Immunity to Trichuris muris in the mouse

Quantitative and qualitative analyses of the serum antibody responses of NIH, C57BL/10, BALB/c, DBA/2 and CFLP mice infected with Trichuris muris have been made using ELISA and immunoprecipitation techniques. No correlation was found between specific serum antibody titres measured using T. muris E/S products and the time of onset of expulsion in the different mouse strains examined. However, there were some differences in the antigen recognition profiles of some sera as determined by immunoprecipitation analyses. In all the strains of mice examined significant increases in detectable specific serum antibody to the parasite E/S products occurred around day 15 to 20 postinfection and continued to rise, as measured up to at least day 40 and even up to day 65. Cortisone acetate treatment during larval development, in infected CFLP mice, in order to establish heavy adult worm burdens, did not reduce specific antibody titres to T. muris E/S products. In responding and tolerant DBA/2 mice there was no marked difference in either the kinetics of specific serum antibody production during primary and secondary infections, or in the antigen specificities of secondary infection sera. The "defect" in mechanism in the tolerant DBA/2 mice, which allows primary infections of T. muris to develop to patency, was shown to be permanent as secondary infections with the parasite could also establish in these animals. An investigation was made of the phenomenon of tolerance in the DBA/2 model- system and in the cortisone treated CBA mice. The capacity of MLNC from different groups of animals to produce IL-2 in vitro upon mitogen stimulation was investigated, on the basis that IL-2 deficit during antigen presentation may result in immune tolerance. Although no differences were found in the responding and tolerant DBA/2 cell-Vpopulations, there was an apparently synergistic interaction between cortisone administration and T. muris infection which dramatically reduced the IL-2 producing capacity of the MLNC. However, IL-2 cannot yet be ruled out as a factor in the inherent tolerance of a proportion of the DBA/2 population as IL-2 receptor expression by the 2 groups of cells assayed was not examined. Basic analyses of the antigens of T. muris were performed. The major protein of adult male homogenate (AMA) was also the major protein of the excretory/secretory (E/S) products and the surface antigen preparations. In addition several common E/S and surface antigens were shown to have proteolytic enzyme activities against gelatin and/or casein. The relationship between T. muris and Trichinella spiralis was examined in greater detail, and the m. wts. of the cross-reacting antigens were determined. Evidence suggested that the stichosomes of these worms may be the source of these antigens. Both Trichuris muris adults and Trichinella spiralis infective larvae each had common major E/S and surface antigens, indeed, both were shown to have surface proteases. These studies were extended to examine the possibility of cross-reactivity between Trichuris muris and T. trichiura; mouse infection sera and human infection sera respectively were able to cross-react with heterologous antigen preparations. The demonstration that anti-Trichinella spiralis 48 kD and 50/55 kD stichocyte antigen MoAbs also reacted with Trichuris trichiura adult homogenate in ELISA supports the suggestion that common stichocyte antigens may exist amongst the trichuroid nematodes Trichuris muris, Trichuris trichiura and Trichinella spiralis. Monoclonal antibodies were produced against the E/S products of Trichuris muris, which were characterized in terms of isotype and antigen specificities. Initial experiments indicated that one of the IgA MoAbs recognizing 34,22,20 and 18 kD E/S proteins may be effective in the passive transfer of immunity

