Schistosoma mansoni Egg-Secreted Antigens Activate Hepatocellular Carcinoma-Associated Transcription Factors c-Jun and STAT3 in Hamster and Human Hepatocytes.

Clinical data have provided evidence that schistosomiasis can promote hepatocellular carcinogenesis. c-Jun and STAT3 are critical regulators of liver cancer development and progression. The aim of the present study was to investigate the hepatocellular activation of c-Jun and STAT3 by Schistosoma mansoni infection. Expression and function of c-Jun and STAT3 as well as proliferation and DNA repair were analyzed by western blotting, electrophoretic mobility-shift assay, and immunohistochemistry in liver of S. mansoni-infected hamsters, Huh7 cells, primary hepatocytes, and human liver biopsies. Hepatocellular activation of c-Jun was demonstrated by nuclear translocation of c-Jun, enhanced phosphorylation (Ser73), and AP-1/DNA-binding in response to S. mansoni infection. Nuclear c-Jun staining pattern around lodged eggs without ambient immune reaction, and directionally from granuloma to the central veins, suggested that substances released from schistosome eggs were responsible for the observed effects. In addition, hepatocytes with c-Jun activation show cell activation and DNA double-strand breaks. These findings from the hamster model were confirmed by analyses of human biopsies from patients with schistosomiasis. Cell culture experiments finally demonstrated that activation of c-Jun and STAT3 as well as DNA repair were induced by an extract from schistosome eggs (soluble egg antigens) and culture supernatants of live schistosome egg (egg-conditioned medium), and in particular by IPSE/alpha-1, the major component secreted by live schistosome eggs. The permanent activation of hepatocellular carcinoma-associated proto-oncogenes such as c-Jun and associated transcription factors including STAT3 by substances released from tissue-trapped schistosome eggs may be important factors contributing to the development of liver cancer in S. mansoni-infected patients. Therefore, identification and therapeutic targeting of the underlying pathways is a useful strategy to prevent schistosomiasis-associated carcinogenesis.

Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production.

BACKGROUND: Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. METHODS: Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. RESULTS: Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. CONCLUSIONS: Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.

Immunology of cystic echinococcosis (hydatid disease).

BACKGROUND: The neglected disease cystic echinococcosis is caused by larval Echinococcus granulosus flatworms, which form bladder-like hydatid cysts in liver, lungs, and other organs. SOURCES OF DATA: Published literature. AREAS OF AGREEMENT: Establishing larvae are susceptible to antibody-dependent killing, as attested by successful animal vaccination, whereas once established they are partially protected by the so-called laminated layer. Host responses are Th2 dominated, with a Th1 component. Diagnostic antigens from cyst fluid are known, but responses appear absent in one-fifth of patients. AREAS OF CONTROVERSY: Is evasion mainly based on induction of Th2 or regulatory responses by the parasite? GROWING POINTS: The parasite induces regulatory responses. The laminated layer has immune-regulatory properties. AREAS TIMELY FOR DEVELOPING RESEARCH: Develop tools for functional genomics; characterize immunologically interesting proteins suggested by genomic information; analyse infection in broader context of granulomatous responses; identify molecules secreted/excreted by intact larvae/cysts towards their outside, including diffusible immune-regulators.

Study on the mitochondrial apoptosis pathways of small cell lung cancer H446 cells induced by Trichinella spiralis muscle larvae ESPs.

Trichinella spiralis (T.spiralis) muscle-larva (ML) excretory-secretory proteins (ESPs) contain antitumour-active substances. ESPs have been shown to inhibit tumour growth. To explore the effects of these proteins on small cell lung cancer cells and the possible mechanisms of their antineoplastic action, H446 SCLC cells were co-cultured with different concentrations of T. spiralis ML ESPs for 12, 24 and 48 h. Our results showed that T. spiralis ML ESPs significantly inhibited H446 cell proliferation, which was dose-and time-dependent. The results of flow cytometry testing indicate a clear apoptosis trend in H446 cells co-cultured with ESPs for 24 h. Reverse transcription polymerase chain reaction and Western blotting results showed increased expression of pro-apoptosis genes Bax, Cyt-C, Apaf-1, caspase-9 and caspase-3, compared with the negative control group, and decreased the expression of anti-apoptosis genes Bcl-2 and Livin. Our results suggest that T. spiralis ML ESPs can induce apoptosis in H446 cells through a mitochondrial pathway, which may be a mechanism of antineoplastic action in T. spiralis ML ESPs.

