Treatment of Echinococcus granulosus hydatid disease with albendazole

The authors report their personal experience of the treatment of 50 patients with hydatid cysts of different localization with 10-12 mg Albendazole per kg per day for three months without intervals. During the treatment patients were submitted to careful clinical, biochemical, radiological and immunological controls. In all 105 hydatid cysts were observed, and the follow-up periods ranged from six to 42 months. The side effects were not severe. Four patients were considered healed, 31 improved, and 11 showed no change. Three patients relapsed after the end of the therapy, and were treated with a further cycle of Albendazole at the same dose rates, with good effect. The observed results are encouraging, and most of the patients did not require surgery.     

Human hydatid disease in Peru is basically restricted to Echinococcus granulosus genotype G1

A molecular PCR study using DNA from 21 hydatid cysts was performed to determine which strain type is responsible for human infection in Peru. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was amplified in 20 out of 21 samples, revealing that all but 1 sample (19/20, 95%) belonged to the common sheep strain (G1). The remaining samples belonged to the camel strain (G6). The G1 genotype was most frequently found in human cases of cystic hydatid disease (CHD) in Peru. Local control measures should focus primarily on decreasing dog and sheep infection rather than intermediate reservoirs.     

Vaccination of dogs against Echinococcus granulosus, the cause of cystic hydatid disease in humans

Dogs are pivotal in Echinococcus granulosus transmission to humans, and dog vaccination provides a very practical and cost-effective prevention strategy. We vaccinated dogs with soluble native proteins isolated from protoscoleces of E. granulosus and induced significant suppression of worm growth and egg production. Accordingly, we tested for vaccine efficacy using recombinant proteins derived from a developmentally regulated gene family (egM) specifically expressed in mature adult E. granulosus worms. Three egM genes--egM4, egM9, and egM123--were subcloned into an expression vector that expressed the molecules as soluble glutathione S-transferase (GST) fusion proteins in Escherichia coli. The 3 fusion proteins were purified for dog vaccination trials (3 doses of 80 microg/protein/dog) in which the dogs were challenged and then necropsied 45 days after infection. Compared with worms in the control dogs that received GST, the 3 recombinant proteins induced a very high level of protection (97%-100%) in terms of suppression of worm growth and, especially, of egg development and embryogenesis. We have thus shown that vaccination of the dog host against E. granulosus is feasible when recombinant proteins are used. Because the egg stage is crucial in the echinococcal life cycle, successful suppression of egg development by vaccination would halt transmission to intermediate hosts, thereby effecting long-term control.     

Chromatographic and antigenic properties of Echinococcus granulosus hydatid cyst-derived glycolipids

The neutral and acidic fraction glycolipids of Echinococcus granulosus metacestode tissue compartments were isolated, defined by their chromatographic and antigenic properties, and assessed as to their efficacy as antigens in the serodiagnosis of human hepatic cystic and alveolar echinococcosis, and other helminthiases. Analyses were accomplished by thin-layer chromatography immunostaining and ELISA. The neutral glycolipid fraction's major carbohydrate epitope was the same as or very similar to that of Taenia crassiceps neutral glyco(sphingo)lipids, as represented by the 'neogala'-series core structure. The blood group-active, carbohydrate epitope P1 was expressed by a number of neutral fraction glycolipid component bands. The reverse-phase, thin-layer chromatography-isolated neutral fraction glycolipid component, designated Ag1, was efficient in the serological discrimination of cystic echinococcosis medium to high-titred sera. Ag1 did not specifically discriminate low-titred sera, i.e., other human helminthiases. The detected sialic acid residues of the acidic fraction glycolipids, on enzymatic cleavage, were identified as N-acylneuraminic acid and terminal. The acidic fraction glycolipids exhibited the paradox of only chemically minor components being antigenic towards cystic and alveolar echinococcosis infection sera. The combined acidic fraction glycolipid components Ra and Rx were capable of serological discrimination between cystic echinococcosis, alveolar echinococcosis and other helminthiases.     

Immunomodulation by hydatid cyst fluid toxin (Echinococcus granulosus)

Since the experimental infection by hydatid cysts ( Echinococcus granulosus ) in mice causes immunomodulation of the host, the effects of hydatid fluid (HF) and fractions of HF were compared in vitro and in vivo . Fractions of HF were obtained using ammonium sulphate precipitation, chloroform/methanol extraction and thin-layer chromatography (TLC). HF proved to be toxic to murine peritoneal macrophages in vitro , and when macrophages were incubated with the different fractions of HF, most toxicity was found in a single TLC-purified fraction with an adjuvant-like effect on the production of specific antibodies against bovine albumin and human red blood cells in mice. Treatment of mice with the toxin caused a drop in the percentage of peripheral blood lymphocytes. Flow-cytometric analysis showed that T-cells from toxin-treated mice had lower membrane-CD3, CD4 and CD8 density, and had higher percentages of CD8⁺ splenocytes and CD4⁺ thymocytes expressing CD25. The toxin caused a down-regulation of CD4 and CD8 expression on thymocytes in vitro , that was dependent on the presence of macrophages. The results may attribute to these toxins a role in the host-parasite relationship of hydatidosis.     

