Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens-Factors Linked to Virulence and Pathogenicity.

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens' virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.

The Cysteine Protease Giardipain-1 from Giardia duodenalis Contributes to a Disruption of Intestinal Homeostasis.

In giardiasis, diarrhoea, dehydration, malabsorption, weight loss and/or chronic inflammation are indicative of epithelial barrier dysfunction. However, the pathogenesis of giardiasis is still enigmatic in many aspects. Here, we show evidence that a cysteine protease of Giardia duodenalis called giardipain-1, contributes to the pathogenesis of giardiasis induced by trophozoites of the WB strain. In an experimental system, we demonstrate that purified giardipain-1 induces apoptosis and extrusion of epithelial cells at the tips of the villi in infected jirds (Meriones unguiculatus). Moreover, jird infection with trophozoites expressing giardipain-1 resulted in intestinal epithelial damage, cellular infiltration, crypt hyperplasia, goblet cell hypertrophy and oedema. Pathological alterations were more pronounced when jirds were infected intragastrically with Giardia trophozoites that stably overexpress giardipain-1. Furthermore, Giardia colonization in jirds results in a chronic inflammation that could relate to the dysbiosis triggered by the protist. Taken together, these results reveal that giardipain-1 plays a key role in the pathogenesis of giardiasis.

Giardia duodenalis enolase is secreted as monomer during trophozoite-epithelial cell interactions, activates plasminogen and induces necroptotic damage.

Enolase, a multifunctional protein expressed by multiple pathogens activates plasminogen to promote proteolysis on components of the extracellular matrix, an important event in early host-pathogen interactions. A secreted form of enolase that is released upon the interaction of trophozoites with epithelial cells has been detected in the secretome of G. duodenalis. However, the role of enolase in the host-pathogen interactions remains largely unknown. In this work, the effects of G. duodenalis enolase (Gd-eno) on the epithelial cell model (IEC-6) were analyzed. Firstly, the coding sequence of Giardia enolase was cloned and the recombinant protein used to raise antibodies that were then used to define the localization and role of enolase in epithelial cell-trophozoite interactions. Gd-eno was detected in small cytoplasmic vesicles as well as at the surface and is enriched in the region of the ventral disk of Giardia trophozoites. Moreover, the blocking of the soluble monomeric form of the enzyme, which is secreted upon interaction with IEC-6 cells by the anti-rGd-eno antibodies, significantly inhibited trophozoite attachment to intestinal IEC-6 cell monolayers. Further, rGd-eno was able to bind human plasminogen (HsPlg) and enhanced plasmin activity in vitro when the trophozoites were incubated with the intrinsic plasminogen activators of epithelial cells. In IEC-6 cells, rGd-eno treatment induced a profuse cell damage characterized by copious vacuolization, intercellular separation and detachment from the substrate; this effect was inhibited by either anti-Gd-eno Abs or the plasmin inhibitor ϵ- aminocaproic acid. Lastly, we established that in epithelial cells rGd-eno treatment induced a necroptotic-like process mediated by tumor necrosis factor α (TNF-α) and the apoptosis inducing factor (AIF), but independent of caspase-3. All together, these results suggest that Giardia enolase is a secreted moonlighting protein that stimulates a necroptotic-like process in IEC-6 epithelial cells via plasminogen activation along to TNFα and AIF activities and must be considered as a virulence factor.

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Giardia duodenalis Virulence - "To Be, or Not To Be".

