Structural basis for the immunomodulatory function of cysteine protease inhibitor from human roundworm Ascaris lumbricoides.

Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.

Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides.

Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.

Immunization of rabbits with nematode Ascaris lumbricoides antigens induces antibodies cross-reactive to house dust mite Dermatophagoides farinae antigens.

There are controversial reports on the relationship between helminthic infection and allergic diseases. Although IgE cross-reactivity between nematode Ascaris antigens and house dust-mite allergens in allergic patients have been reported, whether Ascaris or the mite is the primary sensitizer remains unknown. Here we found that immunization of naïve animals with Ascaris lumbricoides (Al) antigens induced production of antibodies cross-reactive to mite antigens from Dermatophagoides farinae (Df). Sera from Bangladeshi children showed IgE reactivity to Ascaris and mite extracts. IgG from rabbits immunized with Al extract exhibited reactivity to Df antigens. Treatment of the anti-Al antibody with Df antigen-coupled beads eliminated the reactivity to Df antigens. In immunoblot analysis, an approximately 100-kDa Df band was the most reactive to anti-Al IgG. The present study is the first step towards the establishment of animal models to study the relationship between Ascaris infection and mite-induced allergic diseases.

[Anti-tumor effect of the whole worm extract of Ascaris lumbricoides on Lewis lung carcinoma in mice].

Forty-five C57BL/6 mice were randomly divided into five groups (A-E). Group B and D served as the control group of A and C. Each mouse of group A was intraperitoneally injected with 0.1 ml whole worm extract of Ascaris lumbricoides every other day, and 10 days later injected with 0.1 ml Lewis lung carcinoma (LLC) cells at right axillary subcutaneously region. Mice of group B were injected with normal saline and then developed tumor model. Each mouse of group C was injected with 0.1 ml LLC cells, and two days later, injected with 0.1 ml whole worm extract of A. lumbricoides every other day for 5 times. After the tumor model developed, mice in group D were injected with normal saline. Group E was the negative control group. Time intervals between implantation and active growth and tumor weight were recorded. Tumor inhibition rate was calculated. The average time interval between tumor implantation and measurable tumor growth for groups A, B, C and D was (7.0 +/-1.1), (6.0 +/- 0.7), (9.0 +/- 1.2) and (7.0 +/- 0.9) days. Tumor weight of [(338.9 +/- 282.2) mg] (P < 0.05). The tumor inhibition rate group A [(722.2 +/- 413.5) mg] was heavier than that of group B was the highest in group C (33.3%). Tumor weight of group C [(237.8 +/- 101.8) mg] was lighter than that of group D [(356.7 +/- 176.9) mg] (P < 0.05). The results indicated that the tumor formation is affected by the whole worm extract of A. lumbricoides which may have an inhibitory effect on tumour growth.

Trickle or clumped infection process? A stochastic model for the infection process of the parasitic roundworm of humans, Ascaris lumbricoides.

The importance of the mode of acquisition of infectious stages of directly-transmitted parasitic helminths has been acknowledged in population dynamics models; hosts may acquire eggs/larvae singly in a "trickle" type manner or in "clumps". Such models have shown that the mode of acquisition influences the distribution and dynamics of parasite loads, the stability of host-parasite systems and the rate of emergence of anthelmintic resistance, yet very few field studies have allowed these questions to be explored with empirical data. We have analysed individual worm weight data for the parasitic roundworm of humans, Ascaris lumbricoides, collected from a three-round chemo-expulsion study in Dhaka, Bangladesh, with the aim of discerning whether a trickle or a clumped infection process predominates. We found that hosts tend to harbour female worms of a similar weight, indicative of a clumped infection process, but acknowledged that unmeasured host heterogeneities (random effects) could not be completely excluded as a cause. Here, we complement our previous statistical analyses using a stochastic infection model to simulate sizes of individual A. lumbricoides infecting a population of humans. We use the intraclass correlation coefficient (ICC) as a quantitative measure of similarity among simulated worm sizes and explore the behaviour of this statistic under assumptions corresponding to trickle or clumped infections and unmeasured host heterogeneities. We confirm that both mechanisms are capable of generating aggregates of similar-sized worms, but that the particular pattern of ICCs described pre- and post-anthelmintic treatment in the data is more consistent with aggregation generated by clumped infections than by host heterogeneities alone. This provides support to the notion that worms may be acquired in clumps. We discuss our results in terms of the population biology of A. lumbricoides and highlight the significance of our modelling approach for the study of the population dynamics of helminth parasites.