Soluble Egg Antigens of Schistosoma japonicum Induce Senescence of Activated Hepatic Stellate Cells by Activation of the FoxO3a/SKP2/P27 Pathway.

BACKGROUND: Liver fibrosis was viewed as a reversible process. The activation of hepatic stellate cells (HSCs) is a key event in the process of liver fibrosis. The induction of senescence of HSCs would accelerate the clearance of the activated HSCs. Previously, we demonstrated that soluble egg antigens (SEA) of Schistosoma japonicum promoted the senescence of HSCs via STAT3/P53/P21 pathway. In this paper, our study was aimed to explore whether there are other signaling pathways in the process of SEA-induced HSCs aging and the underlying effect of SKP2/P27 signal on senescent HSCs. METHODOLOGY/PRINCIPAL FINDINGS: Human hepatic stellate cell line, LX-2 cells, were cultured and stimulated with SEA. Western blot and cellular immunofluorescence analysis were performed to determine the expression of senescence-associated protein, such as P27, SKP2 and FoxO3a. Besides, RNA interfering was applied to knockdown the expression of related protein. The senescence of HSCs was determined by senescence-associated ß-gal staining. We found that SEA increased the expression of P27 protein, whereas it inhibited the expression of SKP2 and FoxO3a. Knockdown of P27 as well as overexpression of SKP2 both suppressed the SEA-induced senescence of HSCs. In addition, the nuclear translocation of FoxO3a from the nucleus to the cytoplasm was induced by SEA stimulation. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that SEA promotes HSCs senescence through the FoxO3a/SKP2/P27 pathway.

LYMPHO-PROLIFERATIVE RESPONSES TO VARIOUS FASCIOLA HEPATICA WORM'S ANTIGENS: AN IN VITRO STUDY.

Fascioliasis is an important zoonotic disease with approximately 2-4 million people infected worldwide and a further 180 million at risk of infection. F. hepatica can survive within the bile ducts for many years through its ability to suppress the host immunity with Fasciola cathepsin L1 cysteine protease and Glutathione S transferase playing an important role. The aim of the present study is to investigate the in vitro lympho-proliferative responses of hepatic hilar lymphocytes (HLN) of infected sheep in response to different F. hepatica antigens. The suppressive effects of Fasciola excretory/secretory (ES) and tegument (TEG) and their fractions were also investigated. Our results showed that both ES and TEG had significant suppressive effects on lympho-proliferation, up to 74% and 92%, respectively. When these antigens were fractionated, fraction 3 (MW of >10000-30000) of both ES (64%) and TEG (59%) in addition to fraction 4 (MW of ≤ 10000) of TEG (38%) inherited the suppressive effects. Identification of the potential molecule(s) with such suppressive effects on lymphocytes in TEG fraction 4 could reveal vaccine candidates.

Schistosoma mansoni antigens as modulators of the allergic inflammatory response in asthma.

Epidemiologic evidence has accumulated suggesting that helminth infection or their products protect against the development of autoimmune and allergic diseases. The mechanisms underlying this protection may include regulatory cells and cytokines. Both helminth infection and allergic diseases drive the immune system toward the Th2 type response with high production of IgE. However, while this antibody response is associated with the pathogenesis of allergic diseases, IgE production in regions endemic for parasite diseases, such as schistosomiasis, might be associated with a protection against infection. In individuals chronically exposed to Schistosoma sp infection, regulatory cells and cytokines which may develop to protect the host against harmful parasite antigens may also protect the host against allergic diseases. We have demonstrated that helminthic infections are associated with a poor response to allergy skin-prick tests and with low asthma pathology. This review summarizes the immune response that is associated with the pathology of allergic diseases such as asthma and with the resistance to helminth infections. Moreover, it is discussed how helminth infection, particularly Schistosoma mansoni or their products may influence the development of atopic asthma.

