Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator.

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a ß-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Significant apoptosis rather autophagy predominates in astrocytes caused by Toxocara canis larval excretory-secretory antigens.

BACKGROUND/PURPOSE: Toxocariasis is a worldwide parasitic zoonosis and mainly caused by Toxocara canis. Humans can be infected by accidental ingestion of T. canis embryonated ova through contacting with contaminated food, water, or encapsulated larvae in paratenic hosts' viscera or meat. Since humans are the paratenic host of T. canis, the wandering and neuroinvasive larvae can cause mechanical tissue damage and the excretory-secretory antigens (TcES Ag) might induce neuroinflammatory responses in the brain. Human cerebral toxocariasis (CT) has been reported to cause several neurological symptoms and may develop into neurodegenerative diseases. However, the roles of astrocytes involved in the pathogenesis of CT remained largely unclear. METHODS: This study intended to investigate the cytotoxic effects of TcES Ag on astrocytes as assessed by apoptosis and autophagy expression. RESULTS: Our results showed TcES Ag treatment reduced cell viability and caused morphological changes. Expressions of autophagy associated proteins including Beclin 1, phosphor-mTOR and LC3-Ⅱ were not significantly changed; however, p62 as well as the cell survival protein, mTOR, was concomitantly decreased in TcES Ag treatment. Significantly accelerated cleaved caspase-3 and cytochrome c expression as well as enhanced caspase-9 and caspase-8 activation were found in astrocytes with TcES Ag treatment. Caspase-3 activity and apoptotic cells numbers were also increased as detected by fluorescence microscopy. CONCLUSION: We concluded that TcES Ag may trigger astrocytes apoptosis predominantly through intrinsic and extrinsic pathways rather autophagy, revealing a novel role of astrocytes in the pathogenesis of CT.

Clonorchis sinensis excretory-secretory products increase malignant characteristics of cholangiocarcinoma cells in three-dimensional co-culture with biliary ductal plates.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-ß1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

Toll-Like Receptor 7/8 Ligand, S28463, Suppresses Ascaris suum-induced Allergic Asthma in Nonhuman Primates.

S28463 (S28), a ligand for Toll-like receptor 7/8, has been shown to have antiinflammatory properties in rodent models of allergic asthma. The principle goal of this study was to assess whether these antiinflammatory effects can also be observed in a nonhuman primate (NHP) model of allergic asthma. NHPs were sensitized then challenged with natural allergen, Ascaris suum extract. The animals were treated with S28 orally before each allergen challenge. The protective effect of S28 in NHPs was assessed by measuring various asthma-related phenotypes. We also characterized the metabolomic and proteomic signatures of the lung environment and plasma to identify markers associated with the disease and treatment. Our data demonstrate that clinically relevant parameters, such as wheal and flare response, blood IgE levels, recruitment of white blood cells to the bronchoalveolar space, and lung responsiveness, are decreased in the S28-treated allergic NHPs compared with nontreated allergic NHPs. Furthermore, we also identified markers that can distinguish allergic from nonallergic or allergic and drug-treated NHPs, such as metabolites, phosphocreatine and glutathione, in the plasma and BAL fluid, respectively; and inflammatory cytokines, IL-5 and IL-13, in the bronchoalveolar lavage fluid. Our preclinical study demonstrates that S28 has potential as a treatment for allergic asthma in primate species closely related to humans. Combined with our previous findings, we demonstrate that S28 is effective in different models of asthma and in different species, and has the antiinflammatory properties clinically relevant for the treatment of allergic asthma.

Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro.

BACKGROUND: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. METHODOLOGY/PRINCIPAL FINDINGS: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. CONCLUSIONS: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.

Immunomodulatory properties of ES-62, a phosphorylcholine-containing glycoprotein secreted by Acanthocheilonema viteae.