PURPOSE OF REVIEW: Here, we review recent progress made on the genetic characterization of Giardia duodenalis assemblages and their relationship with virulence. We also discuss the implications of virulence factors in the pathogenesis of giardiasis, and advances in the development of vaccines and drugs based on knowledge of virulence markers. RECENT FINDINGS: The use of transcriptomic and proteomic technologies as well as whole genome sequencing (WGS) from single cysts has allowed the assembly of the draft genome sequences for assemblages C and D of G. duodenalis. These findings, along with the published genomes for assemblages A, B, and E, have allowed comparative genomic investigations. In addition, the use of these methodologies for the characterization of the secretomes of trophozoite-epithelial cell interactions for assemblages A/B has led to the identification of virulence markers including energy metabolism enzymes, proteinases, high-cysteine membrane proteins (HCMPs), and variant surface proteins (VSPs). Recently, some drugs and vaccines, targeting virulence factors have been developed, offering possible alternatives to current treatment and prevention options against giardiasis. SUMMARY: Among the nine recognized species of Giardia, G. duodenalis stands out because of its broad spectrum of hosts and its socio-economic importance. This species comprises eight genetic assemblages (A to H), of which A and B are zoonotic, and the other assemblages have narrow host specificities. Assemblages A and B may be considered as the most virulent ones, but the existence of asymptomatic carriers and considerable genetic variability within and among these assemblages hampers the definition of common virulence factors. The attachment of Giardia trophozoites to epithelial cells and structural cytoskeleton components of the adhesive disk, such as giardins or tubulins, is proposed to play key roles, but toxins have not yet been precisely defined. However, recent transcriptomic and proteomic analyses of the secretomes of trophozoites representing assemblages A and B and interacting with particular epithelial cell lines have defined a series of virulence factors, including glycolytic (e.g., enolase) and arginolytic (e.g., arginine deiminase) enzymes, cysteine proteases (e.g., giardipain-1) and VSPs (e.g., VSP9B10A). Other factors, such as HCMPs and tenascins, have been consistently found to be excreted/secreted, but their role(s) in the pathogenesis of giardiasis has not yet been elucidated. Interestingly, recent investigations of single cysts representing assemblages C and D using advanced sequencing and informatic methods have suggested that the transcription/expression profiles of virulence factors vary both within and between assemblages, thus assemblage-specific molecules might allow adaptation to the microenvironment within the host. Importantly, some drugs active against cysteine-rich proteins of Giardia, including giardipain-1, VSPs and arginine deiminase, have been shown to be targeted by cysteine-modifying compounds as disulfiram, L-canavanin and allicin. On the other hand, VSPs are presently considered as key vaccine candidates because they induce protection against Giardia in rodents and dogs. Overall, this review reveals that much more work is needed to identify, characterize, and understand the roles of virulence factors in Giardia and to assess their validity as drug and vaccine targets. Clear, advanced omics and informatic tools should assist in this future endeavor, with a focus on targeting virulence factors that are common and/or unique to distinct assemblages to develop new and effective interventions against Giardia.

Drug resistance in Giardia: Mechanisms and alternative treatments for Giardiasis.

The use of chemotherapeutic drugs is the main resource against clinical giardiasis due to the lack of approved vaccines. Resistance of G. duodenalis to the most used drugs to treat giardiasis, metronidazole and albendazole, is a clinical issue of growing concern and yet unknown impact, respectively. In the search of new drugs, the completion of the Giardia genome project and the use of biochemical, molecular and bioinformatics tools allowed the identification of ligands/inhibitors for about one tenth of ≈150 potential drug targets in this parasite. Further, the synthesis of second generation nitroimidazoles and benzimidazoles along with high-throughput technologies have allowed not only to define overall mechanisms of resistance to metronidazole but to screen libraries of repurposed drugs and new pharmacophores, thereby increasing the known arsenal of anti-giardial compounds to some hundreds, with most demonstrating activity against metronidazole or albendazole-resistant Giardia. In particular, cysteine-modifying agents which include omeprazole, disulfiram, allicin and auranofin outstand due to their pleiotropic activity based on the extensive repertoire of thiol-containing proteins and the microaerophilic metabolism of this parasite. Other promising agents derived from higher organisms including phytochemicals, lactoferrin and propolis as well as probiotic bacteria/fungi have also demonstrated significant potential for therapeutic and prophylactic purposes in giardiasis. In this context the present chapter offers a comprehensive review of the current knowledge, including commonly prescribed drugs, causes of therapeutic failures, drug resistance mechanisms, strategies for the discovery of new agents and alternative drug therapies.

Activity of Thioallyl Compounds From Garlic Against Giardia duodenalis Trophozoites and in Experimental Giardiasis.

Fresh aqueous extracts (AGEs) and several thioallyl compounds (TACs) from garlic have an important antimicrobial activity that likely involves their interaction with exposed thiol groups at single aminoacids or target proteins. Since these groups are present in Giardia duodenalis trophozoites, in this work we evaluated the anti-giardial activity of AGE and several garlic's TACs. In vitro susceptibility assays showed that AGE affected trophozoite viability initially by a mechanism impairing cell integrity and oxidoreductase activities while diesterase activities were abrogated at higher AGE concentrations. The giardicidal activities of seven TACs were related to the molecular descriptor HOMO (Highest Occupied Molecular Orbital) energy and with their capacity to modify the -SH groups exposed in giardial proteins. Interestingly, the activity of several cysteine proteases in trophozoite lysates was inhibited by representative TACs as well as the cytopathic effect of the virulence factor giardipain-1. Of these, allicin showed the highest anti-giardial activity, the lower HOMO value, the highest thiol-modifying activity and the greatest inhibition of cysteine proteases. Allicin had a cytolytic mechanism in trophozoites with subsequent impairment of diesterase and oxidoreductase activities in a similar way to AGE. In addition, by electron microscopy a marked destruction of plasma membrane and endomembranes was observed in allicin-treated trophozoites while cytoskeletal elements were not affected. In further flow cytometry analyses pro-apoptotic effects of allicin concomitant to partial cell cycle arrest at G2 phase with the absence of oxidative stress were observed. In experimental infections of gerbils, the intragastric administration of AGE or allicin decreased parasite numbers and eliminated trophozoites in experimentally infected animals, respectively. These data suggest a potential use of TACs from garlic against G. duodenalis and in the treatment of giardiasis along with their additional benefits in the host's health.

DNA damage induced by metronidazole in Giardia duodenalis triggers a DNA homologous recombination response.

The mechanisms underlying metronidazole (MTZ) resistance in Giardia duodenalis have been associated with decreased activity of the enzymes implicated in its activation including nitroductase-1, thioredoxin reductase and pyruvate-ferredoxin oxidoreductase (PFOR). MTZ activation generates radicals that can form adducts with proteins such as thioredoxin reductase and α- and -ß giardins as well as DNA damage resulting in trophozoite's death. The damage induced in DNA requires a straight forward response that may allow parasite survival. Here, we studied changes in histone H2A phosphorylation to evaluate the DNA repair response pathway after induction of double strand break (DSB) by MTZ in Giardia DNA. Our results showed that the DNA repair mechanisms after exposure of Giardia trophozoites to MTZ, involved a homologous recombination pathway. We observed a significant increase in the expression level of proteins GdDMC1B, which carries out Rad51 role in G. duodenalis, and GdMre11, after 12 h of exposure to 3.2 µM MTZ. This increase was concomitant with the generation of DSB in the DNA of trophozoites treated MTZ. Altogether, these results suggest that MTZ-induced DNA damage in Giardia triggers the DNA homologous recombination repair (DHRR) pathway, which may contribute to the parasite survival in the presence of MTZ.

Proteomic analysis and immunodetection of antigens from early developmental stages of Trichinella spiralis.

Trichinella spiralis is an ubiquitous parasitic nematode that lives in muscle tissue of many hosts and causes trichinellosis in humans. Numerous efforts have been directed at specific detection of this infection and strategies for its control. TSL-1 and other antigens, mainly from muscle larvae (ML), have been used to induce partial protection in rodents. An improvement in protective immunity may be achieved by using antigens from other parasite stages. Further, identification of other parasite antigens may provide insights into their role in the host-parasite interaction. In this study, T. spiralis antigens from early developmental parasite stages, namely ML and pre-adult (PA) obtained at 6h, 18h and 30h post-infection, were identified by proteomic and mass spectrometry analyses. Our findings showed a differential expression of several proteins with molecular weights in the range of 13-224kDa and pI range of 4.54-9.89. Bioinformatic analyses revealed a wide diversity of functions in the identified proteins, which include structural, antioxidant, actin binding, peptidyl prolyl cis-trans isomerase, motor, hydrolase, ATP binding, magnesium and calcium binding, isomerase and translation elongation factor. This, together with the differential recognition of antigens from these parasite stages by antibodies present in intestinal fluid, in supernatants from intestinal explants, and in serum samples from mice infected with T. spiralis or re-infected with this parasite, provides information that may lead to alternatives in the design of vaccines against this parasite or for modulation of immune responses.

Toxocara canis: larval migration dynamics, detection of antibody reactivity to larval excretory-secretory antigens and clinical findings during experimental infection of gerbils (Meriones unguiculatus).

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.

Sequential exposure and assembly of cyst wall filaments on the surface of encysting Giardia duodenalis.

The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3-25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.

Epidemiology of trichinellosis in Mexico, Central and South America.

Trichinella species are widely distributed throughout the world and are found in a large number of carnivorous animals, humans and incidental hosts. The data presented in this review show that Trichinella infection has been reported in both humans and animals in Mexico, Argentina and Chile since the end of the 19th century, and more recently in Bolivia. This parasitic infection is still a public health problem in countries such as Argentina and Chile. Although efforts have focused on the control and prevention of trichinellosis in these countries, there were still human cases and outbreaks of trichinellosis reported. Diagnosis of infection in animals such as pigs still includes, in many endemic areas, the use of trichinoscopy. In Argentina, however, artificial digestion has been recently introduced in slaughterhouses to detect Trichinella infection in pigs. In some endemic areas in Mexico, the use of serological assays for human trichinellosis and pig infections have resulted in improved detection. Most of the outbreaks of human trichinellosis in Mexico, Argentina and Chile have occurred as a result of the consumption of undercooked pork or pork products from animals raised under poor hygienic conditions and which are clandestinely slaughtered. In several studies, rats, dogs and cats have been found to be infected with Trichinella and may be considered a risk for transmission of the infection to pigs or other animals intended for human consumption. Another potential source of transmission of Trichinella to humans is horsemeat; however, information related to horse trichinellosis in Latin-American countries is scarce. In most cases the etiological agent of human trichinellosis in Central and South America has been reported to be Trichinella spiralis; however, only in a few cases has the parasite species been properly identified. Based on the reports available, it is clear that there is a need to carry out better controlled epidemiological studies to determine the true prevalence of the infection in this region of the world. Also, more sensitive methods of diagnosis and improvements in conditions for pig production as well as better sanitary inspection systems, are needed for controlling and preventing transmission of trichinellosis in these countries.

Histopathological investigation of experimental ocular toxocariasis in gerbils.

Male gerbils were inoculated intragastrically with embryonated Toxocara canis eggs. They were euthanised, and their eyes were excised at different days p.i. to identify the number of larvae as well as lesions resulting in these organs. In most animals, larvae were detected from day 5 to day 60 p.i. (end of the study). From days 10 to 20 p.i., larvae and haemorrhage were observed in the choroid and in the ciliary process. At days 30 and 40 p.i., some eyes had larvae surrounded by an infiltrate of neutrophils, oedema, haemorrhages and tissue damage in the retina or the ciliary process. On day 60 p.i., there were granulomatous lesions in the retina. This study provides a model for the study of ocular toxocariasis.

Detection of Trichinella infection in slaughter horses by ELISA and western blot analysis.

In order to determine the presence of Trichinella infections in horses slaughtered at an abattoir in Mexico, 147 serum samples were examined by two immunoenzymatic methods. Specific antibodies were detected by ELISA in 7% of the serum samples at a dilution 1:400 and in 10% at lower dilutions (1:20, 1:40) using Trichinella spiralis muscle larvae (ML) excretory/secretory (E/S) products. Serum samples from four naturally infected horses (confirmed by direct methods) gave negative O.D. values in an ELISA at a 1:400 dilution and only two of them were positive at a 1:20 and 1:40 dilutions. Serum samples from experimentally infected horses reacted by Western blotting with ML components with molecular weights of 47, 52, 59, 67, 72 and 105 kDa which correspond to the TSL-1 antigens. Serum samples from the four naturally infected horses and from the abattoir horses that were positive in ELISA using E/S antigens recognized several ML components, some of them reacted with all the TSL-1 antigens mentioned above and others recognized preferentially two or three of these molecules. Since the serologic assays may not offer the sensitivity required in the diagnosis of horses trichinellosis and the direct methods had not always been useful in the detection of larvae in horsemeat related to trichinellosis outbreaks in Europe, it is proposed that additional assays are performed to determine Trichinella infection in horses. These can include detection of parasite antigens by ELISA and Dot ELISA or PCR, which in turn may also help to determine the presence of the parasite in early and late infections of horses.

[Updates on equine trichinellosis]. / Actualidades sobre la triquinelosis equina.

