Immunization with a Mu-class glutathione transferase from Echinococcus granulosus induces efficient antibody responses and confers long-term protection against secondary cystic echinococcosis.

Cystic echinococcosis, a zoonosis caused by cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) genetic complex, affects humans and diverse livestock species. Although a veterinary vaccine exhibiting high levels of antibody-mediated protection has successfully reached the market, the large genetic diversity among parasite isolates and their particular host preferences, makes still necessary the search for novel vaccine candidates. Glutathione transferases (GSTs) constitute attractive targets for immunoprophylaxis due to their outstanding relevance in helminth detoxification processes, against both exogenous and endogenous stressors. Among the six GSTs known to be expressed in E. granulosus s.l., EgGST1 (Mu-class), EgGST2 (Sigma-class), and EgGST3 (a still non-classifiable isoenzyme), show the highest proteomic expression. Therefore, their recombinant forms -rEgGST1, rEgGST2 and rEgGST3- were herein analyzed regarding their potential to induce long-term antiparasite protection in mice. Only immunization with rEgGST1 induced long-lasting protection; and accordingly, rEgGST1-specific antibodies enhanced the parasite killing through both the classical activation of the host complement system and the antibody-dependent cellular cytotoxicity by macrophages. These results support further testing of rEgGST1 as a vaccine candidate in diverse hosts due to the broad expression of EgGST1 in different parasite stages and tissues.

Recombinant CD5 and CD6 Ectodomains Induce Antiparasitic and Immunomodulatory Effects in Secondary Cystic Echinococcosis.

Scavenger receptors participate in a wide range of biological functions after binding to multiple non-self or altered self-ligands. Among them, CD5 and CD6 are lymphocyte scavenger receptors known to interact with different microbial-associated molecular patterns, and the administration of the recombinant soluble ectodomains of human CD5 (rshCD5) and/or CD6 (rshCD6) has shown therapeutic/prophylactic potential in experimental models of fungal, bacterial and echinococcal infections. The latter is a zoonosis caused by the larval stage of the cestode parasite Echinococcus granulosus sensu lato, which in humans can induce secondary cystic echinococcosis (CE) after the spillage of protoscoleces contained within fertile cysts, either spontaneously or during surgical removal of primary hydatid cysts. Herein, we have analysed the mechanisms behind the significant protection observed in the mouse model of secondary CE following prophylactic administration of rshCD5 or rshCD6. Our results show that both molecules exhibit intrinsic antiparasitic activities in vitro, as well as immunomodulatory functions during early secondary CE, mainly through Th1/Th17 cytokine bias and promotion of peritoneal polyreactive antibodies. These data support the relevance of the parasite components bound by rshCD5 and rshCD6, as well as the potential of their prophylactic administration as a useful strategy to reduce secondary CE in patients.

Effects of human donor milk on gut barrier function and inflammation: in vitro study of the beneficial properties to the newborn.

Introduction: The gastrointestinal and immune systems of premature infants are not fully developed, rendering them more vulnerable to severe complications like necrotizing enterocolitis. Human milk offers a rich array of bioactive factors that collectively contribute to reducing the incidence of gut infections and inflammatory conditions. When a mother's milk is unavailable, preterm infants are often provided with donor human milk processed in Human Milk Banks. However, it remains uncertain whether pasteurized milk confers the same level of risk reduction as unprocessed milk. This uncertainty may stem from the well-documented adverse effects of heat treatment on milk composition. Yet, our understanding of the comprehensive impact on protective mechanisms is limited. Methods: In this study, we conducted a comparative analysis of the effects of raw versus pasteurized milk and colostrum versus mature milk on cellular functions associated with the gut epithelial barrier and responses to inflammatory stimuli. We utilized THP-1 and HT-29 cell lines, representing monocyte/macrophages and gut epithelial cells, respectively. Results: Our observations revealed that all milk types stimulated epithelial cell proliferation. However, only raw colostrum increased cell migration and interfered with the interaction between E. coli and epithelial cells. Furthermore, the response of epithelial and macrophage cells to lipopolysaccharide (LPS) was enhanced solely by raw colostrum, with a milder effect observed with mature milk. In contrast, both raw and pasteurized milk diminished the LPS induced response in monocytes. Lastly, we examined how milk affected the differentiation of monocytes into macrophages, finding that milk reduced the subsequent inflammatory response of macrophages to LPS. Discussion: Our study sheds light on the impact of human milk on certain mechanisms that potentially account for its protective effects against necrotizing enterocolitis, highlighting the detrimental influence of pasteurization on some of these mechanisms. Our findings emphasize the urgency of developing alternative pasteurization methods to better preserve milk properties. Moreover, identifying the key components critically affected by these protective mechanisms could enable their inclusion in donor milk or formula, thereby enhancing immunological benefits for vulnerable newborns.