Concurrent bacterial stimulation alters the function of helminth-activated dendritic cells, resulting in IL-17 induction.

Infection with schistosome helminths is associated with granulomatous inflammation that forms around parasite eggs trapped in host tissues. In severe cases, the resulting fibrosis can lead to organ failure, portal hypertension, and fatal bleeding. Murine studies identified IL-17 as a critical mediator of this immunopathology, and mouse strains that produce high levels of IL-17 in response to schistosome infection show increased mortality. In this article, we demonstrate that schistosome-specific IL-17 induction by dendritic cells from low-pathology C57BL/6 mice is normally regulated by their concomitant induction of IL-10. Simultaneous stimulation of schistosome-exposed C57BL/6 dendritic cells with a heat-killed bacterium enabled these cells to overcome IL-10 regulation and induce IL-17, even in wild-type C57BL/6 recipients. This schistosome-specific IL-17 was dependent on IL-6 production by the copulsed dendritic cells. Coimmunization of C57BL/6 animals with bacterial and schistosome Ags also resulted in schistosome-specific IL-17, and this response was enhanced in the absence of IL-10-mediated immune regulation. Together, our data suggest that the balance of pro- and anti-inflammatory cytokines that determines the severity of pathology during schistosome infection can be influenced not only by host and parasite, but also by concurrent bacterial stimulation.

The pathogenic Th17 cell response to major schistosome egg antigen is sequentially dependent on IL-23 and IL-1ß.

CBA/J mice infected with the helminth Schistosoma mansoni develop severe CD4 T cell-mediated hepatic granulomatous inflammation against parasite eggs associated with a robust Th17 cell response. We investigated the requisites for Th17 cell development using novel CD4 T cells expressing a transgenic TCR specific for the major Sm-p40 egg Ag, which produce IL-17 when stimulated with live schistosome eggs. Neutralization of IL-23 or blockade of the IL-1 receptor, but not IL-6 neutralization, abrogated egg-induced IL-17 secretion by transgenic T cells, whereas exogenous IL-23 or IL-1ß reconstituted their ability to produce IL-17 when stimulated by syngeneic IL-12p40-deficient dendritic cells. Kinetic analysis demonstrated that IL-17 production was initiated by IL-23 and amplified by IL-1ß. Significantly, schistosome-infected IL-12p40-deficient or IL-1R antagonist-treated CBA/J mice developed markedly reduced hepatic immunopathology with a dampened egg Ag-specific IL-17 response. These results demonstrate that the IL-23-IL-1-IL-17 axis has a central role in the development of severe schistosome egg-induced immunopathology.

TpF1 from Treponema pallidum activates inflammasome and promotes the development of regulatory T cells.

Human syphilis is a multistage disease, with diverse and wide-ranging manifestations caused by Treponema pallidum. Despite the fact that a cell-mediated immune response takes part in the course of syphilis, T. pallidum often manages to evade host immunity and, in untreated individuals, may trigger chronic infection. With this study, we demonstrate for the first time, to our knowledge, that Treponema pallidum induces a regulatory T (Treg) response in patients with secondary syphilis and we found that the miniferritin TpF1, produced by the bacterium, is able to expand this response and promote the production of TGF-ß. Accordingly, TpF1 stimulates monocytes to release IL-10 and TGF-ß, the key cytokines in driving Treg cell differentiation. Interestingly, we also found that TpF1 stimulates monocytes to synthesize and release several proinflammatory cytokines, such as TNF-α, IL-6, and IL-1ß, the latter following the activation of the multiprotein complex inflammasome. Collectively, these data strongly support a central role for TpF1 both in the inflammation process, which occurs in particular during the early stage of syphilis, and in the long-term persistence of the spirochete within the host by promoting Treg response and TGF-ß production.

