type controls; additionally, in both cases, the enteropathy was significantly ameliorated. Although Th2 responses were significantly reduced in mast cell-deficient W/Wv mice, those from mMCP-1-deficient mice were similar to wild-type mice. Additionally, levels of TNF-α and nitric oxide were significantly reduced in both W/Wv and mMCP-1 deficient mice. Conclusions: These results imply that mast cells may contribute to the induction of protective Th2 responses and, importantly, that the intestinal inflammation associated with gastrointestinal helminths is partly mediated by mMCP-1.

IgE enhances clearance of Trichinella spiralis and regulates mast cell responses in mice

Rationale Trichinella spirals infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis. As IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. Methods Wild-type and IgE −/− mice were infected with T. spiralis. Blood and tissue eosinophil counts, mast cell numbers, serum MCP-1, gut worm expulsion and tissue cysts were followed. Results Wild-type but not IgE −/− mice mounted a strong IgE response. The splenic mastocytosis observed in BALB/c mice following infection was significantly diminished in IgE −/− mice. Similar levels of peripheral blood eosinophilia occurred in both wild-type and IgE deficient animals. Splenic mast cell numbers and serum levels of mouse MCP-1 were lower in IgE −/− animals and they were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE −/− mice was about twice that in controls and the fraction of larvae that was necrotic was reduced. Conclusions We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of mast cell homeostasis in vivo.

IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis.

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE(-/-)) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE(-/-) mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE(-/-) animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE(-/-) mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.

Necator americanus: maintenance through one hundred generations in golden hamsters ( Mesocricetus auratus). II. Morphological development of the adult and its comparison with humans

Through 100 passages, the human hookworm Necator americanus was adapted to the golden hamster, Mesocricetus auratus, without either the requirement for exogenous steroids or other immunosuppressive agents, nor the requirement to infect hamsters as pups. Adult N. americanus recovered from infected hamsters were morphologically similar to those from infected humans in Sichuan Province, China, although they were smaller and the females produced fewer eggs. The natural history and kinetics of N. americanus infection was different in female and male hamsters. Female hamsters supported low intensity infections that lasted for approximately two months. In contrast, the peak intensity of infection in male hamsters was high, but this situation lasted less than for 4 weeks at which time many of the hookworms were expelled. However, even after the major parasite expulsion, the total number of hookworms consistently remained higher in chronically infected male hamsters compared with female hamsters. The hamster model of N. americanus is potentially useful for studying the development of new anthelminthic drugs and vaccines. Index descriptors and abbreviations: Necator americanus; Hookworm; Golden hamster; Morphology; Reproductive system; Scanning electronic microscopy; Light microscopy

Mast cells disrupt epithelial barrier function during enteric nematode infection.

We have investigated the influence of mast cells on the barrier function of intestinal epithelium during nematode infection. Trichinella spiralis infection induces a strong type 2 cytokine-mediated inflammation, resulting in a critical mucosal mastocytosis that is known to mediate expulsion of the parasites from the intestine. The host response to infection is also characterized by an increase in mucosal leakiness. We show here that intestinal epithelial permeability is markedly elevated during infection, with kinetics that mirror the adaptive immune response to primary and secondary infection. Furthermore, we have identified degradation of the tight junction protein, occludin, thereby providing a mechanism for increased paracellular permeability during helminth infection. We further demonstrate by using anti-c-kit antibody and IL-9 transgenic mice that mast cells are directly responsible for increasing epithelial paracellular permeability and that mice deficient in a mast cell-specific protease fail to increase intestinal permeability and fail to expel their parasite burden. These results provide the mechanism whereby mucosal mast cells mediate parasite expulsion from the intestine.

Is there a common mechanism of gastrointestinal nematode expulsion?

Parasitic gastrointestinal (GI) nematodes are one of the most commonly acquired infections in the world. Although they cause relatively little mortality, infections result in high levels of morbidity that can result in developmental consequences in infected children and cause significant economic loss in infected animals. Over the last 30 years there has been extensive research into the mechanisms controlling the expulsion of gastrointestinal nematodes. Although many of the effector mechanisms that contribute to the loss of the parasite have been defined, we still appear to be some way from understanding the actual cause of parasite loss. Part of this stems from the different responses induced by different gastrointestinal parasites. It is clear that a Th2 response is essential for the expulsion of GI helminths; however, each of the characteristic immunological effector mechanisms induced following infection with these parasites may not be required or may be insufficient in isolation, but together they operate to expel GI helminths. These responses then succeed more efficiently in some cases than in others to induce parasite expulsion. The contribution made by various effector mechanisms to the expulsion of these parasites may therefore be a reflection of both the niche which the parasite inhabits as well as possible evasive/suppressive mechanisms employed by the parasites. In this review the various aspects of parasite expulsion will be described and the controversial issues in the field will be discussed.