Filarial nematodes are parasites that have the ability to persist in their hosts for extended periods of time due to the employment of various mechanisms to divert or down-regulate the host's immune responses. One of these mechanisms is the production of immunomodulatory excretory-secretory (ES) products. This review will discuss the properties of one such product, ES-62, which over the years, has been shown to interact with and modulate the activities of a variety of cells of the immune system including B and T lymphocytes, dendritic cells, macrophages and mast cells. Overall, ES-62 diverts the immune system towards an anti-inflammatory phenotype and consistent with this it has been shown to have therapeutic potential in models of inflammatory disease associated with autoimmunity and allergy.

Effect of Fasciola hepatica proteins on the functioning of rat hepatocytes.

Fasciolosis is a hepatic parasitic infection that affects many mammal species and creates a great economic and veterinary problem. Molecular mechanisms of parasite-hepatocyte interactions have not been precisely characterized yet. Therefore, the aim of the study was to investigate alterations in the metabolic activity of rat liver cells exposed to Fasciola hepatica somatic proteins. Hepatocytes were incubated with 0-1 mg/ml of fluke's somatic proteins for various periods of time. Afterward, changes in hepatocytes metabolic activity were determined with MTT and enzyme leakage tests. Hepatocytes' capacity to synthesize albumin was also investigated. It was observed that protein concentration, as well as longevity of their action, influenced metabolic activity of rat liver cells. Diminution of hepatocytes survival rate, an increase in enzyme leakage and altered synthetic capacity after treatment with parasite's proteins were reported. It is concluded that somatic proteins of F. hepatica may play an important role in liver cell damaging.

Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs.

BACKGROUND: Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. CONCLUSIONS: We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.

Induction of dominant lethal mutations by Ascaris trypsin inhibitor in male mice.

Trypsin inhibitor (ATI) isolated from Ascaris suum, a gastrointestinal nematode parasite, was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ATI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Three groups of adult BALB/c males received 50, 100 or 250mg/kg body weight (bw) single intraperitoneal (ip) injection of ATI in sterile phosphate-buffered saline (PBS). The control group received concurrent injection of PBS. After the administration of ATI or PBS, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, and spermatogonia), every second week two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ATI did not impair mating activity of males. Pregnancy rates were reduced ( approximately 5-10%) by treatment of males with higher doses of ATI, but differences between treatment and control groups were not statistically significant (P>0.05). In the females bred to ATI-treated males, significant increase in preimplantation loss was observed at post-injection week 1 (reflecting exposure to spermatozoa) and 3 (reflecting exposure to mid and early spermatids) for higher doses of the inhibitor (P<0.05 or P<0.01). During mating days 15-21 a statistically significant increase in postimplantation loss and dominant lethal effects were observed for all doses of ATI. At higher doses, dominant lethal effects were restricted to spermatozoa (P<0.01). These data suggest that ATI induces dominant lethal mutations at postmeiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ATI. These preliminary findings show that ATI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

Cytogenetic study of Ascaris trypsin inhibitor in cultured human lymphocytes with metabolic activation.

The trypsin inhibitor (ATI) isolated from gastrointestinal nematode Ascaris suum was tested in vitro for induction of chromosome aberrations and sister chromatid exchanges (SCE). Genotoxicity assessment of purified ATI was carried out on metaphase plates received from peripheral blood lymphocyte macroculture (48 h test of structural chromosome aberrations and 72 h test of SCE) with exogenous metabolic activation. ATI was tested in dose of 25, 50 and 100 microg per ml of culture. Kinetics of cell divisions were determined by the replication index (RI). The mitotic index (MI) was expressed as a number of metaphases per 1000 nuclei analysed. Analysis of chromosome aberrations showed that higher doses of ATI (50 and 100 microg/ml) significantly increased the frequency of chromosome aberrations (mainly of chromatid gaps and breaks) compared to the negative control. All concentrations of ATI caused a statistically significant reduction in the MI and RI. In comparison with the negative control, a significant increase in the SCE frequency was observed in all applied doses of ATI. Thus, in the presence of S9 activation, the Ascaris trypsin inhibitor showed potential clastogenic activity and inhibition of the dynamics of lymphocyte divisions.

C-type lectin Mermaid inhibits dendritic cell mediated HIV-1 transmission to CD4+ T cells.