Study of tissue transglutaminase spliced variants expressed in THP-1 derived macrophages exhibiting distinct functional phenotypes.

Tissue transglutaminase (TG2) expressed in monocytes and macrophage is known to participate in processes during either early and resolution stages of inflammation. The alternative splicing of tissue transglutaminase gene is a mechanism that increases its functional diversity. Four spliced variants are known with truncated C-terminal domains (TGM2_v2, TGM2_v3, TGM2_v4a, TGM2_v4b) but scarce information is available about its expression in human monocyte and macrophages. We studied the expression of canonical TG2 (TGM2_v1) and its short spliced variants by RT-PCR during differentiation of TPH-1 derived macrophages (dTHP-1) using two protocols (condition I and II) that differ in Phorbol-12-myristate-13-acetate dose and time schedule. The production of TNF-α and IL-1ß in supernatant of dTHP-1, measured by ELISA in supernatants showed higher proinflammatory milieu in condition I. We found that the expression of all mRNA TG2 spliced variants were up-regulated during macrophage differentiation and after IFN-γ treatment of dTHP-1 cells in both conditions. Nevertheless, the relative fold increase or TGM2_v3 in relation with TGM2_v1 was higher only with the condition I. M1/M2-like THP-1 macrophages obtained with IFN-γ/IL-4 treatments showed that the up-regulation of TGM2_v1 induced by IL-4 was higher in relation with any short spliced variants. The qualitative profile of relative contribution of spliced variants in M1/M2-like THP-1 cells showed a trend to higher expression of TGM2_v3 in the inflammatory functional phenotype. Our results contribute to the knowledge about TG2 spliced variants in the biology of monocyte/macrophage cells and show how the differentiation conditions can alter their expression and cell function.

Hypoxia and inflammation conditions differentially affect the expression of tissue transglutaminase spliced variants and functional properties of extravillous trophoblast cells.

PROBLEM: Persistent hypoxia and inflammation beyond early pregnancy are involved in a bad outcome because of defective trophoblast invasiveness. Tissue transglutaminase (TG2) coregulates several cell functions. An aberrant expression and/or transamidation activity could contribute to placental dysfunction. METHOD OF STUDY: The first-trimester trophoblast cell line (Swan-71) was used to study TG2 expression and cell functions in the absence or presence of inflammatory cytokines (TNF-α, IL-1ß) or chemical hypoxia (CoCl2 ). We analyzed The concentration of cytokines in the supernatant by ELISA; Cell migration by scratch assay; NF-κB activation by detection of nuclear p65 by immunofluorescence or flow cytometry using a Swan-71 NF-κB-hrGFP reporter cell line. Tissue transglutaminase expression was analyzed by immunoblot and confocal microscopy. Expression of spliced mRNA variants of tissue transglutaminase was analyzed by RT-PCR. Transamidation activity was assessed by flow cytometry using 5-(biotinamido)-pentylamine substrate. RESULTS: Chemical hypoxia and TGase inhibition, but not inflammatory stimuli, decreased Swan-71 migration. IL-6 production was also decreased by chemical hypoxia, but increased by inflammation. Intracellular TGase activity was increased by all stimuli, but NF-κB activation was observed only in the presence of proinflammatory cytokines. TG2 expression was decreased by CoCl2 and TNF-α. Translocation of TG2 and p65 to nuclei was observed only with TNF-α, without colocalization. Differential relative expression of spliced variants of mRNA was observed between CoCl2 and inflammatory stimuli. CONCLUSION: The observed decrease in total TG2 expression and relative increase in short variants under hypoxia conditions could contribute to impaired trophoblast invasion and impact on pregnancy outcome.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

Predictive value of blood measurement of Complement System proteins and metabolic components for early detection of obstetric complications linked to poor placental function.