Direct effects of IL-4/IL-13 and the nematode Nippostrongylus brasiliensis on intestinal epithelial cells in vitro.

Nematode infections induce the upregulation of mucin- and glycosylation-related genes in intestinal epithelial cells in vivo. However, the factor(s) that induce these changes in epithelial cells have not been fully elucidated. In the present study, we analysed the effects of the Th2 cytokines IL-4 and IL-13 and the excretory-secretory (ES) product of the nematode Nippostrongylus brasiliensis on the gene expression of the major mucin core peptide MUC2, the sialyltransferase ST3GalIV (Siat4c) and the sulphotransferase HS3ST1 in intestinal epithelium-derived IEC-6 cells by quantitative reverse transcription (RT)-PCR. The administration of IL-4 and IL-13 resulted in a significant upregulation of ST3GalIV and HS3ST1 gene transcription, but had no effect on MUC2, in IEC-6 cells. RT-PCR studies also demonstrated the constitutive expression of IL-13Ralpha1 and IL-4R in IEC-6 cells. On the other hand, the ES product induced upregulation of ST3GalIV, but not HS3ST1 or MUC2, while coadministration of IL-13 and the ES product induced a slight but significant upregulation of MUC2. Co-incubation of live N. brasiliensis adult worms with IEC-6 cells resulted in the upregulation of ST3GalIV and MUC2. These results suggested that HS3ST1 gene expression is strictly regulated by IL-4/IL-13, while ST3GalIV and MUC2 gene expressions are regulated by redundant mechanisms.

Toll-like receptor 2 and Muc2 expression on human intestinal epithelial cells by Gymnophalloides seoi adult antigen.

Goblet cell hyperplasia and mucin hypersecretion are important for the expulsion of the intestinal trematode, Gymnophalloides seoi , from mice. However, regulatory mechanisms underlying these processes remain elusive. To better understand the effects of G. seoi antigen on the host's intestinal epithelial cells, we determined whether G. seoi induces expression of Toll-like receptors (TLRs) and mucin-related (MUC) genes on a human intestinal epithelial cell line (HT29 cells). We treated HT29 cells with G. seoi or other adult helminth antigens and measured mRNAs of TLRs and MUCs. We also performed reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry to determine whether TLR and MUC expression is regulated by interferon (IFN)-gamma, interleukin-4, or monoclonal antibodies (mAbs) against G. seoi 46 kDa antigen. Gymnophalloides seoi antigen significantly induced expression of TLR2 and MUC2 in HT29 cells, and IFN-gamma was found to upregulate TLR2 expression on the surface of the cells. The expression of MUC2 was increased by IFN-gamma, but was decreased significantly via the combination of mAbs-to-human TLRs and G. seoi antigen. These results demonstrated that G. seoi antigen upregulates TLR2 and MUC2 expression on human intestinal epithelial cells. These effects reflect a helminth-induced, IFN-gamma-dependent, and innate mucosal immune mechanism in this human intestinal cell line.

Helminth antigens modulate immune responses in cells from multiple sclerosis patients through TLR2-dependent mechanisms.

To better understand the link between parasite infections and the course of multiple sclerosis (MS), we studied the role of TLRs in helminth product recognition by dendritic cells (DCs) and B cells. Baseline expression of TLR2 was significantly higher in infected-MS patients compared with uninfected MS subjects or healthy controls. Moreover, cells exposed to TLR2 agonists or to soluble egg Ag (SEA) from Schistosoma mansoni resulted in significant TLR2 up-regulation. SEA suppressed the LPS-induced DCs production of IL-1beta, IL-6, IL-12, and TNF-alpha and enhanced TGF-beta as well as IL-10 production. Similarly, after exposure to SEA, anti-CD40-activated B cells increased IL-10 production. Both processes were MyD88 dependent. In addition, SEA down-regulated the expression of LPS-induced costimulatory molecules on DCs in a MyD88-independent manner. DCs stimulation by SEA and TLR2 agonists induced increasing phosphorylation of the MAPK ERK1/2. Neither stimulus showed an effect on p38 and JNK1/2 phosphorylation, however. Addition of the ERK1/2 inhibitor U0126 was associated with dose-dependent inhibition of IL-10 and reciprocal enhancement of IL-12. Finally, cytokine effects and changes observed in DCs costimulatory molecule expression after SEA exposure were lost when TLR2 expression was silenced. Overall, these findings indicate that helminth molecules exert potent regulatory effects on both DCs and B cells through TLR2 regulation conducted via different signaling pathways. This knowledge could prove critical in developing novel therapeutic approaches for the treatment of autoimmune diseases such as MS.

