The therapeutic potential of the recombinant antigen from Dirofilaria immitis (rDiAg) for immune-mediated pregnancy loss.

The mammalian fetuses are semi-allograft for mothers. Therefore the failure of immunological tolerance often causes pregnancy loss. Recently, the effects of helminthes therapy for immune mediated diseases have been reported. In the present study we employed the murine model to examine the therapeutic potential of the recombinant antigen from a nematoda parasite, Dirofilaria immitis for immune mediated pregnancy loss. Recombinant D. immitis polyproteins (rDiAg) had been cloned and selected by us for the strongest immuno-regulatory activities in parasite antigens. Female CBA/J mice were injected with sterilized rDiAg or PBS solution using micro-osmotic pumps before mating. Pregnant CBA/J mice were sacrificed on day 13.5 for scoring the number of resorbed and viable fetuses for histological and immunological analysis. The serum cytokine concentrations were measured using suspension array system. The resorption rate of mock-treated mice was 42.9% (resorbed fetus 12/total fetus 28). The resorption rate was decreased to 11.1% (resorbed fetus 3/total fetus 27) with rDiAg treatments. The IL-4, IL-23 and TNF-α concentrations in serum were significantly lower in rDiAg-treated mice than mock-treated mice. The serum IL-17 level was also reduced in rDiAg-treated mice but the difference was not significant. The rDiAg treatment reduced the resorption rates of CBA/J×DBA/2J mouse model, which mimic human pregnancy failures with allo-immune backgrounds. Our observations suggest as the first time of therapeutic potentials of the rDiAg for pregnancy loss.

Toxocara canis: search for a potential drug amongst beta-carboline alkaloids--in vitro and mouse studies.

The goal of this study was to search for new treatments for Toxocara canis using both in vitro and in vivo experiments. We specifically looked for a treatment for T. canis larva migrans, and examined beta-carboline alkaloids (17 compounds) with various structural modifications, both in in vitro and in vivo experiments. In the in vitro experiments, screening for nematocidal activity on the T. canis second stage larvae, cytotoxic activity, and immune activity in the host were undertaken. Compound 17 was selected, as it exhibited nematocidal activity for T. canis larvae and did not have any cytotoxic or immunosuppressive activity in the host. The effectiveness of compound 17 was then examined using T. canis larvae infected mice in in vivo experiments. To evaluate the anthelmintic effect, the relative mobility value for the larvae was examined in addition to the number of larvae in the brain, skeletal muscle, and liver. Compound 17 was also examined in both free and liposome-entrapped (LE) forms. Polyethylene glycol (PEG)-LE compound 17 showed an anthelmintic effect in which the number of larvae in the brain was decreased compared free albendazole. PEG-LE compound 17 also effectively suppressed the mobility of the larva in brain and skeletal muscle. The experimental procedure employed assisted in the discovery of this potential candidate and is a promising approach for finding alternative therapeutic regimens for T. canis larva migrans.

The effect of free and polyethylene glycol-liposome-entrapped albendazole on larval mobility and number in Toxocara canis infected mice.

As part of our exploratory drug research on the larva migrans that causes roundworm in dogs and cats, this study was carried out to clarify the effect of free and liposome-entrapped (LE) albendazole in Toxocara canis infected mice. In infected mice, evaluation of mobility and number of larva were examined in detail in the brain, skeletal muscle and liver. Larva mobility was evaluated by using the relative mobility (RM) value. Albendazole was LE as one of the drug delivery systems (DDSs). Polyethylene glycol (PEG) was added to the liposome in order to avoid evoking a response by the reticuloendothelial system (RES). By using the albendazole PEG-LE delivery system, it was possible to target the larvae in the mouse brain and liver resulting in a decrease in the number of larvae. In the skeletal muscle of the infected mice, the intraperitoneal dosages of PEG-LE albendazole did not cause a complete decrease in the number of larvae, even though free albendazole did cause the number to decrease. Therefore, it is necessary to take into consideration the migrating stage of the larvae before the initiation of any drug administration.