Dendritic cells (DCs) are important in HIV-1 transmission; DCs capture invading HIV-1 through the interaction of the gp120 oligosaccharides with the C-type lectin DC-SIGN and migrate to the lymphoid tissues where HIV-1 is transmitted to T cells. Thus, the HIV-1 envelope glycoprotein gp120 is an attractive target to prevent interactions with DCs and subsequent viral transmission. Here, we have investigated whether the structural homologue of DC-SIGN, the nematode C-type lectin Mermaid can be used to prevent HIV-1 transmission by DCs. Our data demonstrate that Mermaid interacts with high mannose structures present on HIV-1 gp120 and thereby inhibits HIV-1 binding to DC-SIGN on DCs. Moreover, Mermaid inhibits DC-SIGN-mediated HIV-1 transmission from DC to T cells. We have identified Mermaid as a non-cytotoxic agent that shares the glycan specificity with DC-SIGN and inhibits DC-SIGN-gp120 interaction. The results are important for the anti-HIV-1 microbicide development directed at preventing DC-HIV-1 interactions.

Toxocara canis larval excretory/secretory proteins impair eosinophil-dependent resistance of mice to Nippostrongylus brasiliensis.

Survival of parasitic helminths within a host requires immune evasion and excretory/secretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis. Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro. Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined.

Prenatal toxicity of Ascaris pepsin inhibitor in mice.

The developmental toxicity of pepsin inhibitor isolated from Ascaris suum, a gastrointestinal nematode parasite, was evaluated. An embryo-fetal development study was conducted in BALB/c mice. Groups of 21 mated females were treated by intraperitoneal injection (0.3 ml/30 g body weight) with 0.9% NaCl solution vehicle or isolated Ascaris pepsin inhibitor (API) at dose levels of 50, 100, 150 or 200mg/kg body weight/day on gestation days (GD) 6-15. Maternal food consumption, body weight, and clinical signs were monitored throughout gestation. Cesarean sections were performed on GD 18 and gravid uterine weight, implantation sites, early and late resorptions, live and dead fetuses were collected. Live fetuses were weighed and examined for external, visceral and skeletal variations and malformations. Maternal body weight gain, gravid uterine weight, food consumption were significantly decreased after injection of higher doses of API (100-200mg/kg/day). All doses of API exhibited an embryotoxic effect (high rate of intrauterine resorption). The percentage of postimplantation loss in the groups with administered API was higher (over 4-11 times) than that in control group. Fetotoxicity was observed in all treatment groups in a dose-related manner and it was evidenced by increased dead fetuses, decreased fetal weight, increased visceral variations and reduced skeletal ossification. Fetal hydronephrosis and internal hydrocephalus were noted at 150, and 200mg/kg/day. In summary, the maternal toxicity no-observed-adverse-effect level (NOAEL) was 50mg/kg/day and the low-observed-adverse-effect level (LOAEL) was 100mg/kg/day under the conditions of this study. However, the developmental toxicity LOAEL was 50mg/kg/day based on decreased fetal body weight and prenatal mortality.

In vitro genotoxic and cytotoxic effects of protein somatic products from helminths on donor blood lymphocytes.

Protein somatic products of adult helminths Hymenolepis nana and Toxocara canis and secretory-excretory somatic product of Trichinella spiralis larvae in vitro produced genotoxic and cytotoxic effects on donor blood lymphocytes, which manifested in accumulation of single-chain breaks, alkaline-labile sites in nuclear DNA, and apoptotic cells. This effect depended on the concentration of parasitic protein products during coculturing.

Effect of Ascaris chymotrypsin inhibitor on fetal development of mice.

BALB/c mice were given daily doses of 40-80 mg of Ascaris chymotrypsin inhibitor /AChI/ per kg body weight from 12th until 15th day of gestation (stage of fetal development). It has been found that injection of AChI disturbed the course of pregnancy (bleeding from uterus, abortions, decreased body weight gain as compared to control /p < 0.05/). AChI exhibited embryotoxic effects (a high rate of intrauterine deaths, decreased number of living fetuses and mean fetal weight, delayed skeletal ossification, induced pathological changes of fetal organs and tissues). Congenital malformation (hydronephrosis) was noted in fetuses after injection of higher doses of the chymotrypsin inhibitor from Ascaris.

