ES-62 is a phosphorylcholine-containing, 62 kDa glycoprotein derived from the excretory-secretory product of Acanthocheilonema viteae, which is effective for the prevention and treatment of immune dysregulation diseases through triggering activation of immune cells, such as dendritic cells, mononuclear macrophages and regulatory B cells and mediating immune responses. Recently, the role of the ES-62 protein in the management of allergic, autoimmune and metabolic diseases has been paid much attention. This review summarizes the regulatory role of the ES-62 protein in immune dysregulation diseases and the underlying mechanisms, so as to provide insights into future experimental studies.
［摘要］ ES-62 (excretory secretory-62) 是源自魏氏棘唇线虫 (Acanthocheilonema viteae) 排泄分泌物中的一种由磷酰胆碱 修饰的四聚体糖蛋白, 可通过诱导树突状细胞、单核-巨噬细胞和调节性 B 细胞等免疫细胞耐受介导免疫调节反应, 以预 防或治疗免疫失调性疾病。近年来, ES-62 蛋白在过敏性、自身免疫性及代谢性疾病等多种免疫失调性疾病中的作用成 为研究热点。本文综述了 ES-62 蛋白在各种免疫失调性疾病中的调节作用及相关机制, 为进一步研究提供理论依据及 实验参考。.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

Mechanisms of thioredoxin peroxidase secretion by Opisthorchis felineus were studied in vivo and in vitro. Specific antibodies were obtained and used for western blotting and immunohistochemical detection in Syrian hamster model of opisthorchiasis. Secreted thioredoxin peroxidase protein was accumulated in the worm incubation medium under conditions of oxidative stress and in bile duct cells of hamsters with chronic opisthorchiasis.

BACKGROUND: Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ) is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP) of F. hepatica in the presence of TCBZ anthelmintic.
METHODS: F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test) and untreated (control)] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma\'s non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
RESULTS: Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05). SDS-PAGE showed different patterns of protein bands between treated and control samples.
CONCLUSIONS: The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

Parasitic helminths release molecules into their environment, which are generally referred to as excretory-secretory products or ES. ES derived from a wide range of nematodes, trematodes and cestodes have been studied during the past 30-40 years, their characterization evolving from simple biochemical procedures such as SDS-PAGE in the early days to sophisticated proteomics in the 21st century. Study has incorporated investigation of ES structure, potential as vaccines, immunodiagnostic utility, functional activities and immunomodulatory properties. Immunomodulation by ES is increasingly the area of most intensive research with a number of defined helminth products extensively analyzed with respect to the nature of their selective effects on cells of the immune system as well as the molecular mechanisms, which underlie these immunomodulatory effects. As a consequence, we are now beginning to learn the identities of the receptors that ES employ and are increasingly acquiring detailed knowledge of the signalling pathways that they interact with and subvert. Such information is contributing to the growing idea that the anti-inflammatory properties of a number of ES products makes them suitable starting points for the development of novel drugs for treating human inflammatory disease.

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.