Immunity to Trichuris muris in the mouse

Quantitative and qualitative analyses of the serum antibody responses of NIH, C57BL/10, BALB/c, DBA/2 and CFLP mice infected with Trichuris muris have been made using ELISA and immunoprecipitation techniques. No correlation was found between specific serum antibody titres measured using T. muris E/S products and the time of onset of expulsion in the different mouse strains examined. However, there were some differences in the antigen recognition profiles of some sera as determined by immunoprecipitation analyses. In all the strains of mice examined significant increases in detectable specific serum antibody to the parasite E/S products occurred around day 15 to 20 postinfection and continued to rise, as measured up to at least day 40 and even up to day 65. Cortisone acetate treatment during larval development, in infected CFLP mice, in order to establish heavy adult worm burdens, did not reduce specific antibody titres to T. muris E/S products. In responding and tolerant DBA/2 mice there was no marked difference in either the kinetics of specific serum antibody production during primary and secondary infections, or in the antigen specificities of secondary infection sera. The "defect" in mechanism in the tolerant DBA/2 mice, which allows primary infections of T. muris to develop to patency, was shown to be permanent as secondary infections with the parasite could also establish in these animals. An investigation was made of the phenomenon of tolerance in the DBA/2 model- system and in the cortisone treated CBA mice. The capacity of MLNC from different groups of animals to produce IL-2 in vitro upon mitogen stimulation was investigated, on the basis that IL-2 deficit during antigen presentation may result in immune tolerance. Although no differences were found in the responding and tolerant DBA/2 cell-Vpopulations, there was an apparently synergistic interaction between cortisone administration and T. muris infection which dramatically reduced the IL-2 producing capacity of the MLNC. However, IL-2 cannot yet be ruled out as a factor in the inherent tolerance of a proportion of the DBA/2 population as IL-2 receptor expression by the 2 groups of cells assayed was not examined. Basic analyses of the antigens of T. muris were performed. The major protein of adult male homogenate (AMA) was also the major protein of the excretory/secretory (E/S) products and the surface antigen preparations. In addition several common E/S and surface antigens were shown to have proteolytic enzyme activities against gelatin and/or casein. The relationship between T. muris and Trichinella spiralis was examined in greater detail, and the m. wts. of the cross-reacting antigens were determined. Evidence suggested that the stichosomes of these worms may be the source of these antigens. Both Trichuris muris adults and Trichinella spiralis infective larvae each had common major E/S and surface antigens, indeed, both were shown to have surface proteases. These studies were extended to examine the possibility of cross-reactivity between Trichuris muris and T. trichiura; mouse infection sera and human infection sera respectively were able to cross-react with heterologous antigen preparations. The demonstration that anti-Trichinella spiralis 48 kD and 50/55 kD stichocyte antigen MoAbs also reacted with Trichuris trichiura adult homogenate in ELISA supports the suggestion that common stichocyte antigens may exist amongst the trichuroid nematodes Trichuris muris, Trichuris trichiura and Trichinella spiralis. Monoclonal antibodies were produced against the E/S products of Trichuris muris, which were characterized in terms of isotype and antigen specificities. Initial experiments indicated that one of the IgA MoAbs recognizing 34,22,20 and 18 kD E/S proteins may be effective in the passive transfer of immunity

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5'-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP

Synergistic Ca^(2+) Responses by Gα_i- and Gα_q-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβ_γ and Gα_q

Cross-talk between Gα_i- and Gα_q-linked G-protein-coupled receptors yields synergistic Ca^(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca^(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca^(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα_q. Thus, both Gβγ and Gα_q contribute to activation of PLCβ3 in cells for Ca^(2+) synergy. We conclude that Ca^(2+) synergy between Gα_i-coupled and Gα_q-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent

Signaling and crosstalk by C5a and UDP in macrophages selectively use PLCbeta 3 to regulate intracellular free calcium

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G-protein coupled receptors (GPCRs) in raising intracellular free Ca2+. To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca2+ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5’-diphosphate (UDP), platelet activating factor (PAF) or lysophosphatidic acid (LPA). The C5a response was Gai-dependent, while the UDP, PAF, and LPA responses were Gaqdependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2+ from intracellular stores as well as sustained Ca2+ levels. C5a and UDP synergized in generating inositol-1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) ß. Macrophages expressed transcripts for three PLCß isoforms (PLCß2, PLCß3, and PLCß4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLCß3, while UDP used PLCß3 but also PLCß4. Neither ligand required PLCß2. Synergy between C5a and UDP likewise depended primarily on PLCß3. Importantly, the Ca2+ signaling deficiency observed in PLCß3-deficient BMDM was reversed by reconstitution with PLCß3. Neither PI-3 kinase nor PKC was required for synergy. In contrast to Ca2+, PI3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI3- kinase. PLCß3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA

A Dual Receptor Crosstalk Model of G-Protein-Coupled Signal Transduction

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Gαi-coupled C5a receptor and the Gαq-coupled UDP receptor signaling systems that results in synergistic calcium release

Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas

Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (, , ) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types