Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection

Co-Administration of a Plasmid DNA Encoding IL-15 Improves Long-Term Protection of a Genetic Vaccine against Trypanosoma cruzi

Background: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. the goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone.Methodology: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-gamma ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-gamma, TNF-alpha, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-gamma responses and survived a lethal challenge given within the first 3 months following immunization. the addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro restimulation.Conclusion: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.National Institutes of HealthFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Millennium Institute for Gene TherapySt Louis Univ, Dept Internal Med, St Louis, MO 63103 USAUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol, Escola Paulista Med, São Paulo, BrazilSt Louis Univ, Dept Mol Microbiol, St Louis, MO 63103 USAUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Microbiol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol, Escola Paulista Med, São Paulo, BrazilNational Institutes of Health: RO1 AI040196CNPq: 420067/2005-1Web of Scienc

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

The impact of nutritional status during fetal life on the overall health of adults has been recognized(1); however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed(2,3). Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer(LTi) cells(2-5). Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor ROR gamma t. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring

Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion

Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis

Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.Slovene Research Agency [P1-0104]; US National Institutes of Health [R37 CA090426]; Slovene Human Resources Development and Scholarship FundOpen Access JournalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The tyrosine phosphatase PTPN22 discriminates weak self peptides from strong agonist TCR signals

T cells must be tolerant of self antigens to avoid autoimmunity but responsive to foreign antigens to provide protection against infection. We found that in both naive T cells and effector T cells, the tyrosine phosphatase PTPN22 limited signaling via the T cell antigen receptor (TCR) by weak agonists and self antigens while not impeding responses to strong agonist antigens. T cells lacking PTPN22 showed enhanced formation of conjugates with antigen-presenting cells pulsed with weak peptides, which led to activation of the T cells and their production of inflammatory cytokines. This effect was exacerbated under conditions of lymphopenia, with the formation of potent memory T cells in the absence of PTPN22. Our data address how loss-of-function PTPN22 alleles can lead to the population expansion of effector and/or memory T cells and a predisposition to human autoimmunity.</p

Themis sets the signal threshold for positive and negative selection in T-cell development

Producción CientíficaThis work shows that the Themis protein has a critical role in positive and negative thymocyte selection by dampening responses to low-affinity ligands but without affecting responses to high-affinity ligands, thus enabling positive selection of weakly self-reactive thymocytes. Themis is a protein expressed in T cells. Mice lacking it have severely reduced numbers of single-positive thymocytes and peripheral T cells, although the mechanism by which Themis controls T-cell development or function remains obscure. Nicholas Gascoigne and colleagues show here that Themis has a crucial role in thymocyte selection by regulating the signalling threshold between positive and negative selection. It dampens responses to low-affinity ligands but does not affect responses to high-affinity ligands, thus enabling positive selection of weakly self-reactive thymocytes. Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate ‘positive selection’, causing maturation of CD4- or CD8αβ-expressing ‘single-positive’ thymocytes from CD4+CD8αβ+ ‘double-positive’ precursors1. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in ‘negative selection’ by activation-induced apoptosis or ‘agonist selection’ of functionally differentiated self-antigen-experienced T cells2,3. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis4,5,6,7,8,9 to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens