Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Extracellular vesicles secreted by Echinococcus multilocularis: important players in angiogenesis promotion.

The involvement of Echinococcus multilocularis, and other parasitic helminths, in regulating host physiology is well recognized, but molecular mechanisms remain unclear. Extracellular vesicles (EVs) released by helminths play important roles in regulating parasite-host interactions by transferring materials to the host. Analysis of protein cargo of EVs from E. multilocularis protoscoleces in the present study revealed a unique composition exclusively associated with vesicle biogenesis. Common proteins in various Echinococcus species were identified, including the classical EVs markers tetraspanins, TSG101 and Alix. Further, unique tegumental antigens were identified which could be exploited as Echinococcus EV markers. Parasite- and host-derived proteins within these EVs are predicted to support important roles in parasite-parasite and parasite-host communication. In addition, the enriched host-derived protein payloads identified in parasite EVs in the present study suggested that they can be involved in focal adhesion and potentially promote angiogenesis. Further, increased angiogenesis was observed in livers of mice infected with E. multilocularis and the expression of several angiogenesis-regulated molecules, including VEGF, MMP9, MCP-1, SDF-1 and serpin E1 were increased. Significantly, EVs released by the E. multilocularis protoscolex promoted proliferation and tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. Taken together, we present the first evidence that tapeworm-secreted EVs may promote angiogenesis in Echinococcus-infections, identifying central mechanisms of Echinococcus-host interactions.

High Expression of Tim-3 in Alveolar Echinococcosis Mediates Depletion of CD8+ T Cell Function.

OBJECTIVE: CD8+ T cells can participate in immune action by secreting various cytokines, which have a killing effect on certain viruses, tumor cells, and other antigenic substances. However, in studies such as chronic viral infections and some parasitic infections, CD8+ T lymphocyte showed functional depletion, and its immune dysfunction was an important reason for the persistence of infection. Tim-3 has been shown to be a negative regulator of CD8+ T cell function, causing depletion of CD8+ T cells in cancer and chronic infection. However, the relationship between Tim-3 and CD8+ T cells in Echinococcus multilocularis infection is not clear. METHODS: In this study, we analyzed peripheral blood CD8+ T cells from 62 alveolar echinococcosis (AE) patients and 30 healthy controls. RESULTS: Compared with the healthy control group, the proportion of CD8+ T cells in the peripheral blood of AE patients increased significantly, while the levels of perforin, granzyme B and IFN-γ in peripheral blood CD8+ T cell related factors of metabolically active alveolar echinococcosis (MAAE) patients decreased significantly. Later detection revealed that the expression of Tim-3 on CD8+ T cells in the peripheral blood of MAAE patients was significantly higher than that of metabolically inactive alveolar echinococcosis (MIAE) patients and healthy controls. The expression levels of function-related factors perforin, granzyme B and IFN-γ in CD8+ Tim-3+ T cell were significantly lower in the CD8+Tim-3- T cells of AE patients. In vitro, the secretion of CD8+ T cell-associated factors was significantly restored by inhibiting Tim-3 expression. CONCLUSION: Therefore, the depletion of CD8+ T lymphocyte in patients with alveolar echinococcosis disease is considered to be related to the high expression of Tim-3 on the surface.

Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

BACKGROUND AND AIMS: Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. APPROACH AND RESULTS: Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. CONCLUSIONS: Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Gene expression in human liver fibrosis associated with Echinococcus granulosus sensu lato.

Liver fibrosis is a dynamic process that occurs in response to chronic liver disease resulting from factors such as chronic infections, autoimmune reactions, allergic responses, toxins, radiation, and infectious agents. Among the infectious agents, multicellular parasites cause chronic inflammation and fibrosis. Twenty-five patients with different stages of cystic echinococcosis (CE) were enrolled in the study. The expression of ACTA2, COL3A1, IFN-γ, MMP2, MMP9, TGF-ß1, and TNF-α genes was determined by qRT-PCR in healthy and fibrotic liver tissue of the CE patients. TGF-ß1 expression was evaluated by immunohistochemistry, and histology was conducted to assess the development of liver fibrosis. Expression of MMP9, ACTA2, COL3A1, and MMP2 was found significantly higher in the fibrotic tissue compared to healthy tissue. We observed a significant correlation between TGF-ß1 and TNF-α gene expressions and liver fibrosis. The mRNA level of IFN-γ was lower in the fibrotic than in the healthy hepatic tissue. Immunohistochemistry analysis revealed TGF-ß1 upregulation in the fibrotic tissue. Histology showed inflammation and fibrosis to be significantly higher in the fibrotic tissue. The findings of this study suggest that Echinococcus granulosussensu lato can promotes fibrosis through the overexpression of TGF-ß1, MMP9, ACTA2, COL3A1, and MMP2. The downregulation of IFN-γ mRNA in fibrotic samples is probably due to the increased production of TGF-ß1 and the suppression of potential anti-fibrotic role of IFN-γ during advanced liver injury caused by E. granulosussensu lato.

Regulation of hepatic microRNAs in response to early stage Echinococcus multilocularis egg infection in C57BL/6 mice.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

Linking murine resistance to secondary cystic echinococcosis with antibody responses targeting Echinococcus granulosus tegumental antigens.

Successful establishment of a parasite infection depends partially on the host intrinsic susceptibility to the pathogen. In cystic echinococcosis (CE), a zoonotic disease caused by the cestode parasite Echinococcus granulosus, the infection outcome in the murine model of secondary CE varies according to the mouse strain used. In this regard, intrinsic differences in susceptibility to the infection were previously reported for Balb/c and C57Bl/6 mice, being C57Bl/6 animals less permissive to secondary CE. Induction of parasite-specific antibodies has been suggested to play relevant roles in such susceptibility/resistance phenomena. Here, we report an in deep comparison of antibody responses induced in both mouse strains. Firstly, only C57Bl/6 mice were shown to induce specific-antibodies with efficient anti-parasite activities during early secondary CE. Then, through ImmunoTEM and Serological Proteome Analysis (SERPA), an evaluation of specific antibody responses targeting parasite tegumental antigens was performed. Both strategies showed that infected C57Bl/6 mice -unlike Balb/c animals- narrowed their IgG recognition repertoire against tegumental antigens, targeting fewer but potentially more relevant parasite components. In this sense, tegumental antigens recognition between Balb/c and C57Bl/6 mice, either by natural and/or induced antibodies, was analyzed through SERPA and MALDI-TOF/TOF studies. A total of 13 differentially recognized proteins (DRPs) uniquely targeted by antibodies from C57Bl/6 mice were successfully identified, wherein a subset of 7 DRPs were only recognized by infection-induced antibodies, suggesting their potential as natural protective antigens. In this regard, immunoinformatic analyses showed that such DRPs exhibited higher numbers of possible T cell epitopes towards the H-2-IAb haplotype, which is present in C57Bl/6 mice but absent in Balb/c animals. In summary, our results showed that the genetic predisposition to generate better T-dependent antibody responses against particular tegumental antigens might be a key factor influencing host susceptibility in the murine model of secondary CE.

Proteomic Analysis on Exosomes Derived from Patients' Sera Infected with Echinococcus granulosus.

Cystic echinococcosis (CE), a zoonotic disease caused by Echinococcus granulosus at the larval stage, predominantly develops in the liver and lungs of intermediate hosts and eventually results in organ malfunction or even death. The interaction between E. granulosus and human body is incompletely understood. Exosomes are nanosized particles ubiquitously present in human body fluids. Exosomes carry biomolecules that facilitate communication between cells. To the best of our knowledge, the role of exosomes in patients with CE is not reported. Here, we isolated exosomes from the sera of patients with CE (CE-exo) and healthy donors and subjected them to liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis identified 49 proteins specifically expressed in CE-exo, including 4 proteins of parasitic origin. The most valuable parasitic proteins included tubulin alpha-1C chain and histone H4. And 8 proteins were differentially regulated in CE-exo (fold change>1.5), as analyzed with bioinformatic methods such as annotation and functional enrichment analyses. These findings may improve our understanding about the interaction between E. granulosus and human body, and may contribute to the diagnosis and prevention of CE.

The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis.

BACKGROUND: Scavenger Receptors (SRs) from the host's innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses. METHODOLOGY/PRINCIPAL FINDINGS: We report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis. CONCLUSIONS/SIGNIFICANCE: Taken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis.

Increase of Vascular Endothelial Growth Factor and Decrease of MCP-1 and Some Updated Epidemiology Aspects of Cystic Echinococcosis Human Cases in Calabria Region.