Analysis of host components in hydatid cyst fluid and immunoblot diagnosis of human Echinococcus granulosus infection.

To improve serodiagnosis of cystic hydatidosis, immunoblotting studies were performed to look for a highly specific parasite antigen(s). First, commercially available hydatid cyst fluid antigen preparations were characterized by SDS-PAGE and by immunoblotting with sera specific for parasite and host animal proteins. One preparation, designed for use in complement fixation tests, did not appear to be suitable for immunoblotting because of the low concentrations of parasite antigens. Several host proteins, including serum albumin and IgG, were detected in the cyst fluid. Sera from patients with Echinococcus granulosus infections and other parasitic diseases were examined by immunoblotting for antibodies against specific cyst fluid parasite antigens. Several parasite antigens were variably recognized. Only one antigen, a 40 kDa protein, was recognized by all E. granulosus-infected patients. Reactivity against this antigen was also observed in all sera from E. multilocularis, cysticercosis, and schistosomiasis patients as well as in some filariasis cases. Two E. granulosus antigens, molecules of 12.5 and approximately 17 kDa, were only recognized by antibodies from some E. granulosus patients.     

Echinococcus granulosus: assays for hydatid immunoregulatory factors using established lymphoid cell lines

Mitosis, mitochondrial metabolic rate and proliferation were measured in established lymphoid cell lines exposed to chromatographic fractions of equine Echinococcus granulosus hydatid fluid. In several cell lines, one or more of the three parameters were modified by the exposure. As an assay for potential immunoregulatory activity, the method was simple and repeatable. The following novel observations were made: (1) Mitotic reaction was found among lines of T-cell, B-cell and macrophage origin; (2) mitosis was accompanied by proliferation in the B-cell lines, B9 and A20, and in the macrophage lines, HL-60 and P388d. With mitotically responsive T-cells, proliferation was slight in CTLL-2 and absent in D10, implying cell-cycle modification; (3) mitotic responsiveness tended to occur in cell lines with mature characteristics; (4) among cytokine-dependent cell lines, hydatid fluid FPLC fraction 1 mimicked IL-1 and several fractions mimicked IL-2 and IL-6 in the maintenance of mitosis; and (5) there was significant statistical interaction between the influences of mammalian cytokines and hydatid fluid fractions, implying that the propensity of antigenically unprimed lymphoid cells to be regulated by E. granulosus is conditioned by cytokine activity.     

Assessment of in vivo complement activation on the Echinococcus granulosus hydatid cyst wall

The larval stage of the parasite Echinococcus granulosus causes hydatid disease. The hydatid cyst is potentially capable of activating host complement, since it is a large, persistent, carbohydrate-rich structure, coated with host immunoglobulins, and localized in the host's internal organs. Nonetheless, in vitro studies have suggested that the cyst surface, the hydatid cyst wall (HCW), is a poor complement activator. In this study, we assessed the occurrence of in vivo complement activation on the hydatid cyst by measuring the levels of two complement activation products, C3d and complexes bearing a C9 activation neoepitope (TCC/MAC), in extracts from HCW of human origin. Low amounts of C3d and TCC/MAC were found in HCW in comparison with their levels in normal human plasma and activated human sera, suggesting that in vivo complement activation on HCW is efficiently down-regulated. This regulation may contribute to limit host inflammation which has been observed to correlate with parasite degeneration and death.     

Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non-reducing SDS-PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH₅PPT (100% vs 83% in pH5PPT-ELISA and 58% in pH₅PPT-IB). AgB-IB achieved higher sensitivity than AgB-ELISA (80% vs 63%). All AgB-IB positive sera recognized the 12 kDa subunit. Qualitative AgB-IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients' PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.     

Long term follow-up of human hydatid disease (Echinococcus granulosus) treated with a high-dose mebendazole regimen

Fifteen patients with inoperable hydatid disease (Echinococcus granulosus) were treated with an initial 6-week high-dose mebendazole regimen with a follow-up ranging from 3-7 years. Ten of 15 patients showed both objective and clinical improvement, although two of these 10 relapsed 1-6 years after completing therapy. Simple, single cysts in the lung and liver showed the best response. Multiple, complex cysts and bone cysts showed little or no objective improvement. One patient developed reversible neutropenia. Overall results were no better than those obtained by others with smaller doses.     