Functional genes and proteins of Trichinella spp.

Research of Trichinella proteins has been conducted with emphasis on excretory-secretory (E-S) products of muscle larvae because of two reasons. The first is that it has prominent and narrow specific antigenicity, and the second is that it may play some role in nurse cell formation after being secreted into host muscle cells. Proteomic analysis of E-S proteins was further advanced by the aid of new analytical methods such as gene cloning, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, and expressed sequence tags database analysis. As the research progressed, the interest of researchers moved to identification of function of E-S products, which has shed further light on the intriguing relationships between parasites and hosts. Major constituents of the E-S products include 43-, 53-, and 45-kDa glycoprotein derived from the stichosome. Many proteins were discovered in E-S products after the 43-, 53-, and 45-kDa proteins although the relationships among them remain unclear. Some of the new proteins were partially defined in terms of their function including nuclear antigens, MyoD-like protein, TsJ5 protein, etc. There are better-characterized proteins based on the gene molecular method, which allow easier identification of the function of proteins of interest. Such examples were demonstrated by proteinases, proteinase inhibitors, heat shock proteins, glycosidases, etc.

Th2 differentiation is unaffected by Jagged2 expression on dendritic cells.

Expression of the Jagged Notch ligands by dendritic cells (DCs) has been suggested to play a role in instructing Th2 responses. Supporting this hypothesis, we found that Jagged2 but not Jagged1 expression, correlates with the ability of DCs to induce Th2 responses. Jagged2 expression is up-regulated in response to the helminth soluble Schistosoma mansoni egg Ag, which conditions DCs to induce Th2 responses, and is markedly down-regulated following exposure to TLR agonists that generally promote Th1 responses. Conversely, Jagged1 expression is markedly induced by TLR ligation. Despite these correlations, suppression of expression of Jagged2 using retrovirally delivered small interfering RNA failed to affect the ability of DCs to induce Th2 cell differentiation either in vitro or in vivo. Moreover, retrovirally induced expression of Jagged2 did not enhance the ability of DCs to induce Th2 cell responses. Our data indicate that Jagged2 expression by DCs is not sufficient or required for Th2 cell differentiation.

Schistosome biology and proteomics: progress and challenges.

The recent availability of schistosomal genome-sequence information allows protein identification in schistosome-derived samples by mass spectrometry (proteomics). Over the last few years, several proteome studies have been performed that addressed important questions in schistosome biology. This review summarizes the applied experimental approaches that have been used so far, it provides an overview of the most important conclusions that can be drawn from the performed studies and finally discusses future challenges in this research area.

Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo.

During infection with the helminth parasite Schistosoma mansoni, the deposition of eggs coincides with the onset of IL-4 production and Th2 development. Although IL-4 is known as a potent inducer of Th2 differentiation, the mechanism by which schistosome eggs induce IL-4 production is not clear. In this study, we demonstrate that the S. mansoni egg Ag (SmEA) induces IgE-dependent IL-4 production by basophils derived from Heligmosomoides polygyrus-infected or OVA/alum-immunized mice in the absence of pathogen-specific IgE. The effect is mediated by the secretory glycoprotein IPSE/alpha-1, because IPSE/alpha-1-depleted SmEA no longer induces cytokine production. Conversely, recombinant IPSE/alpha-1 is sufficient to induce IL-4 production. Importantly, the injection of SmEA or recombinant IPSE/alpha-1 into H. polygyrus-infected 4get/KN2 IL-4 reporter mice rapidly induces the dose-dependent IL-4 production by basophils in the liver, a major site of egg deposition. Thus, IPSE/alpha-1 induces basophils to produce IL-4 even in the absence of Ag-specific IgE.