We aim to investigate some of the pathogenetic mediators of the human echinococcosis and to obtain updated epidemiological findings on cases of echinococcosis in Calabria, Southern Italy. Echinococcosis diagnosis was based on imaging, serological investigations, and molecular assay. Indeed, real-time PCR indicated the presence of G2/G3 genotypes of Echinococcus granulosus complex. Regarding pathogenesis, a relevant novel tool of immune depression should be deemed the reduced level of serum MCP-1. Also, we found a previously unreported VEGF, possibly associated with neovascularization requested by the parasite cyst metabolism. Cytokine profiles suggest a bias of the immunity toward Th2 and Treg responses. Nitric oxide levels exhibited a significant decrease one week after therapy versus basal level measured before surgery and/or chemotherapy. An increase of serum total IgE class and IgG4 subclass was found in Echinococcus-positive patients versus controls. Our data demonstrated an endemic spreading, at least in the province of Catanzaro and neighboring Calabria territories, for such parasitosis with the novel issue of the number of female overcoming male cases. In conclusion, the novel findings of this study were the increased VEGF and the reduced serum MCP-1 in the studied cases, as well as the number of Echinococcus-infected females overcoming the infected males.

Monoclonal Antibodies Production Against a 40KDa Band of Hydatid Cyst Fluid.

BACKGROUND AND OBJECTIVES: Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus. Hydatid cyst fluid, cyst membrane and Protoscolices, contain a complex mixture of antigens that can induce immune responses in the host. Anti-cancer properties of Protoscolices and hydatid cyst fluid has been shown. In order to identify antigens of hydatid cyst fluid that have anti-cancer effect, in this study production of monoclonal antibodies against one of the hydatid cyst fluid band (40KDa) has been investigated. There are many published patents about applications of monoclonal antibodies. METHODS: In this experimental study, 40KDa band of hydatid cyst fluid that has cross reaction with sera of patients with breast cancer was used as antigen. A group of mice were immunized with this antigen, and then their spleen cells were extracted and fused with SP2 cells. Monoclonal antibodies production was checked in wells with signs of cell growth using ELISA and western blotting. The reaction of the produced monoclonal antibodies with breast cancer cells was tested using flow cytometry method. Finally, effect of the monoclonal antibodies on growth of breast cancer cells was investigated in vitro. RESULTS: The results of this study showed that in the first plate antibody against 40KDa was detected in several wells. In the second plate monoclonal antibodies with high titer was detected in one well. The produced monoclonal antibodies reacted with the surface of breast cancer cells. However, they had no significant effect on growth of breast cancer cells in culture medium. CONCLUSION: Monoclonal antibodies against hydatid cyst fluid 40KDa band were produced. These antibodies reacted with the surface of breast cancer cells but had no significant effect on growth of these cells.

Larval Echinococcus multilocularis infection reduces dextran sulphate sodium-induced colitis in mice by attenuating T helper type 1/type 17-mediated immune reactions.

The tumour-like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune-mediated processes. Parasite-mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS) -induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT-PCR. Colitis severity was assessed (by board-certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi-quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS-induced gut inflammation by concomitant E. multilocularis infection, which correlated with down-regulation of T helper type 1 (Th1)/Th17 T-cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS-induced gut inflammation upon down-regulation of Th1/Th17 cytokine expression and attenuation of CD11b+ cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS-induced colitis in mice by attenuating Th1/Th17-mediated immune reactions.

Impairment of Macrophage Presenting Ability and Viability by Echinococcus granulosus Antigens.