Ascaris lumbricoides: alteration of the erythrocyte superficial charge using the Partition Method in aqueous two-phase system.

Sialic acid is responsible for the negative charge of the erythrocyte. The decrease of sialic acid has hemodynamical and hemorheological importance. The aim was to study the effect of A. lumbricoides on the erythrocyte superficial charge using the Partition Method in aqueous two-phase system in order to indirectly evaluate the alteration of sialic acid in the red cells. We worked with five parasite extracts (AE) and larvae concentrate (LC). Erythrocyte superficial charge was studied by working with non-treated (Controls) and treated erythrocytes. The treatment consisted of incubating the erythrocytes with AE or LC for 30 minutes at 4 degrees C, 20 degrees C and 37 degrees C. The red cells were separated in a sensitive charge two-phase system (Dx/ PEG). The partition coefficient (P) of treated and untreated erythrocytes were calculated. The results showed a P decrease at the three temperatures for red cells treated with four of the AE. The remaining extract did change P values at any of the temperatures studied. The erythrocytes treated with LC showed a decrease in the P value at 37 degrees C and 4 degrees C but no change was observed at 25 degrees C. Statistical analysis concluded that P values were significantly lower in treated erythrocytes than in their corresponding untreated ones (p < 0.05). The Partition Method showed that this parasite alters the erythrocyte superficial charge which may indicate that it can catch sialic acid.

Ascaris lumbricoides: alteration of the erythrocyte superficial charge using the Partition Method in aqueous two-phase system / Ascaris lumbricoides: alteración de la carga superficial eritrocitaria usando el Método de Partición en sistemas bifásicos acuosos

Sialic acid is responsible for the negative charge of the erythrocyte. The decrease of sialic acid has hemodynamical and hemorheological importance. The aim was to study the effect of A. lumbricoides on the erythrocyte superficial charge using the Partition Method in aqueous two-phase system in order to indirectly evaluate the alteration of sialic acid in the red cells. We worked with five parasite extracts (AE) and larvae concentrate (LC). Erythrocyte superficial charge was studied by working with non-treated (Controls) and treated erythrocytes. The treatment consisted of incubating the erythrocytes with AE or LC for 30 minutes at 4 ºC, 20 ºC and 37 ºC. The red cells were separated in a sensitive charge two-phase system (Dx/ PEG). The partition coefficient (P) of treated and untreated erythrocytes were calculated. The results showed a P decrease at the three temperatures for red cells treated with four of the AE. The remaining extract did change P values at any of the temperatures studied. The erythrocytes treated with LC showed a decrease in the P value at 37 ºC and 4 ºC but no change was observed at 25 ºC. Statistical analysis concluded that P values were significantly lower in treated erythrocytes than in their corresponding untreated ones (p < 0.05). The Partition Method showed that this parasite alters the erythrocyte superficial charge which may indicate that it can catch sialic acid.

La disminución de ácido sialico, responsable de la carga negativa del eritrocito, tiene importancia hemodinámica y hemorreológica. El objetivo fue estudiar el efecto de A. lumbricoides sobre la carga superficial eritrocitaria aplicando el método de partición en sistemas bifásicos acuosos, a los fines de evaluar de manera indirecta la alteración de acido sialico de los eritrocitos. Se trabajó con 5 extractos del parásito adulto (EA) y con un concentrado de larvas (500-600 larvas/mL) (CL). Se estudió la carga superficial eritrocitaria, trabajando con eritrocitos no tratados y tratados. El tratamiento consistió en la incubación de los eritrocitos con EA o CL durante 30 minutos a 4 ºC, 25 ºC y 37 ºC. Los eritrocitos fueron sometidos a la separación en un sistema bifásico carga sensible constituido por Dx / PEG. Se calculó el coeficiente de reparto (P), de los eritrocitos sin tratar y tratados. Los resultados mostraron disminución de P a las 3 temperaturas, en hematíes tratados con 4 de los EA. El EA restante no modificó los valores de P a ninguna de las temperaturas estudiadas. CL produjo la disminución de P a 37 ºC y 4 ºC, pero no se observó modificación a 25 ºC. Los análisis estadísticos concluyeron que los valores de P son significativamente menores en los eritrocitos tratados que en los respectivos eritrocitos sin tratar (p < 0.05). El método de partición demostró que A. lumbricoides altera la carga superficial eritrocitaria lo que indicaría que el parásito, tanto en su estado adulto como en sus fases larvales, puede captar acido sialico.