Interleukin-4 Receptor–Stat6 Signaling in Murine Infections with a Tissue-Dwelling Nematode Parasite

Interleukin-4 (IL-4) has been shown to be crucial in parasite expulsion in several gastrointestinal nematode infection models. Data from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of IL-4 in the clearance of a primary infection of Brugia from the murine host. Our data from infections of IL-4 receptor(-/-) and Stat6(-/-) mice further indicate that IL-4 exerts its effects by activating the Stat6 molecule in host target cells, a finding which links clearance requirements of a gastrointestinal tract-dwelling nematode with those of a tissue-dwelling nematode. Additionally, we show that the requirements for IL-4 receptor binding and Stat6 activation extend to accelerated clearance of a secondary infection as well. The data shown here, including analysis of cell populations at the site of infection and infection of immunoglobulin E (IgE)(-/-) mice, lead us to suggest that deficiencies in eosinophil recruitment and isotype switching to IgE production may be at least partially responsible for slower parasite clearance in the absence of IL-4.

Mast cell-independent impairment of host defense and muscle contraction in T. spiralis-infected W/W(V) mice.

In response to nematode infection, the host presumably attempts to create an unfavorable environment to prevent larval penetration of the host and to expedite parasite expulsion from the gut. In this study, we have used W/W(V) mice with or without mast cells after bone marrow reconstitution (BMR-W/W(V)) to examine the role of mast cells in the host response. W/W(V), BMR-W/W(V), and wild-type (+/+) mice were infected with Trichinella spiralis. Infected W/W(V) mice exhibited less tissue damage and experienced a delay in worm expulsion and a greater degree of larval penetration of the gut leading to encystment in skeletal muscle. Tissue injury was greater and worm expulsion was normalized in BMR-W/W(V) mice, but larval penetration remained unchanged. Spontaneous contractile activity of jejunal muscle was disrupted in W/W(V) mice, as was the contractile response to carbachol. These abnormalities were also present in BMR-W/W(V) mice. These results indicate that mast cells mediate tissue damage and contribute to the timely expulsion of nematodes from the gut during primary infection.

The role of mucins in host-parasite interactions: Part II - helminth parasites.

Some parasites express mucin-like molecules. These have possible roles in attachment and invasion of host cells and in the avoidance of host immune processes. Enzymes of parasite origin might also facilitate infection, either by degrading host mucus barriers or by generating binding sites on host cells. Host mucins have roles in preventing parasite establishment or in parasite expulsion. They, in turn, might be exploited by parasites, either as sources of fuel or binding sites, or as host-finding targets. Here, we describe the biochemical properties of mucins and mucin-like molecules in relation to interactions (established and putative) between helminth parasites and their hosts.

Critical role for signal transducer and activator of transcription factor 6 in mediating intestinal muscle hypercontractility and worm expulsion in Trichinella spiralis-infected mice.

Intestinal nematode infections in rats or mice are accompanied by intestinal muscle hyper contractility that may contribute to parasite expulsion from the gut. Previous studies demonstrated that both the expulsion of nematode parasites and the associated muscle hyper contractility are dependent on CD4(+) T helper cells. Nevertheless, the precise immunological mechanism underlying changes in intestinal muscle function remains to be determined. In this study, we investigated the role of interleukin 4 (IL-4) and signal transducer and activator of transcription factor 6 (STAT6) in the development of intestinal muscle hypercontractility and worm expulsion by infecting IL-4 and STAT6-deficient mice with Trichinella spiralis. Worm expulsion was almost normal in IL-4-deficient mice but substantially delayed in STAT6-deficient mice. Consistent with delayed worm expulsion, we also observed a marked attenuation of carbachol-induced muscle contraction in STAT6-deficient mice but only a moderate decrease in muscle hypercontractility in IL-4-deficient mice. In addition, we also observed severe impairment of T helper type 2 cytokine responses and intestinal mucosal mastocytosis in STAT6-deficient mice, although some degree of intestinal tissue eosinophilia was evident in these animals. These results are consistent with the hypothesis that STAT6-dependent changes in intestinal muscle function contribute to host protection in nematode infection.

Immunopathology of intestinal helminth infection.

The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Parasite expulsion is associated with intestinal pathology in several model systems and both of these phenomena are T cell dependent. However, while immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been elucidated. In contrast, the intestinal pathology observed in many other disease models closely resembles that seen in helminth infections, but has been attributed to Th1 cytokines. We have used infection with the nematode Trichinella spiralis in mice defective for cytokines to demonstrate that although parasite expulsion is indeed IL-4 dependent, contrary to expectations, the enteropathy is also regulated by IL-4. Furthermore, abrogation of severe pathology in iNOS deficient and TNF receptor defective animals does not prevent parasite expulsion. TNF and iNOS are therefore involved in intestinal pathology in nematode infections, apparently under regulation by IL-4 and Th2 mediated responses. Therefore, it appears that the IL-4-dependent protective response against the parasite operates by a mechanism other than merely the gross degradation of the parasite's environment brought about by the immune enteropathy. However, it remains important to elucidate the protective mechanisms involved in parasite expulsion, which are still unclear.

The efficacy and tolerability of triclabendazole in Cuban patients with latent and chronic Fasciola hepatica infection.