Preliminary evaluation of maternotoxic effect of Ascaris extract in mice.

Administration intraperitoneally of the Ascaris suum extract--ASE-(0.6-1.4 g of Ascaris proteins/kg/day) at a late stage of organogenesis (8-12 days of gestation) disturbed course of mouse pregnancy. It has been found that injections of higher doses of ASE to pregnant mice caused the symptoms manifesting maternal toxicity (decreased body weight gain/p < 0.001/as compared to control, intrauterine resorption of litter, vaginal hemorrhages, female mortality and altered behaviour). There is a linear interrelationship between the logarithm of the dose of ASE and mortality of pregnant mice. The DL50 value of Ascaris proteins for pregnant mice was 1.02 g/kg/day (confidence interval 0.97-1.07 g/kg/day). ASE exerted embryotoxic effects: significantly decreased the number of surviving fetuses per litter and the mean body weight of fetuses, increased the number of fetal resorptions.

Comparison of the effect of Ascaris trypsin inhibitor in various periods of mouse pregnancy.

Trypsin inhibitor isolated from Ascaris suum and injected into pregnant BALB/c mice (five times, in doses: 300 or 400 mg/kg/day) in various periods of pregnancy (early and late organogenesis) disturbed the development of fetuses. The nature and intensity of prenatal disturbances are determined by the inhibitor dose and time of injection. It has been found that administration of the inhibitor from 5-th until 9-th day of gestation did not delay or prevent implantation, but caused a high rate of intrauterine deaths and also specific congenital malformations (exnencephaly and hydrocephalus). Additionally, other types of defects were noted in fetuses after injection of the inhibitor between 8-th and 12-th day of pregnancy (cleft palate, fusion of ribs). Independent of the time of injection during gestation the inhibitor exhibited embriotoxic effects (e.g. decreased the number of live fetuses per litter and mean fetal weight).

Preliminary evaluation of maternotoxic effect of Ascaris alpha-chymotrypsin inhibitor in mice.

Administration intraperitoneally of the Ascaris alpha-chymotrypsin inhibitor (40-300 mg/kg/day) at a late stage of organogenesis (8-12 days of gestation) disturbed course of mouse pregnancy. The low doses of alpha-chymotrypsin inhibitor (40-80 mg/kg/day) significantly decreased the number of live fetuses per litter, increased the number of fetal resorptions. The symptoms of maternal toxicity that occurred after administration of the highest doses of the inhibitor (80-300 mg/kg/day) to pregnant mice included: decreased body weight gain as compared to control, vaginal hemorrhage, intrauterine resorption of litters, abortions, altered behaviour of animals immediately after injection and death. There is a linear interrelationship between the logarithm of the doses of the inhibitor and mortality of pregnant mice. The DL50 value of the inhibitor for female was 116 mg/kg/day (confidence interval: 95.5-140.0 mg/kg/day).

The alterations in genetic apparatus of somatic and generative cells of the host caused by helminths metabolites.

Alterations in the genetic apparatus of mice bone marrow cells and testicles caused by Hymenolepis nana, Ascaris suum and Toxocara canis metabolites have been investigated by means of micronuclear test application. The activity of spermatogenesis has been investigated at experimental hymenolepidosis, migrating ascariasis and toxocarosis with the use of 3H-timidine. Helminths metabolites have been established to exert a mutagenic effect on somatic cells of bone marrow, spermatogonies and also on the generative cells (spermatides) of helminths in invaded mice. The concurrent increase in micronucleus number in erythrocytes, spermatogonies and in spermatides (to a lesser degree) of invaded mice has been revealed. The decrease in spermatogenesis activity has been established in experimental hymenolepidosis, migrating ascariasis and toxocarosis in invaded mice.