Using an experimental model of hepatic Echinococcus multilocularis infection in C57BL/6J mice, intraperitoneal administration of 0.8 μg of recombinant IL‐12 to mice with an established infection was shown to reduce the parasite burden as soon as two weeks after the end of treatment. At that time, in vitro Echinococcus multilocularis‐induced spleen T cell proliferative responses as well as IFN‐γ and IL‐5 production were higher in IL‐12 treated mice than in untreated mice. Administration of 0.8 μg of IL‐12 at the time of infection was shown to be without effect on the parasite establishment. However, this treatment greatly inhibited the subsequent metacestode development. Indeed, ten weeks after infection, it induced a complete healing in 37.5% of mice. At that time, the development of metastases was inhibited in 68.75% of IL‐12‐treated mice. This reduction of parasite burden was mainly associated with a strong proliferation of spleen cells to E. multilocularis antigen and with a high IFN‐γ production. Altogether, our results show that IL‐12 is of crucial importance in inhibiting the larval growth after the metacestode establishment in the liver and suggest that this cytokine could be of potential value in the treatment of human alveolar echinococcosis.

As no antiparasitic drug is definitively efficient in patients with alveolar echinococcosis, the effects of exogenous IFN-gamma on murine Echinococcus multilocularis infection were assessed with regards to the parasite burden, parasite-specific immune responses, and the urinary level of the collagen cross-link pyridinolines. They were analyzed after 3-week treatments with 1 or 5 micrograms of IFN-gamma per day twice a week. The treatment with 1 microgram transiently reduced the liver metacestode load, and the metastase weight as far as 6 weeks after the end of treatment. It slightly increased Th 1-type T cell responses and reduced the excretion of pyridinolines. These results should encourage further study to assess whether the decrease in liver fibrosis leads to an improvement of the efficacy of albendazole therapy. In contrast, the treatment with 5 micrograms increased the liver metacestode load and was less efficient than that with 1 microgram in decreasing pyridinoline excretion. These results incitate to follow up carefully patients with alveolar echinococcosis who are treated with IFN-gamma.

The course of Echinococcus multilocularis infection was studied in four different strains of mice after intrahepatic inoculation of a metacestode homogenate. Among these strains of mice, A/J and BALB/c mice were characterized, respectively, as the most resistant and susceptible strains. Although there was no significant difference between the mean surface of hepatic metacestodes of these two strains of mice at any examination time, 13 weeks after infection, the mean metastatic burden of A/J mice was significantly lower than that of BALB/c mice. Moreover, at this time, some BALB/c mice spontaneously died from their infection whereas all A/J mice remained in good health. The relative resistance of A/J mice to parasite development was associated with a strong and sustained in vitro spleen cell proliferative response to a crude E. multilocularis extract as well as with a high parasite‐induced production of IFN‐γ and IL‐2. The susceptibility of BALB/c mice was on the contrary associated with a high IL‐4 production. Interestingly, the parasite extract also stimulated a significant IL‐4 production by spleen cells of uninfected susceptible BALB/c mice, but not by control A/J mouse spleen cells. Altogether, these results suggest that the relative resistance of A/J mice to E. multilocularis growth is associated with the development of T cell responses characterized by the production of high levels of Th1 cytokines.

To determine whether the development of hepatic Echinococcus multilocularis infection is influenced by major histocompatibility-linked genes, metacestode growth and host immune responses were compared in 4 C57BL10 congenic murine strains of H-2b, H-2d, H-2k and H-2q haplotypes. Although the H-2q strain appeared slightly more resistant than the other strains, the 4 strains of mice developed comparable spleen cell proliferative response and Th1Th2 cytokine production at 13 weeks p.i. A kinetic analysis, performed in 2 of these congenic strains, showed a similar pattern of parasite growth in these mice and failed to detect any significant difference in the production of parasite-specific IgM, IgG1 and IgG2a antibodies. Consequently, this study indicates that the control of secondary alveolar echinococcosis is not H-2 gene-linked.