Development of membrane-based tests for the detection of urinary antigens and antibodies in human toxoplasmosis: preliminary studies in Ghanaian patients.

Two membrane-based ELISA systems were used in detecting Toxoplasma antigens and anti-Toxoplasma antibodies in urine samples collected from 54 ophthalmology (22 suggestive active and 32 suggestive past infection) patients and 26 pregnant women attending obstetrics/gynaecology clinic (OGP), suspected of toxoplasmosis by eye examination, past medical records and questionnaire, respectively, in Ghana from mid-February to April 2002. The antigen detecting ELISA was able to demonstrate antigen in 100% (22/22) ophthalmology (active infection) and 62.5% (20/32) ophthalmology (past infection) patients, and 42% (11/26) of OGP which included 3 that were sero-negative prior to and during this study, giving an overall prevalence of 66.3% (53/80). The urinary antigen positive samples also included 6 that were negative for both the Dye Test (DT) and latex agglutination test (LAT). Antigen was not detected in the urine of 22 normal (sero-negative for antibodies to Toxoplasma) individuals. The membrane-based urinary antibody detecting sandwich ELISA also detected anti-Toxoplasma antibodies in 100% (22/22) of ophthalmology (active infection) and 81.3% (26/32) of ophthalmology (past infection) patients, a total of 89% (48/54); and 80.8% (21/26) of OGP with an overall prevalence of 86.3% (69/80), including 7 ophthalmology patients' samples that were sero-negative for both DT and LAT. Antibody sero-positivity of the samples was determined by DT as 87% (47/54) in ophthalmology patients and 73.1% (19/26) in pregnant women, LAT as 85.2% (46/54) and 65.4% (17/26), and an overall prevalence as 82.5% (66/80) and 78.8% (63/80), respectively. The membrane-based ELISA systems appear promising but need to be investigated further for its efficacy as reliable diagnostic tests.

The high prevalence of asymptomatic Toxocara infection among schoolchildren in Manado, Indonesia.

We performed a serological survey of Toxocara canis infection in junior high school students from three districts in northern Sulawesi. Almost all of the 117 subjects from two rural districts near Manado allowed dogs in their houses, and there was an 84.6% prevalence of T. canis infection in this group. Fifty-three subjects (45.3%) had serum samples with a high titer of specific anti-Toxocara antibody. By contrast, 41 students tested in one urban district showed a 12.2% prevalence. To confirm the clinical symptoms of visceral larva migrans (VML) and ocular larva migrans (OLM) caused by Toxocara, we administered a questionnaire survey, serological liver function tests, and an ophthalmoscopic examination in 34 subjects having high anti-Toxocara antibodies. One rural district showed a high prevalence; 58 out of 71 subjects (81.7%) had a high titer of anti-Toxocara antibodies according to a plate-ELISA test, although none showed clinical signs. Five of these subjects exhibited hypereosinophilia. These results indicated that T. canis infection in northern Sulawesi is latent in many more cases than previously estimated, and suggest that people living in environments polluted by Toxocara eggs become easily infected with T. canis and show a high prevalence of infection.

Molecules of parasites as immunomodulatory drugs.

Parasite molecules offer unique advantages for the treatment of immunologicical disorders, and several candidate molecules have been shown to be effective. In our studies, it was shown that a factor inducing immunoglobulin E from filarial nematode parasites was suppressive in animal models of immunological disorders such as allergy and insulin dependent diabetes mellitus (IDDM). The Th1/Th2 paradigm of CD4+ T helper cell subsets can provide the basis for the development of new types of drugs and of novel strategies for the treatment of allergic and autoimmune disorders by parasite molecules. In our experimental system, parasite molecules from a filarial nematode parasite led to the down-regulation of the allergic reaction in animal models. In the majority of hosts, infection with helminths is associated with markedly reduced cellular immune reactions and polarization of T cell responses to Th2 and Th3 types. Some studies have suggested that the stimulation of host immunoregulatory networks with parasite molecules leading to the synthesis of anti-inflammatory cytokines (interleukin10, transforming growth factor-beta (TGF-beta and others) can provide new therapy for immunological disorders. It is known that parasites produce some types of molecule that mimic host molecules such as CD40 ligand, TGF-beta and macrophage migration inhibitory factor. These molecules are also candidates for medicinal agents. This review describes many of the latest possibilities in this field and shows how they can be best put to use for the development of medicinal agents, molecular target identification, and for prioritization.