Trichinellosis is a zoonosis caused by parasites of the genus Trichinella. Transmission of trichinellosis to humans has been shown to occur mainly by the ingestion of meat from pigs, bears of foxes parasitized with muscle larvae of this parasite. However, in Europe, the major human outbreaks of the disease have occurred due to the ingestion of parasitized horse meat. Although the larvae were not isolated from the horse meat, the identification of larvae as T. nativa, T. britovi and T. spiralis was done in biopsy samples obtained from infected individuals. More recently T. spiralis muscle larvae have been isolated and identified, for the first time, in muscle tissue of horses slaughtered at an abattoir in the State of Mexico. Furthermore, in ELISA assays using total extracts or TSL-1 antigens, circulating antibodies against Trichinella have been detected in horses slaughtered at abattoirs from various countries in Europe and Mexico. On the other hand, the experimental infection of horses with parasites of the genes Trichinella has been achieved by several research groups and data obtained regarding the kinetics of antibody production in these animals are important in the development of sensitive and specific diagnostic assays for horse trichinellosis. This will allow to determine the frequency of this infection in horses which are used for animal and human feeding. These assays will also be very helpful for designing strategies to control transmission on the disease by horse meat.

Detection of Trichinella spiralis muscle larvae in naturally infected horses.

Human trichinellosis outbreaks related to horsemeat consumption have been reported in France and Italy in recent years. In order to determine if Trichinella is present in horses slaughtered at an abattoir in the State of Mexico, diaphragm muscle tissue samples (22-37 g) from 80 horses were examined by artificial digestion. Four of these samples had larvae that were characterized as Trichinella sp. by morphological criteria and as Trichinella spiralis by the polymerase chain reaction.

[Characterization of surface antigens of the nematode parasite Trichinella spiralis: study of its role in protection mechanisms and their usefulness in the diagnosis of trichinosis]. / La caracterización de los antígenos de superficie del nemátodo parásito Trichinella spiralis: estudio de su participación en los mecanismos de protección y su utilidad en el diagnóstico de la triquinosis.

Among the most important aspects in the study of trichinosis are the development of specific and sensitive diagnostic methods for the detection of the infection by the parasite as well as the characterization of antigens from Trichinella spiralis that induce protection in the host. In the context, the characterization of surface stichosome and excretory secretory antigens of T. spiralis muscle larvae has been an important issue due to the high immunogenicity of such components in most hosts so far studied. In this work, we have been able to identify and characterize molecules from both compartments of muscle larvae. These components have been used in assays for specific detection of T. spiralis infections particularly in pigs, as well as in assays to evaluate their role in the induction of protection in mice.

[Current aspects of trichinosis diagnosis]. / Aspectos actuales sobre el diagnóstico de la triquinelosis.

An important approach to the study of trichinellosis is the development of sensitive diagnostic methods that allow the detection of Trichinella spiralis. Recently, ELISA assays that use surface/stichosome and/or secretion/excretion antigens from muscle larvae have been proved to be highly sensitive and specific in the diagnosis of the infection. Furthermore, the high immunodominance of carbohydrate residues on these molecules in a broad host range make them useful diagnostic markers for trichinellosis.

Thy-1 functions as a recognition/signaling molecule during mouse T lymphocyte development.

Thy-1 is a prototype of mammalian glycosyl phosphatidylinositol (GPI)-anchored molecules and belongs to the Ig superfamily. This cell surface glycoprotein is expressed on mouse T lymphocytes, neurons and hematopoietic stem cells. Despite detailed structural studies, little is known about the physiological role(s) of Thy-1. We discuss here our results on the role of Thy-1 in immature T lymphocytes during intrathymic maturation. It was observed that Thy-1-mediated adhesion of mouse thymocytes to thymic stromal cells occurs through interaction with an unknown ligand. The interaction occurs by a Ca(2+)-independent mechanism and does not require TCR/CD3 surface expression. To evaluate the signal transduction upon Thy-1 ligation in immature thymocytes, we cultured mouse thymocytes with monoclonal antibodies specific for Thy-1, immobilized onto the tissue culture plates. Monoclonal antibodies directed at determinants located in a defined epitope domain, but not others, triggered marked physiological cell death (apoptosis) of immature thymocytes, as evidenced by morphological and biochemical data. This apoptosis is independent of the cell surface expression of TCR/CD3. It is a developmentally regulated process since the period in which thymocytes are sensitive to Thy-1-dependent apoptosis corresponds to the developmental "window" during which massive death of immature thymocytes takes place within the thymus. Therefore, we propose that Thy-1 could function as a cell survival/death regulator in mouse T lymphocyte development.