Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×107 PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3+ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.

Morphological Characteristics of Alveolar and Cystic Echinococcosis Lesions in Human Liver and Bone

Among echinococcoses diseases of human interest, two have a global public health impact: cystic and alveolar echinococcosis caused by Echinococcus granulosus sensu lato and Echinococcus multilocularis, respectively. Cystic and alveolar echinococcosis are neglected infectious diseases epidemiologically and are clinically vastly different with distinct microscopic features. Because of the rareness of these zoonotic diseases, pathologists have limited diagnostic experience in the analysis of the lesions caused by Echinococcus tapeworms. Here, we describe the main microscopic features to be considered to characterize these lesions: laminated layer, central necrosis, growth pattern, and delineation from adjacent tissue. Moreover, immunohistology using monoclonal antibodies is of great diagnostic help in reaching a definitive diagnosis by identifying the laminated body and small particles of E. multilocularis (spems) and small particles of E. granulosus (spegs).

Antigen Similarity In Hydatid Cyst Wall And Human Bone Tumours: A Short Report

Background: less studies have been done on bone cancers which are complex despite lower incidance. Hydatidosis is a parasitic disease that may influence host immunity by mimicking cancer cells antigens. So, this study aimed to elavuate the similarity of the immunogenic antigens between hydatid cyst and different bone cancers.Method: Cyst wall of hydatid cysts were collected and their antigens were separated with SDS-PAGE gel electrophoresis (SDS-PAGE). Serum samples obtained from patients with bone cancers and the anitigenicity of isolated anitgens were evaluated inwith E. granulosus( Larval form )infection and healthy individuals using western-blot approaches. Results: The crude extract of the laminated layer showed two specific antigens, 53 KDa and 70 KDa, after stainging the membrane with Coomassie blue. Both antigens reacted with the serum of different bone cancers but only the 53 KDa band reacted with all sera.Conclusion: It seems people with bone tumours may have extra antibodies in their serum comparing to healthy and hydatidosis which may be an autoantibodies; and the presence of this antibody against 70 KDa band protein in sera of patients with various types of bone cancers, may be helpful in diagnostic test or designing of preventive approaches in future.

Response patterns in adventitial layer of Echinococcus granulosus sensu stricto cysts from naturally infected cattle and sheep

Cystic echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus granulosus sensu lato. The disease is characterized by the development of cystic structures inside viscera of the intermediate host, mainly liver and lungs. These cysts are formed by three layers: germinal, laminated, and adventitial layer, the latter being the local host immune response. Metacestodes that develop protoscoleces, the infective stage to the definitive host, are termed fertile, whereas cysts that do not produce protoscoleces are termed non-fertile. Sheep usually harbor fertile cysts while cattle usually harbor non-fertile cysts. Adventitial layers with fibrotic resolution are associated to fertile cysts, whereas a granulomatous reaction is associated with non-fertile cysts. The aim of this study was to analyze cellular distribution in the adventitial layer of fertile and non-fertile E. granulosus sensu stricto cysts found in liver and lungs of cattle and sheep. A total of 418 cysts were analyzed, 203 from cattle (8 fertile and 195 non-fertile) and 215 from sheep (64 fertile and 151 non-fertile). Fertile cysts from cattle showed mixed patterns of response, with fibrotic resolution and presence of granulomatous response in direct contact with the laminated layer, while sheep fertile cysts always displayed fibrotic resolution next to the laminated layer. Cattle non-fertile cysts display a granulomatous reaction in direct contact with the laminated layer, whereas sheep non-fertile cysts display a granulomatous reaction, but in direct contact with the fibrotic resolution. This shows that cattle and sheep cystic echinococcosis cysts have distinct local immune response patterns, which are associated to metacestode fertility.

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue

Background Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis. Methodology/Principal findings Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative. Conclusions/Significance We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.

Molecular characterization and serodiagnostic potential of Echinococcus granulosus hexokinase

Background Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus (sensu stricto), is a life-threatening but neglected zoonosis. Glycolytic enzymes are crucial molecules for the survival and development of E. granulosus. The aim of this study was to investigate the molecular characterization, immunogenicity, tissue distribution and serodiagnostic potential of E. granulosus hexokinase (EgHK), the first key enzyme in the glycolytic pathway. Methods EgHK was cloned and expressed in Escherichia coli. Specific serum antibodies were evaluated in mice immunized with recombinant EgHK (rEgHK). The location of EgHK in the larval stage of E. granulosus was determined using fluorescence immunohistochemistry, and the potential of rEgHK as a diagnostic antigen was investigated in patients with CE using indirect enzyme-linked immunosorbent assay (ELISA). Results Recombinant EgHK could be identified in the sera of patients with CE and in mouse anti-rEgHK sera. High titers of specific immunoglobulin G were induced in mice after immunization with rEgHK. EgHK was mainly located in the tegument, suckers and hooklets of protoscoleces and in the germinal layer and laminated layer of the cyst wall. The sensitivity and specificity of the rEgHK-ELISA reached 91.3% (42/46) and 87.8% (43/49), respectively. Conclusions We have characterized the sequence, structure and location of EgHK and investigated the immunoreactivity, immunogenicity and serodiagnostic potential of rEgHK. Our results suggest that EgHK may be a promising candidate for the development of vaccines against E. granulosus and an effective antigen for the diagnosis of human CE.

Ultrastructure of Human Fertile Hydatid Cysts Using a Scanning Electron Microscope (SEM)

Introduction: Echinococcosis and hydatidosis caused by the metacestode of Echinococcus granulosus are among the most important zoonotic diseases in the world. This study aims to study the ultrastructure of fertile hydatid cysts that infect humans using a scanning electron microscope (SEM). Materials and Methods: Twenty samples of human fertile hydatid cysts were collected from the human liver and lung after performing surgery operations and examined with an SEM. Results: The results of the electron microscopy with different magnifications revealed that the laminated layer (LL) consists of sheets that appeared more compact and aligned. The brood capsules appeared, consisting of a net of finger-shaped structures that emerged from bulges of various sizes and shapes. Conclusion: Under a transmission electron microscope, it was found that the LL had a coherent and flexible structure, settling on a three-dimensional microscopic network of hydrophilic fibers, with high humidity. These fibers were arranged irregularly and had a diameter of about 10 nm; therefore, the fibers adjacent to the germinal layer (GL) were possibly attached to microtriches of tegument, which reached a thickness of 1 mm in the LL.

Analysis of Vertical Combustion and Carbonization Patterns of Floor Materials When Using a Needle Flame

This study analyzed flame growth characteristics and carbonization patterns when floor materials were burned vertically using a needle flame produced for this study. It was found that PVC flooring was fire retardant and the area under direct flame contracted inward. Vertical combustion causes solidification in the form of a lump at the bottom and also generates soot in a pattern that progresses upwards. This study found that laminated flooring exhibited no fire retarding characteristics and that the laminated layer of its upper surface was destroyed by fire, causing irregular delamination. The carbonization ranges at the left and right sides were determined to be symmetrical. A vertical combustion test of a sample carpet showed that it exhibited no fire-retarding characteristics. It was observed that if heat accumulated in the carpet, the flame formed an ascending air current, and that when flammable materials were present around the flame, they further accelerated the diffusion of the flame. The carbonization pattern at the carpet surface exposed to direct flame revealed that the carpet surface had melted and had flown downwards and that many tiny holes formed on it.