Preeclampsia-eclampsia syndrome (PES) is associated with severe obstetric complications and there are no efficient methods available for an early detection. We studied blood concentration of some immunological and metabolic markers in association with obstetric outcome in healthy pregnant women and patients with obstetric risk factors, by ELISA and biochemical tests. Patients with complications showed higher levels of CRP and C4 positively correlated with Triglycerides and Cholesterol concentrations. Our results provide evidence that Immunological and metabolic alterations contribute to obstetric complications and that biomarkers linked to these alterations could be useful for an early detection of these problems.

Linking murine resistance to secondary cystic echinococcosis with antibody responses targeting Echinococcus granulosus tegumental antigens.

Successful establishment of a parasite infection depends partially on the host intrinsic susceptibility to the pathogen. In cystic echinococcosis (CE), a zoonotic disease caused by the cestode parasite Echinococcus granulosus, the infection outcome in the murine model of secondary CE varies according to the mouse strain used. In this regard, intrinsic differences in susceptibility to the infection were previously reported for Balb/c and C57Bl/6 mice, being C57Bl/6 animals less permissive to secondary CE. Induction of parasite-specific antibodies has been suggested to play relevant roles in such susceptibility/resistance phenomena. Here, we report an in deep comparison of antibody responses induced in both mouse strains. Firstly, only C57Bl/6 mice were shown to induce specific-antibodies with efficient anti-parasite activities during early secondary CE. Then, through ImmunoTEM and Serological Proteome Analysis (SERPA), an evaluation of specific antibody responses targeting parasite tegumental antigens was performed. Both strategies showed that infected C57Bl/6 mice -unlike Balb/c animals- narrowed their IgG recognition repertoire against tegumental antigens, targeting fewer but potentially more relevant parasite components. In this sense, tegumental antigens recognition between Balb/c and C57Bl/6 mice, either by natural and/or induced antibodies, was analyzed through SERPA and MALDI-TOF/TOF studies. A total of 13 differentially recognized proteins (DRPs) uniquely targeted by antibodies from C57Bl/6 mice were successfully identified, wherein a subset of 7 DRPs were only recognized by infection-induced antibodies, suggesting their potential as natural protective antigens. In this regard, immunoinformatic analyses showed that such DRPs exhibited higher numbers of possible T cell epitopes towards the H-2-IAb haplotype, which is present in C57Bl/6 mice but absent in Balb/c animals. In summary, our results showed that the genetic predisposition to generate better T-dependent antibody responses against particular tegumental antigens might be a key factor influencing host susceptibility in the murine model of secondary CE.

Impact of Holder pasteurization on immunological properties of human breast milk over the first year of lactation.

BACKGROUND: The timing of milk donations to human milk banks ranges from a few days to more than 1 year after delivery, and the Holder method is used for pasteurization. We evaluated the effect of temporal variation and thermal treatment on the immunological properties of milk. METHODS: We analyzed 73 milk samples, raw and after pasteurization, donated at different lactation stages. We studied antibodies, lysozyme, cytokines, soluble receptors, and factors with impact on barrier function. We also evaluated in vitro the capacity of milk to modulate nuclear factor-κB (NF-κB) signaling in an HT-29 epithelial cell line stimulated with tumor necrosis factor-α (TNF-α). RESULTS: With few exceptions, immune components exhibited their highest levels in colostrum, and were stable in the various stages of mature milk. Pasteurization altered the immunological composition of milk, and very drastically for some components. Raw milk of the first year reduced NF-κB activation in HT-29 cells treated with TNF-α to approximately the same extent, and Holder pasteurization significantly affected this capacity. CONCLUSIONS: Overall, the present work reports that mature donated milk is equally valuable over the first year of lactation, but warns about drastic losses of anti-inflammatory properties during Holder pasteurization that could be critical for the health of preterm infants.

Development of novel fluorescent particles applicable for phagocytosis assays with human macrophages.

Phagocytosis is a fundamental process for removal of pathogens and for clearance of apoptotic cells. The objective of this work was the preparation of fluorescent microspheres by a simple method and the evaluation of its applicability in phagocytosis assays by using different human derived cells, differentiated THP-1 cell line and blood monocytes, with flow cytometry measurements for functionality assays. Our results show that microparticles are efficiently internalised in a non-opsonised form and in dose-dependent manner by both cellular types. Concerning mechanism we determined that tTG-ß3 integrin signaling could be involved in the uptake of these particles.

Mechanistic insights into EgGST1, a Mu class glutathione S-transferase from the cestode parasite Echinococcus granulosus.

Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pKa of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 105 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST.