BACKGROUND: Despite advances toward an improved understanding of the evasive mechanisms leading to the establishment of cystic echinococcosis, the discovery of specific immunosuppressive mechanisms and related factors are of great interest in the development of an immunotherapeutic approach. OBJECTIVE: To elucidate immunosuppressive effects of bioactive factors contained in chromatographic fractions from hydatid cystic fluid (HCF) of Echinococcus granulosus. METHODS: Hydatid cystic fluid was fractionated by reverse phase chromatography. Non-specific Concanavalin A-driven proliferation of spleen cells was used to determine specific inhibitory fractions. Trypan blue exclusion test and flowcytometry analysis were performed to check whether highly inhibitory fractions of HCF have apoptotic effect on peritoneal macrophages. Western blot analysis was used to determine proteolytic effects of parasitic antigens on major histocompatibility complex (MHC) class II (I-a) contained in membrane proteins extract from macrophages. RESULTS: High concentrations of HCF and few of chromatographic fractions suppressed spleen cells proliferation. Fractions 7 and 35 were the highest inhibitory fractions. Specifically fraction 35 and to a lesser extent HCF induced apoptosis in peritoneal naive macrophages. However, HCF and the fraction 7 proteolytically altered the expression of MHC class II molecules on peritoneal macrophages. The proteolytic molecule was identified to be a serine protease. Macrophages taken at the chronic and end phase from cystic echinococcosis-infected mice were able to uptake and process C-Ovalbumine-FITC. These cells expressed a drastically reduced level of (I-a) molecules. CONCLUSION: Our study present new aspects of immune suppression function of E. granulosus. Further molecular characterization of apoptotic and proteolytic factors might be useful to develop immunotherapeutic procedure to break down their inhibitory effects.

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels.

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.

Post-immunization immunohistochemical expression of Caspase 3 and p53 apoptotic markers in experimental hydatidosis.

The aim of this study was to investigate post-immunization apoptotic changes in experimental hydatidosis, using Caspase 3 and p53 immunohistochemical markers. Two groups of rabbits were immunized with a crude antigen (group 1) or a partially purified antigen (group 2) and were compared to an infected non-immunized control group. More effective immune responses were obtained in group 2 than group 1, signified by fewer and smaller cystic lesions and more severe destructive changes. Normal growth of cysts was attained in the control group, with no expression of apoptotic markers. Significantly higher expression of Caspase 3 and p53 were observed in group 1 compared to group 2, as indicated by OD and area percentage, respectively (Group 1 Caspase 3: 0.89±0.21, 93.5%±6.2; Group 1 p53: 0.46±0.18, 53.26%±11.6; Group 2 Caspase 3: 0.52±0.15, 49.23%±11.7; Group 2 p53: 0.19±0.4, 18.17%±7.3). Vaccine-induced immune responses and cellular damage may underlie the expression of apoptotic markers that appeared to result in a degenerative and atrophic course of action upon immunization. The results of the current study emphasize the importance of immunization for the stimulation of protective immune responses and in preventing mechanisms of evasion to ensure normal cell growth. A cost/benefit control program that implements proper vaccine preparations should be further assessed for complete elimination of severe infections in endemic areas.

Post-immunization immunohistochemical expression of Caspase 3 and p53 apoptotic markers in experimental hydatidosis / Expressão imuno-histoquímica de marcadores de apoptose Caspase 3 e p53 pós-imunização em hidatidose experimental

Abstract The aim of this study was to investigate post-immunization apoptotic changes in experimental hydatidosis, using Caspase 3 and p53 immunohistochemical markers. Two groups of rabbits were immunized with a crude antigen (group 1) or a partially purified antigen (group 2) and were compared to an infected non-immunized control group. More effective immune responses were obtained in group 2 than group 1, signified by fewer and smaller cystic lesions and more severe destructive changes. Normal growth of cysts was attained in the control group, with no expression of apoptotic markers. Significantly higher expression of Caspase 3 and p53 were observed in group 1 compared to group 2, as indicated by OD and area percentage, respectively (Group 1 Caspase 3: 0.89±0.21, 93.5%±6.2; Group 1 p53: 0.46±0.18, 53.26%±11.6; Group 2 Caspase 3: 0.52±0.15, 49.23%±11.7; Group 2 p53: 0.19±0.4, 18.17%±7.3). Vaccine-induced immune responses and cellular damage may underlie the expression of apoptotic markers that appeared to result in a degenerative and atrophic course of action upon immunization. The results of the current study emphasize the importance of immunization for the stimulation of protective immune responses and in preventing mechanisms of evasion to ensure normal cell growth. A cost/benefit control program that implements proper vaccine preparations should be further assessed for complete elimination of severe infections in endemic areas.