Various types of Dirofilaria immitis polyproteins selectively induce a Th2-Type immune response.

Dirofilaria immitis polyproteins (DiAgs) are found as 15-kDa monomeric and 30-kDa dimeric forms in excretory-secretory products of the adult worm. We evaluated the ability of various types of recombinant DiAg (rDiAg; V1 and V2 as monomers and V1V2, V2V1, V1V1, and V2V2 as dimers) to influence Th1/Th2 immune responses. V1-, V1Vx- and V2-, V2Vx-driven nonspecific immunoglobulin E (IgE) production peaked at 21 and 14 days after administration, respectively. Dimer-induced IgE response was an interesting biphasic pattern with the second peaks on days 35 (V2Vx) or 42 (V1Vx). Absolute amounts of nonspecific IgE production induced with monomers were larger than those observed with dimers at the first peak. The magnitude of cell expansion and interleukin-10 (IL-10) production in mesenteric lymph node (MLN) B-cell induced with rDiAgs was linked to the levels of the first IgE peak in vivo and IgE produced by rDiAg plus IL-4-stimulated B cells in vitro. All rDiAgs failed to augment IgG2c production. V2 and V2Vx elicited IL-4 production by MLN cells more rapidly than V1 and V1Vx. The inhibitory effect of rDiAg on gamma interferon (IFN-gamma) production was stronger in monomers than in dimers. Neutralization of IL-10 restored IFN-gamma production, whereas the expression of IL-4 and IgE was partly prevented by depletion of IL-10. These results indicate that monomer rather than dimer is an efficient form of DiAg and suggest that the difference of IgE-inducing capacity among these DiAgs is closely associated with the pattern of both B-cell activation and IL-4 production.

Cerebellar ataxia due to Toxocara infection in Mongolian gerbils, Meriones unguiculatus.

We assessed the usefulness of gerbils as an experimental model for neurologic toxocarosis. Mongolian gerbils, Meriones unguiculatus, infected with Toxocara canis or Toxocara cati (1000 eggs/gerbil) showed progressive neurologic disorders from 50 days after infection in T. canis-infected gerbils or from 120 days after infection in T. cati-infected gerbils. The incidence of the onset was 6 of the 13 gerbils (49%) in the T. canis-gerbils and 5 of the 7 gerbils (71%) in the T. cati-gerbils. Histopathologically, the cerebellum was the most affected in both groups. We observed loss of Purkinje cells, glial nerve fibers, and nerve sheaths. We also found foci consisting of aggregated macrophages scattered in the white matter of the cerebellum. The affected gerbils showed ataxia and ultimately died of cachexia. Our findings suggest that irreversible neurologic toxocarosis in gerbils can be induced by infection with either T. canis or T. cati.

Inhibitory effect of isoquinoline alkaloids on movement of second-stage larvae of Toxocara canis.

To find new anthelmintics against parasites living in host tissues, we used an in vitro assay to screen isoquinoline alkaloids for nematocidal activity on the larva of dog roundworm, Toxocara canis. To evaluate the efficacy of anthelmintics in vitro, Tsuda et al. previously introduced the concept of Relative Mobility (RM) of Toxocara larvae. After improvement of the assay system using image data processing, we generated a new index, RM(50), the concentration at which RM=50%. However, except for pyrantel, the RM(50) of most existing anthelmintics could not be calculated because of low activity. Of the isoquinoline alkaloids tested, emetine, sanguinarine, 6-methoxydihydrosanguinarine (6-MS), chelerythrine and berberine showed strong nematocidal activities. However, these compounds were highly cytotoxic; thus, the prospect of their direct application is low. We then tested the cytotoxicity (IC(50)) of other isoquinoline alkaloids in HL60 tissue-culture cells. We continued our search for new anthelmintics by examining in detail the relationship between RM(50) and IC(50). We determined that an ideal target compound would exhibit a low RM(50)/IC(50) ratio. Allocryptopine, dehydrocorydaline and papaverine were identified as potentially effective anthelmintics.