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.     

小鼠细粒棘球蚴体外促血管生成作用研究

目的分析细粒棘球蚴体外对人脐静脉内皮细胞血管生成能力的影响及其促血管生成因子转录水平,初步探讨细粒棘球蚴的促血管生成作用。方法将继发感染小鼠体内的细粒棘球蚴体外培养后的上清作用于体外培养的人脐静脉内皮细胞,观察其成管现象并用NIH Image J软件的Angiogenesis模块进行分析。同时,在细粒棘球蚴基因组中寻找同宿主(小鼠)的基质金属蛋白酶9(Matrix metalloproteinase-9,MMP-9)和高迁移率族蛋白B1(High mobility group box B1,HMGB1)的同源蛋白,并对其转录水平进行分析。结果鼠源细粒棘球蚴囊培养上清能显著促进人脐静脉内皮细胞的成管(F=73.03,P0.001),在细粒棘球蚴囊壁和原头节中存在小鼠MMP-9和HMGB1同源蛋白的表达,且MMP-9在原头节中的转录水平高于棘球蚴囊(t=-11.65,P0.001),而HMGB1在棘球蚴囊中的转录水平高于原头节(t=6.43,P=0.003)。结论细粒棘球蚴囊培养上清可能含有促进血管生成的虫源分子,但其确切机制还有待进一步研究。     

Growth kinetics of leukocyte cell lines cultured with hydatid fluid of Echinococcus granulosus equinus

Oxidation of 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and tritiated thymidine uptake were used to measure, respectively, the viable cell count and the S-phase activity of D10, B9, A20 and p388d leukocytic cell lines cultured in the presence of varying concentrations of fluid from fertile and infertile hydatid cysts of Echinococcus granulosus equinus. Exposure to hydatid fluid raised or lowered the entry of D10 cells into S-phase depending on the concentration of the fluid and of the supporting cytokines. Enlargement of the S-phase population was unaccompanied by increase in viable count indicating that the mitotic cycles induced by hydatid products were not completed. A similar conclusion was reached in respect of the p388d monocytic line, and both changes appear consistent with those occurring in histopathology in vivo. By contrast, the main effect on B9 and A20 B cells was inhibition of entry into S-phase.     

Contribution of C5-mediated mechanisms to host defence against Echinococcus granulosus hydatid infection

The aim of this work was to investigate the contribution of complement C5-mediated mechanisms, with an emphasis on inflammation, to host defences against Echinococcus granulosus hydatid disease. Thus, we compared the systemic and local inflammatory responses induced by the parasite, and the outcome of infection, between congenic C5-sufficient (B10.D2 n/SnJ) and C5-deficient (B10.D2 o/SnJ) mice challenged with protoscoleces. Indirect evidence of in-vivo complement activation during the establishment phase was obtained; infection induced serum amyloid P and eosinophil responses which were dependent on C5. Early recruitment of polymorphonuclear cells was not dependent on the presence of C5. The higher capacity of C5-sufficient mice to recruit eosinophils was also observed during the cystic phase of infection, and mice recruiting more eosinophils developed lower parasite masses. Analysis of the outcome of infection after 8 months showed that C5-sufficient mice were more resistant to infection than C5-deficient mice in terms of individuals with no cysts; this trend was not statistically significant. In addition, C5-deficient mice developed higher numbers of large (> 5 mm in diameter) cysts and higher cyst weights than C5-sufficient mice indicating that C5-mediated mechanisms are detrimental for parasite growth. Taken together, our results suggest that complement, through C5-mediated effectors, contributes to host defences by both restricting the establishment of infection and controlling the growth of established cysts. This contribution may, at least partially, be associated with the ability of C5a to promote eosinophil infiltration.     

Cellular and humoral responses to antigenic subunits of Echinococcus granulosus cyst fluid in hydatid patients

The hydatid fluid antigenic subunits which stimulate the proliferative response of T cells in PBMC isolated by hydatid patients were identified. In the same subjects, the serum IgG antibody profiles were determined by immuno-blotting to compare T cell activation and IgG production. The study was carried out on 18 patients with clinically and serologically diagnosed hydatidosis and on ten healthy blood donors. To assess the proliferative response to the different antigenic subunits, sheep hydatid fluid was separated by the SDS-PAGE and the fractions were blotted onto nitrocellulose and solubilized. The comparison between immunoblotting and PBMC proliferation assay shows that the 55 and 65kDa subunits of antigen 5 and the 12kDa subunit of antigen B are the most reactive subunits in the two techniques. The 16 and 20 kDa subunits of antigen B are more reactive in PBMC proliferation assay than in immunoblotting, thus assigning a specific role to antigen B in cellular response.