Echinococcus granulosus: Evidence of a heterodimeric glutathione transferase built up by phylogenetically distant subunits.

In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits.

Natural and induced antibodies contribute to differential susceptibility to secondary cystic echinococcosis of Balb/c and C57Bl/6 mice.

Antibodies are key immune players in several helminth infections and animal models have been central for the identification of their mechanisms of protection. Murine secondary cystic echinococcosis is a useful model for studying Echinococcus granulosus immunobiology, being the immune profile mounted by the experimental host a determinant of parasite success or failure in infection establishment. In the present study, we analyzed infection outcome using Balb/c and C57Bl/6 mice strains, and compared their antibody responses in terms of quality and intensity. Our results showed that Balb/c is a highly susceptible strain to secondary cystic echinococcosis, while C57Bl/6 mice are quite resistant. Moreover, significant differences between strains were observed in natural and induced antibodies recognizing E. granulosus antigens, both at the systemic and peritoneal levels. Natural cross-reacting IgM, IgG2b and IgG3 antibodies were detected in sera from both strains but with different intensities, and - remarkably - natural IgG2b showed to be an intrinsic correlate of protection in both mice strains. Interestingly, naïve C57Bl/6 serum displayed a higher protoscolicidal activity, and heterologous - but not homologous - transference of C57Bl/6 naïve serum into Balb/c mice, significantly reduced their infection susceptibility. In the peritoneal cavity, different levels of natural cross-reacting IgM and IgG3 antibodies were detected in both mice strains, while cross-reacting IgG2b was detected only in C57Bl/6 mice. On the other hand, infected mice from both strains developed isotype-mixed antibody responses, with Balb/c mice biasing their response towards high avidity IgG1 and C57Bl/6 mice showing a predominance of mixed IgM/IgG2c/IgG2b/IgG3. In this regard, IgG1 levels showed to be a correlate of susceptibility in both mice strains. In conclusion, our results suggest that antibodies - either natural or induced - play a role in the susceptibility degree to murine secondary cystic echinococcosis.

Enamel organ proteins as targets for antibodies in celiac disease: implications for oral health.

Enamel defects in permanent and deciduous teeth may be oral manifestations of celiac disease. Sometimes they are the only sign that points to this underdiagnosed autoimmune pathology. However, the etiology of these specific enamel defects remains unknown. Based on previously reported cross-reactivity of antibodies to gliadin with the enamel proteins, amelogenin and ameloblastin, we analyzed (using immunohistochemistry) the ability of anti-gliadin IgG, produced during untreated disease, to recognize enamel organ structures. We used swine germ teeth as a tissue model because they are highly homologous to human teeth in terms of proteins and development biology. Strong staining of the enamel matrix and of the layer of ameloblasts was observed with serum samples from women with celiac disease; high IgG reactivity was found against both gliadin peptides and enamel matrix protein extract, but there was no IgG reactivity against tissue antigens. In line with these findings, the gamma globulin fraction from gliadin-immunized BALB/c mice showed a similar staining pattern to that of amelogenin-specific staining. These results strongly suggest a pathological role for antibodies to gliadin in enamel defect dentition for both deciduous and permanent teeth, considering that IgG can be transported through the placenta during fetal tooth development.

Identification of novel glutathione transferases in Echinococcus granulosus. An evolutionary perspective.

Glutathione transferase enzymes (GSTs) constitute a major detoxification system in helminth parasites and have been related to the modulation of host immune response mechanisms. At least three different GSTs classes have been described in Platyhelminthes: Mu, Sigma and Omega. Mining the genome of Echinococcus multilocularis and the ESTs databases of Taenia solium and E. granulosus identified two new GSTs from the cestode E. granulosus, named EgGST2 and EgGST3. It also revealed that the Omega class of GSTs was absent from the Taenidae family. EgGST2 and EgGST3 are actively expressed in the parasite. In order to know the origin of these new GSTs, in silico analyses were performed. While EgGST2 is classified as belonging to the Sigma class, the data obtained for EgGST3 allowed a less clear interpretation. The study of the evolutionary relatedness based on the C-terminal domain sequence, gene structure conservation and three-dimensional structure predictions, suggests that EgGST3 is derived from the Platyhelminthes' Sigma-class cluster. Interestingly, the N-terminal domain displays some characteristic Omega-class residues, including a Cys residue that is likely to be involved in the catalytic mechanism. We discuss different evolutionary scenarios that could explain the observed patterns.

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus.

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.