SUMMARYSpecific and non‐specific parasite‐induced changes in lymphocyte responses were analysed in C57BL/6J mice after intrahepatic infection with Echinococcus multilocularis. Spleen cells harvested at selected times after infection were in vitro stimulated with mitogens or a crude soluble parasitic extract (EmAg) at an optimized dose. Cell proliferative responses to Con‐A were not modified by the infection over the first 22 weeks. In contrast, LPS‐induced responses were decreased from the 13th week. A strong CD4 + proliferative T‐cell response to the parasitic extract of infected mouse spleen cells was observed at the early stage of infection. This response then progressively decreased but remained significantly higher than that of control mice until the 19th week of infection. Cytokine production was investigated after in vitro EmAg stimulation of spleen cells. IFN‐γ, IL‐2. IL‐5 were produced within the first weeks after infection whereas the detection of IL‐10 was slightly delayed. Thus, the promotion of the disease does not appear associated with the expansion of one rather than another T‐cell subset in C57BL/6J mice. A general immunosuppression affecting both mitogenic and parasite‐specific T‐cell responses was observed at the end of the infection.

The aim of this study was to apply the enzymatic MTT (3,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] — formazan colorimetry for quantifying the viability of Echinococcus multilocularis whole cysts, after maintenance in vivo or in vitro. The enzymatic activities of young cysts freshly removed from rodents were linearly correlated with the parasite cyst weight. A comparative evaluation of the MTT assay and the in vitro viability assessments showed that the number of animals used for drug-screening purposes would be reduced by 35.8%. In this way, the use of different parasite samples removed from the same host is required, because of their different ages and their subsequent different abilities to reduce MTT. Cysts removed from mice exhibited higher colorimetric values than those removed from jirds. Thus, small entire cysts obtained from mice were maintained in the CMRL 1066 culture medium. Their enzymatic activities were evaluated at different times. The results indicate that, in such conditions, the optimal period of time for testing the effect of drugs against the metacestodes is limited to the 10 days following their transfer from mouse to culture flasks. The MTT assay encourages further studies to improve the viability of the whole cysts in vitro, using other standardizable culture conditions.

The loading of poly (D,L-lactide) nanoparticles with ABZ has led us to evaluate the potential of this new colloidal drug delivery system against E. multilocularis, using a murine model of hepatic alveolar echinococcosis. ABZ-loaded nanoparticles had a monodisperse size distribution between 100 and 150 nm. The efficiency of drug loading to nanoparticles was over 97%. In vitro, at an ABZ concentration of 1.0 μg ml−1, the formulation had no toxicity for peritoneal macrophages harvested from uninfected mice, In vivo, the ABZ-loaded nanoparticles exhibited no signs of toxicity at any of the doses tested. Intravenous injections of 6 mg kg−1 of bound ABZ to infected mice had an equivalent antiparasitic effect on the metacestode growth to that of a treatment with 1500 mg kg−1 of orally administered free ABZ. The parasite hepatic superficial size as well as the peritoneal metastatic burden was significantly reduced by these 2 courses of treatment, as compared to those of untreated mice. Our results should encourage further study in order to explain the absence of dose-dependent efficacy of ABZ-loaded nanoparticles demonstrated in the present study.

A specific proliferation of the peripheral blood mononuclear cells (PBMC) stimulated by antigens of Echinococcus sp. has been shown in patients with cystic as well as alveolar echinococcosis. However, the development of a major granulomatous reaction around the parasitic larvae is a characteristic feature of the local immune response to E. multilocularis while humoral immune responses seem to predominate in E. granulosus infection. The aim of this study was to analyse the specific proliferation of the PBMC from 36 patients with alveolar echinococcosis, and 23 controls, induced by a crude preparation of E. multilocularis (EmcAg) as well as by two E. multilocularis specific antigen preparations, the Em2 antigen and the protoscolex (ESAg) antigen. The significant correlation observed between the proliferation index either by Em2 and ES or by Emc suggests that both antigens account for an important part of the lymphocyte proliferative response. The strong effect of these species specific antigens on lymphocyte proliferation is confirmed by the comparison of the results obtained in this study to those obtained in a previous study of specific cellular immunity to E. granulosus antigens in patients with cystic echinococcosis. The proliferation indices were significantly elevated in all those 7 patients with a proven AE who were sero-negative using the Emc ELISA as well as in the 12 patients also seronegative, but using the Em2 ELISA. In 5 seronegative patients who had had a complete resection of the parasitic lesions two years before the lymphocyte proliferation evaluation, the PI was above the threshold value for two dilutions of EmcAg. PI under the threshold values were obtained only in patients with residual lesions. These results suggest that E. multilocularis specific antigens promote the proliferation of lymphocytes which could be TH1 cells, responsible for the intense periparasitic granulomatous reaction characteristic of alveolar echinococcosis.