Excretory-secretory products from Fasciola hepatica induce eosinophil apoptosis by a caspase-dependent mechanism.

Eosinophils (Eo) are known to be important effector cells in the host defense against helminth parasites. Excretory-secretory products (ESP) released by helminths have shown wide immunomodulatory properties, such as the induction of cellular apoptosis. We investigated the ability of ESP from Fasciola hepatica to induce Eo apoptosis. In this work, we observed that ESP induced an early apoptosis of rat peritoneal eosinophils and that this phenomenon was time- and concentration-dependent. Furthermore, we demonstrated that activation of protein tyrosine kinases (TyrK) and caspases were necessary to mediate the Eo apoptosis induced by the ESP, and that carbohydrate components present in these antigens were involved in this effect. We have described for the first time the ability of ESP from F. hepatica to modify the viability of Eo by apoptosis induction. Besides that, we have observed Eo apoptosis in the liver of rats 21 days after F. hepatica infection. The diminution in Eo survival in early infection could be a parasite strategy in order to prevent a host immune response.

A helminth glycan induces APC maturation via alternative NF-kappa B activation independent of I kappa B alpha degradation.

Activation of APCs via TLRs leads to activation of NF-kappaB, a key transcription factor in cells of the immune system most often associated with induction of Th1-type and proinflammatory responses. The neoglycoconjugate lacto-N-fucopentaose III (12-25 molecules)-dextran (LNFPIII-Dex) activates dendritic cells (DCs) via TLR4, as does LPS. However, unlike LPS, LNFPIII-Dex-activated cells induce Th2-type CD4+ T cell responses. This observation led us to ask whether LNFPIII-activated APCs were differentially activating NF-kappaB, and if so, could this partly account for how DCs mature in response to these two different pathogen-associated molecular patterns (PAMPs). In this study, we show that LNFPIII-Dex stimulation of APCs induces rapid, but transient NF-kappaB translocation and activity in the nucleus, in comparison with the persistent activation induced by LPS. We then demonstrate that transient vs persistent NF-kappaB activation has important implications in the development of the APC phenotype, showing that the second wave of NF-kappaB translocation in response to LPS is required for production of the proinflammatory mediator NO. In contrast to LPS, LNFPIII-stimulated APCs that only transiently activate NF-kappaB do not induce degradation of the known IkappaB family members or production of NO. However, cells stimulated with LNFPIII rapidly accumulate p50, suggesting that an alternative p105 degradation-dependent mechanism is primarily responsible for NF-kappaB activation downstream of LNFPIII. Finally, we show that while NF-kappaB translocation in LNFPIII-stimulated APCs is transient, it is required for the development of the DC 2 phenotype, confirming a crucial and multifaceted role for NF-kappaB in innate immune responses.

Lung-migrating digenean parasites: in vitro influence on nitric oxide production from normal rat pulmonary macrophages.

Nitric oxide (NO) is one of the most versatile players in the immune system. Most parasites induce inflammation in the host associated with NO production. Here, we compare the in vitro effect of Schistosoma bovis somatic (SbS) and excretory-secretory (SbES) antigens, and excretory-secretory Paragonimus mexicanus adult worm (PmES) molecules on rat alveolar macrophages NO production measured by the Griess method and by RT-PCR. Additionally, we address the divergence of the NO stimulatory/inhibitory effects of these two parasites. Polymyxin B was used to assess possible LPS contamination. In vitro incubation of rat alveolar macrophages with PmES (10 microg/ml) and SbS (50 microg/ml), but not with SbES extracts, resulted in NO production and an increase in iNOS cell mRNA. This production was specific and inhibited by L-NAME and L-canavanine. Different effects were observed when cells were incubated with P. mexicanus and S. bovis antigens.