Current chemotherapy for the treatment of infections caused by the liver fluke Fasciola hepatica is not satisfactory. Therefore, the efficacy and tolerability of triclabendazole (TCZ) was assessed for this indication. Eighty-two patients (51 female, 31 male, age 15-81 yr, mean 42 yr) with chronic or latent F. hepatica infection refractory to previous anti-helminthic chemotherapy were enrolled in a 60-day open, non-comparative trial. Patients received 20 mg/kg TCZ as two doses of 10 mg/kg administered after food 12 hr apart. Efficacy of treatment was assessed by stool microscopy, determination of Fasciola excretory-secretory antigen (FES) in feces, and by ultrasonography (US) which were systematically performed pre-therapy and on Days 1-7, 15, 30, and 60 post-therapy. For continuous safety assessment, patients were hospitalized during the first week after therapy and then monitored at home for the appearance of any adverse events. Clinical chemistry and hematology tests were carried out on Days 1, 3, 7, 15, and 60, and whenever an adverse effect occurred possibly related to therapy. Seventy-one (92.2%) of the 77 patients who completed the 60-day follow-up period became egg-negative. Efficacy of therapy was supported by the disappearance or decrease of FES antigen and of ultrasonography abnormalities. In the 6 remaining patients, parasitological cure was achieved by another single TCZ dose of 10 mg/kg on Day 60. A total of 74 adverse events possibly related to therapy was reported by 54 patients. The most important adverse event was colic-like abdominal pain (40 patients [49%]) consistent with the expulsion of the parasite through the bile ducts as confirmed by US on Days 2-7. Most adverse events (53) were graded as mild, 20 as moderate, and only 1 as severe (a biliary colic responding to spasmolytic therapy within two hours). Triclabendazole 20 mg/kg is an effective therapy for the treatment of F. hepatica infection in patients who have failed to respond to other antihelminthic agents. Biliary colics reflecting the expulsion of dead or damaged parasites usually occur during Day 3-7 and respond well to spasmolytic therapy.

Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis

The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague-Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the 'insoluble' mucin complex revealed a relative decrease (4-7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague-Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcalpha2-3(GalNAcbeta1-4)Galbeta1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sd(a)- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcbeta1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.

Acquired resistance in rainbow trout against Gyrodactylus derjavini

Investigations were conducted on the host response in rainbow trout and the associated changes in mucous cell density during infection with the skin monogenean Gyrodactylus derjavini. Parasite populations increased on all naive hosts and peaked 4-5 weeks p.i. after which infection levels decreased. Introduction of naive fish into responding host populations resulted in heavy infections of the naive fish, whereas parasite expulsion continued in the responding host groups showing an acquired, non-sterile immunity. This non-sterile immunity lasted at least a month as these hosts were refractory to reinfection despite being exposed to a high infection pressure. Mucous cell hyperplasia was seen in some groups during the intermediary phase of infection, but at the termination of the study a significant depletion was evident. Passive immunization of naive host (with sera from immune hosts) did not confer protection. This indicates differences between host responses to G. derjavini compared to responses against other pathogens where such a passive immunity has been described.

Acquired resistance in rainbow trout against Gyrodactylus derjavini

AbstractInvestigations were conducted on the host response in rainbow trout and the associated changes in mucous cell density during infection with the skin monogenean Gyrodactylus derjavini. Parasite populations increased on all naive hosts and peaked 4–5 weeks p.i. after which infection levels decreased. Introduction of naive fish into responding host populations resulted in heavy infections of the naive fish, whereas parasite expulsion continued in the responding host groups showing an acquired, non-sterile immunity. This non-sterile immunity lasted at least a month as these hosts were refractory to reinfection despite being exposed to a high infection pressure. Mucous cell hyperplasia was seen in some groups during the intermediary phase of infection, but at the termination of the study a significant depletion was evident. Passive immunization of naive host (with sera from immune hosts) did not confer protection. This indicates differences between host responses to G. derjavini compared to responses against other pathogens where such a passive immunity has been described.

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive.

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.

Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice.

The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1- and Th2-inducing conditions.

The evolutionary ecology of host-specificity: experimental studies with Strongyloides ratti.

Factors constraining the evolution of host-specificity were investigated using a gastrointestinal parasitic nematode, Strongyloides ratti. S. ratti is a natural parasite of rats which can also reproduce, with decreased success, in laboratory mice. Observed host-specificity arose from lower establishment, reduced per capita fecundity and more rapid expulsion of parasites from mice relative to rats. Variation in the efficacy of thymus-dependent immunity between host species (rats and mice) was insufficient to explain the majority of the observed differences in parasite establishment and reproductive success. The role of natural selection in determining host-specificity was addressed using experimental selection followed by reciprocal fitness assays in both host species. Experimental selection failed to modify the host-specificity of S. ratti to any measurable degree, suggesting either a lack of genetic variation for this trait or the involvement of as yet unidentified factors underlying the differences in S. ratti fitness in rats and mice respectively. These results are discussed in relation to competing theoretical models of ecological specialization, host immunology and previous attempts to experimentally alter the host-specificity of parasitic nematodes.

Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.