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach.

BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma. OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides. METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program. RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6. CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Antibody responses to Ascaris-derived proteins and glycolipids: the role of phosphorylcholine.

In addition to proteins, glycolipids can be targets of antibody responses and contribute to host-pathogen interaction. Following the structural analysis of Ascaris lumbricoides-derived glycolipids, the antibody responses of a group of children with no, light and heavy infections were analysed. The role of the phosphorylcholine moiety, present on Ascaris glycoproteins and glycolipids, in antibody reactivity of these infected individuals was determined. Children carrying heavy infections showed highest IgG reactivity to glycolipids compared to lightly or non-infected children. Substantial IgG antibody reactivity to both (glyco)proteins and glycolipids was found to be directed to the phosphorylcholine moiety as determined by either removal of this group or a competition assay. This was most pronounced for glycolipids, where removal of the phosphorylcholine moieties by hydrofluoric acid treatment abrogated IgG antibody reactivity. Measurement of IgG4 and IgE isotypes showed no IgG4 reactivity to Ascaris glycolipids, but raised IgE responses were detected in subjects with light or no Ascaris infections, suggesting that IgE responses to glycolipids may play a role in controlling parasite burden. Differences found in antibody profiles to glycolipids and (glyco)proteins, indicate that these different classes of compounds may have distinct roles in shaping of and interacting with humoral immune responses.

[The second stage larvae of Ascaris lumbricoides].

OBJECTIVE: To induce the apoptosis of human alveolar epithelial cells (A549) by the extraction of the second stage larvae of Ascaris lumbricoides and investigate the extraction concentration and inducing time related to the apoptosis. METHODS: Following to the results of Microculture Tetrazolium Test (MTT), five concentrations of the extraction of the second stage larvae were chosen to induce the apoptosis of A549 cells. Meanwhile, control groups without the inducement were set up. For each group, observation was made at five time points since the start of inducement, to assess the existence of apoptosis and percentage of cells showing characteristics of apoptosis. HE stain and diphenylamine reaction methods were used to assess the cell apoptosis. Agarose gel electrophoresis of DNA and flow cytometry were also employed to confirm the apoptosis for some groups. RESULTS: Observations indicated that the apoptosis ratio of A549 cells induced by the extraction at different concentrations were significantly higher than that of the control cells (P < 0.05). At most time points of observation, the apoptosis ratio increased with the increase of concentration, indicating a positive correlation between them. For each concentration group, the apoptosis ratio increased as the inducing time prolonged until a peak appeared at 5 h of the inducement. CONCLUSION: The extraction of the second stage larvae of Ascaris lumbricoides can induce apoptosis of human alveolar epithelial cells in vitro with a concentration-dependent pattern. With regard to the relationship of apoptosis to the time of inducement, two trends were revealed and the relationship also influenced by the concentration of the larvae extraction.

Inhibition of human napsin A.

The newly-discovered human aspartic proteinase, napsin A was not susceptible to protein inhibitors from potato, squash or yeast but was weakly inhibited by the 17 kDa polypeptide from Ascaris lumbricoides and potently by isovaleryl and lactoyl-pepstatins. A series of synthetic inhibitors was also investigated which contained in the P(1)-P(1)' positions the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues and in which the residues occupying P(4)-P(3)' were varied systematically. On this basis, the active site of napsin A can be readily distinguished from other human aspartic proteinases.

Lung-stage protein profile and antigenic relationship between Ascaris lumbricoides and Ascaris suum.

