Strongyloides venezuelensis-derived venestatin ameliorates asthma pathogenesis by suppressing receptor for advanced glycation end-products-mediated signaling.

INTRODUCTION: EF-hand Ca2+-binding proteins such as S100 protein family members are recognized by the receptor for advanced glycation end-products (RAGE) and are involved in the pathogenesis of asthma/allergic airway inflammation (AAI). Venestatin, an EF-hand Ca2+-binding protein, which is secreted by the parasitic helminth Strongyloides venezuelensis, binds with RAGE and suppresses RAGE-mediated inflammatory responses after parasite invasion. In this study, we evaluated the effect of venestatin on pathogenesis in a house dust mite (HDM) murine model of asthma/AAI. METHODS: Mice were intranasally treated with HDM, HDM with recombinant venestatin, or HDM with synthetic peptides, which were designed based on the EF-hand Ca2+-binding domain of venestatin. Pro-inflammatory responses in the lungs of mice were assessed. RESULTS: HDM treatment induced inflammatory cell infiltration, phosphorylation of the mitogen-activated protein kinase and inhibitor κB, and production of the cytokines tumor necrosis factor-α and interleukin-5 in the lungs. Co-administration of recombinant venestatin with HDM suppressed these pro-inflammatory responses. Treatment with synthetic peptides reduced inflammatory cell infiltration in a RAGE-dependent manner. CONCLUSION: The EF-hand domain of venestatin may have potential therapeutic benefits in asthma.

Secretome analysis of Strongyloides venezuelensis parasitic stages reveals that soluble and insoluble proteins are involved in its parasitism.

BACKGROUND: Parasites excrete and secrete a wide range of molecules that act as the primary interface with their hosts and play critical roles in establishing parasitism during different stages of infection. Strongyloides venezuelensis is a gastrointestinal parasite of rats that is widely used as a laboratory model and is known to produce both soluble and insoluble (adhesive) secretions during its parasitic stages. However, little is known about the constituents of these secretions. RESULTS: Using mass spectrometry, we identified 436 proteins from the infective third-stage larvae (iL3s) and 196 proteins from the parasitic females of S. venezuelensis. The proteins that were secreted by the iL3s were enriched with peptidase activity, embryo development and the oxidation-reduction process, while those of the parasitic females were associated with glycolysis, DNA binding (histones) and other unknown functions. Trypsin inhibitor-like domain-containing proteins were identified as the main component of the adhesive secretion from parasitic females. An absence of secretion signals in many of the proteins indicated that they are secreted via non-classical secretion pathways. CONCLUSIONS: We found that S. venezuelensis secretes a wide range of proteins to establish parasitism. This includes proteins that have previously been identified as being involved in parasitism in other helminths as well as proteins that are unique to this species. These findings provide insights into the molecular mechanisms underlying Strongyloides parasitism.

First molecular identification of Strongyloides fuelleborni in long-tailed macaques in Thailand and Lao People's Democratic Republic reveals considerable genetic diversity.

Strongyloides fuelleborni is a soil-transmitted nematode parasite of non-human primates. The worm is prevalent also in human populations in Africa and South-East Asia. In this study, we amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides adult males recovered from faecal samples from long-tailed macaques (Macaca fascicularis) in Thailand and Lao PDR. The prevalence in Thailand was 31.1% (55/177) and in Lao PDR it was 62.1% (41/66), with an overall prevalence of 39.5% (96/243). All 18S rRNA sequences that we obtained (n = 96) showed 100% identity with published S. fuelleborni sequences. The 96 cox1 sequences that we obtained represented 32 new haplotypes. When included with the 17 previously known haplotypes from S. fuelleborni, the cox1 sequences fell into four clusters, which had clear geographical structure. This is the first molecular confirmation of S. fuelleborni in long-tailed macaques in Thailand and Lao PDR. Clearly, awareness needs to be raised of the zoonotic potential of S. fuelleborni. A monitoring programme should be organized, taking into account the role of reservoir hosts (i.e. monkeys) in the natural background of human strongyloidiasis caused by S. fuelleborni.

Ion-exchange protocol to obtain antigenic fractions with potential for serodiagnosis of strongyloidiasis.