Resumo O objetivo do presente estudo foi investigar mudanças de apoptose pós-imunização em hidatidose experimental, usando os marcadores imuno-histoquímicos Caspase 3 e p53. Dois grupos de coelhos foram imunizados com antígeno bruto (grupo 1) e com antígeno parcialmente purificado (grupo 2). Estes grupos foram comparados a um grupo controle infectado e não-imunizado. Respostas imunes mais eficientes foram obtidas do grupo 2, que apresentou lesões císticas menores e menos frequentes, e mudanças destrutivas mais graves. Cistos cresceram normalmente no grupo controle, sem expressão dos marcadores de apoptose. Expressões significativamente mais altas de Caspase 3 e p53 foram observadas no grupo 1 quando comparado ao grupo 2, como indicado por DO e área de percentagem, respectivamente (Grupo 1 Caspase 3: 0,89±0,21, 93,5%±6,2; Grupo 1 p53: 0,46±0,18, 53,26%±11,6; Grupo 2 Caspase 3: 0,52±0,15, 49,23%±11,7; Grupo 2 p53: 0,19±0,4, 18,17%±7,3). Respostas imunológicas induzidas por vacinas e danos celulares podem ser a base para a expressão dos marcadores de apoptose cujos desfechos demonstraram ação degenerativa e atrófica durante imunização. Os resultados do presente estudo enfatizam a importância da imunização para o estímulo de respostas imunes de proteção e para mecanismos de prevenção de evasão para garantir crescimento celular normal. Um programa de controle de custo/benefício que implemente preparações de vacinas adequadas deve ser analisado em mais detalhe para a completa eliminação de infecções graves em áreas endêmicas.

Intra-cystic concentrations of albendazole-sulphoxide in human cystic echinococcosis: a systematic review and analysis of individual patient data.

Cystic echinococcosis (CE) is a widespread zoonosis caused by the species complex Echinococcus granulosus. Albendazole (ABZ)-the first-line anthelminthic drug for medical treatment of CE-is metabolized in vivo to the active derivative ABZ-sulphoxide (ABZ-SO). Target-site ABZ-SO concentrations in the hydatid cyst mediate the anthelminthic effect in CE. Primary outcome of this systematic review of individual patient data was the intra-cystic ABZ-SO concentration stratified by cyst size, location, calcification status and use of praziquantel. Studies reporting intra-cystic ABZ-SO concentrations in humans were identified by a systematic search. A pooled analysis of individual patient data was performed to assess intra-cystic concentrations. Pharmacokinetic data of 121 individual cysts were analysed. There was no correlation between plasma and intra-cystic ABZ-SO concentrations (rho = -0.03, p = 0.76). Intra-cystic drug concentrations were also not associated with sex and treatment duration. Use of praziquantel in combination with ABZ was associated with higher plasma (median 540 vs. 240 µg/L; p = 0.04) but not intra-cystic ABZ-SO concentrations (median 220 vs. 199 µg/L; p = 0.36). Relative drug concentrations in hepatic cysts were higher than in other cysts (0.8 vs. 0.4; p = 0.05). Intra-cystic concentrations were higher in calcified than non-calcified cysts (median 897 vs. 245 µg/L; p = 0.03). There was a trend towards higher intra-cystic concentrations in smaller sized cysts (ß = -17.2 µg/L/cm; 95th CI, -35.9 to 1.6; p = 0.07). This study demonstrates that mean intra-cystic drug concentrations are similar to plasma concentrations on a population level. However, in individual patients plasma concentrations are not directly predictive for intra-cystic concentrations. The use of booster drugs was not associated with higher intra-cystic ABZ-SO concentrations in this analysis.

Hidatidosis paravesical. Presentación de un caso clínico / Bladder hidatidosis. Presentation of a clinical case

La hidatidosis primaria pélvica o paravesical es una forma excepcional de presentación de la enfermedad, mucho más común en pulmón, hígado y riñón. La clínica suele corresponder a trastornos irritativos miccionales. El diagnóstico de imagen es por ecografía, TAC y RNM. La serología es complementaria. El tratamiento médico es con albendazol pero la solución definitiva del quiste hidatídico paravesical es la cirugía. Se presenta un caso clínico de quiste hidatídico paravesical tratado con cirugía abierta, con una breve revisión de la literatura (AU)

Primary pelvic or paravesical hydatid disease is an exceptional presentation of the disease, more common in lung, liver and kidney. The symptoms are typically for irritative micturition disorders. The image diagnosis is by ultrasound, CT and MRI. The serology is complementary. Albendazole is pharmacological treatment but the final solution for the paravesical hydatid cyst is surgery. We present a case of paravesical hydatid cyst treated with open surgery, with a brief review of the literatura (AU)