The protein profile and antigenic properties of lung-stage larvae of Ascaris lumbricoides and A. suum were studied using 2-dimensional electrophoresis and immunoblot analysis, respectively. The protein profiles of the 2 parasites were identical except for the presence of only 1 major protein spot specific for each. There was a complete cross-reactivity between the 2 parasites at the immunological level, and no specific antigen was recognized using specific antibody raised against the 2 parasites in rabbits.

The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties.

The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.

High-resolution structure of Ascaris trypsin inhibitor in solution: direct evidence for a pH-induced conformational transition in the reactive site.

BACKGROUND: The Ascaris trypsin inhibitor (ATI) is a member of a new family of serine protease inhibitors isolated from the helminthic worm Ascaris lumbricoides var suum. This family comprises five chymotrypsin/elastase inhibitors and one trypsin inhibitor. Members are characterized by the presence of five disulfide bonds (two of which are located on either side of the reactive site) in a single small protein domain of 61-62 residues. RESULTS: The solution structure of ATI has been determined at pH 2.4 and pH 4.75 by NMR spectroscopy. Iterative refinement permitted the unambiguous assignment of the pairing of the five disulfide bridges (Cys5-Cys38, Cys15-Cys33, Cys18-Cys29, Cys22-Cys60, and Cys40-Cys54) which were previously unknown. The structure includes four short beta-strands arranged in two approximately perpendicular beta-sheets. The reactive site loop is bounded by two disulfide bridges (Cys15-Cys33 and Cys18-Cys29) and is part of the long loop (residues 15-25) connecting strands beta 1 and beta 2. Comparison of the nuclear Overhauser enhancement data at the two pH values revealed significant differences centered around the reactive site. While the reactive site at pH 2.4 closely resembles that of other protease inhibitors, at pH 4.75 the reactive site loop undergoes a major conformational rearrangement involving the psi backbone torsion angles of the P2, P1 and P1' residues (residues 30-32). This is associated with a change in the positions of the side chains of Arg31 and Glu32. CONCLUSIONS: The overall three-dimensional structure of ATI posesses an unusual fold and, with the exception of the reactive site, shows no similarity to other serine protease inhibitors. The observation that the reactive site of the low pH form of ATI is similar to that of other serine proteases suggests that this is the active form of the protein.

Vitamin A absorption in children with ascariasis.

The effect of Ascaris lumbricoides infection on retinol absorption was investigated in young children from a slum area of Dhaka City, Bangladesh. Twenty-four children aged 4-10 years were screened and in every case eggs of either Ascaris, Trichuris or hookworm were isolated from the stool. The average serum retinol was 0.91 (SD 0.35) mumol/l and sixteen children had levels below 1.05 mumol/l. This compared with a serum retinol concentration of 1.70 (SD 0.52) mumol/l in five reference children from a more privileged social background. An oral dose of retinol (41.8 mumol) was given to ten children in whom the concentration of Ascaris eggs in the stool varied. Less than 1% of the supplement could be recovered in the stools collected over the following 48 h. Ascaris worms were isolated from the stool and assayed for retinol content. In no case was retinol detected in the worms. These findings do not support the contention that infection with Ascaris predisposes to malabsorption of vitamin A.

Proteolysis inhibitors from Ascaris lumbricoides: fact or fiction?

The results of our own 25 years investigations on biological activity of proteolytic inhibitors of Ascaris lumbricoides with the help of various experimental models were discussed. As it results from these studies alpha-chymotrypsin and trypsin inhibitors from A. lumbricoides are not fiction but are substances with significant biological activity, which may be important in regulation of the host-parasite system as well as parasite pathogenic factor.

[The presence of antioxidative defense enzymes in the ascarid Ascaris lumbricoides]. / Nalichie fermentov antiokisilitel'noi zashchity u askardiy Ascaris lumbricoides.

Superoxide dismutase, glutathione peroxidase and glutathione have been discovered in muscles of the ringworm A. lumbricoides. Superoxide dismutase activity was also revealed in eggs and larvae. Using polyacrylamide gel electrophoresis and electrofocusing, it was shown that this enzyme exists in ringworm muscles in multiple molecular forms. Oxygen poisoning of the ringworm and aerobic incubation of muscle homogenates in the presence of Fe+2 do not result in accumulation of malonyldialdehyde. Biochemical defence mechanisms are discussed against oxygen poisoning of the organisms which live under the conditions of reduced and changing pO2.