A Dirofilaria immitis polyprotein up-regulates nitric oxide production.

We investigated the effect of recombinant Dirofilaria immitis polyprotein (rDiAg) on nitric oxide (NO) production by peritoneal macrophages. rDiAg induced NO production by macrophages from wild-type and lipopolysaccharide-hyporesponsive C3H/HeJ, but not CD40(-/-), mice. These results suggest that CD40 is involved in rDiAg-driven NO production by murine macrophages.

Involvement of IL-2/IL-2R system activation by parasite antigen in polyclonal expansion of CD4(+)25(+) HTLV-1-infected T-cells in human carriers of both HTLV-1 and S. stercoralis.

The intermediate state of HTLV-1 infection, often found in individuals dually infected with Strongyloides stercoralis (S. stercoralis) and HTLV-1, is assumed to be a preleukemic state of adult T-cell leukemia (ATL). To investigate the effects of S. stercoralis superinfection on the natural history of HTLV-1 infection, we characterized peripheral blood samples of these individuals in Okinawa, Japan, an endemic area for both HTLV-1 and S. stercoralis and we studied effects of the parasite antigen on T-cells. The dually infected individuals showed a significantly higher provirus load and an increase in CD4(+)25(+) T cell population, with a significant, positive correlation. This increase was attributable to polyclonal expansion of HTLV-1-infected cells, as demonstrated by inverse-long PCR analysis of the integration sites. S. stercoralis antigen activated the IL-2 promoter in reporter gene assays, induced production of IL-2 by PBMC in vitro, and supported growth of IL-2 dependent cell lines immortalized by HTLV-1 infection or the transduction of Tax. Taken collectively, these results indicate that S. stercoralis infection induces polyclonal expansion of HTLV-1-infected cells by activating the IL-2/IL-2R system in dually infected carriers, an event which may be a precipitating factor for ATL and inflammatory diseases.

Recombinant Dirofilaria immitis polyprotein that stimulates murine B cells to produce nonspecific polyclonal immunoglobulin E antibody.

Nonspecific immunoglobulin E (IgE) production is an event characteristically observed in parasitic helminth infections, but its mechanisms are still unclear. To define these mechanisms, we prepared a recombinant Dirofilaria immitis protein (rDiAg) and assessed its effect on nonspecific IgE production. rDiAg preferentially induced nonspecific IgE production, without eliciting specific IgE production, as well as a Th2-type cytokine profile (high interleukin-4 [IL-4] and IL-10 production but low gamma interferon production) in BALB/c mice. rDiAg significantly elicited the proliferative response of naive B cells. This response was not abolished by polymyxin B, an inhibitor of lipopolysaccharide (LPS), and rDiAg normally expanded splenic B cells from LPS nonresponder C3H/HeJ mice. Thus, the mitogenic effect of rDiAg was not due to LPS contamination. rDiAg also enhanced levels of CD23 expression on splenic B cells. Splenic B cells produced marked levels of IgE when cultured with the combination of rDiAg and IL-4 (rDiAg-IL-4), whereas peritoneal B cells produced negligible levels of IgE. rDiAg-IL-4-induced IgE production by splenic B cells was synergistically increased by coculture with peritoneal B cells. rDiAg-driven IL-10 secretion was higher in peritoneal B cells than in splenic B cells. IgE production by splenic B cells cocultured with peritoneal B cells was decreased to a level comparable to that by splenic B cells in the presence of a neutralizing anti-IL-10 monoclonal antibody. Collectively, these results suggest that rDiAg-induced polyclonal expansion and IgE class switching of splenic B cells contribute to nonspecific IgE production and that these responses are enhanced by peritoneal B-cell-derived IL-10.