The aim of this study was to fractionate and partially characterize the antigenic extract of filariform larvae of Strongyloides venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays. Somatic antigen (SA) and its fractions DEAE S1 and DEAE S2 - which interacted with the resin - were evaluated by 1-dimensional electrophoresis to obtain protein profiles. SA and its fractions were tested in serum samples for IgG detection by ELISA. Serum samples (n = 155) were analysed: 50 from strongyloidiasis patients (G1), 55 from patients with other parasitic infections (G2) and 50 from healthy volunteers. Sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR) were calculated. The DEAE S2 fraction provided a high diagnostic value for IgG detection (Se 92·0%, Sp 91·4%, AUC 0·981, LR+ 10·75, LR - 0·09). In conclusion, the DEAE S2 fraction would probably be a source of immunodominant polypeptides for IgG detection in human strongyloidiasis serodiagnosis.

F-actin accumulates in the vulva of female Strongyloides venezuelensis.

Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis.

Usefulness of concanavalin-A non-binding fraction of Strongyloides venezuelensis larvae to detect IgG and IgA in human strongyloidiasis.

Glycosylated components from Strongyloides have an important role in parasite establishment and host recognition of these substances. Considering the sugar-binding capacity of lectins, such as concanavalin-A (Con-A), IgG and IgA detection in serum samples from strongyloidiasis patients was tested using different antigenic preparations. The total saline extract (SE) of Strongyloides venezuelensis filariform larvae was fractionated in Con-A column to obtain Con-A unbound (Con-A UF) and Con-A bound (Con-A BF) fractions. Sensitivity (Se), specificity (Sp), area under the ROC curve (AUC), likelihood ratio (LR), and correlation coefficients were calculated. Con-A UF showed the highest diagnostic parameters for IgG detection (Se 95.0%, Sp 92.5%, AUC 0.99, LR 12.7) and high correlation (r = 0.700) with SE. Con-A fractions did not clearly demonstrate any usefulness for IgA detection. In conclusion, the results obtained demonstrate that Con-A UF is an important source of specific peptides efficient to detect IgG in strongyloidiasis immunodiagnosis.

Hydrophobic fractions from Strongyloides venezuelensis for use in the human immunodiagnosis of strongyloidiasis.

The objective of the present research was to evaluate detergent and aqueous phases of total saline (TS) and alkaline extracts of Strongyloides venezuelensis for human strongyloidiasis immunodiagnosis. Total extracts and detergent and aqueous antigenic fractions were separated using Triton X-114 and were examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) tests to detect immunoglobulin G (IgG). Serum samples were obtained from 120 individuals: 40 strongyloidiasis patients (group I), 40 patients with other parasitic diseases (group II), and 40 apparently healthy individuals (group III). Each extract provided a different profile of antigenic components as recognized by IgG in IB. The detergent fraction of the TS extract demonstrated the highest sensitivity and specificity for ELISA and IB. The results indicated that the detergent saline fraction, purified from S. venezuelensis, furnished the most valid results for the strongyloidiasis immunodiagnosis and could be employed as an alternative antigen and as a useful source of specific polypeptides.

Development in vitro of free-living infective larvae to the parasitic stage of Strongyloides venezuelensis by temperature shift.

Free-living infective larvae Strongyloides venezuelensis were cultured in Dulbecco's modified Eagle's medium at 25 and 37 degrees C, and development to the parasitic stage was evaluated using morphological, protein and antigenicity criteria. Few larvae cultured at 25 degrees C showed development whereas, in most of the larvae cultured at 37 degrees C, there appeared characteristic changes such as a bulb-like head and droplets under the cuticle with an increase of body width of the larvae. The results obtained from two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) revealed that the protein spot patterns of the larvae cultured at 25 and 37 degrees C were differentiated by 17 specific spots. In addition, Western blot analysis combined with 2D-PAGE for reaction with serum obtained from an infected rat revealed that protein spots showing immunodominant antigen at 37 degrees C were almost the same as those of the larvae recovered from the rats rather than those of the larvae at 25 degrees C. These results strongly suggested that a temperature shift from 25 to 37 degrees C has an important role in the development of free-living infective larvae to the parasitic stage of S. venezuelensis. The culture system established in the present study was useful for biological and biochemical studies in the development from/of the free-living to the parasitic stage of Strongyloides species.

Protein analysis of Strongyloides venezuelensis by two-dimensional polyacrylamide gel electrophoresis.

Infective larvae, larvae in the lung and adult-stage worms in the small intestine of Strongyloides venezuelensis were analysed for protein by two-dimensional gel electrophoresis. The infective larvae were differentiated from the other two stages of parasite with 13 stage-specific spots, whereas the larvae in the lung and the adult-stage worms were identical to each other in spot patterns except for 6 spots.