Phospholipase C epsilon-1 (PLCƐ1) mediates macrophage activation and protection against tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCƐ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCƐ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCƐ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.

Dose-fractionation studies of a Plasmodium phosphatidylinositol 4-kinase inhibitor in a humanized mouse model of malaria.

UCT594 is a 2-aminopyrazine carboxylic acid Plasmodium phosphatidylinositol 4-kinase inhibitor with potent asexual blood-stage activity, the potential for interrupting transmission, as well as liver-stage activities. Herein, we investigated pharmacokinetic/pharmacodynamic (PK/PD) relationships relative to blood-stage activity toward predicting the human dose. Dose-fractionation studies were conducted in the Plasmodium falciparum NSG mouse model to determine the PK/PD indices of UCT594, using the in vivo minimum parasiticidal concentration as a threshold. UCT594 demonstrated concentration-dependent killing in the P. falciparum-infected NSG mouse model. Using this data and the preclinical pharmacokinetic data led to a low predicted human dose of <50 mg. This makes UCT594 an attractive potential antimalarial drug.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

Stage-specific feed intake restriction differentially regulates placental traits and proteome of goats.

A total of twenty-four healthy twin-bearing Liuyang black goats were allocated to two trials. In Trial 1, twelve goats received either the control diet (CG, n 6, 100 % feed) or restricted diet (RG, n 6, 60 % feed of CG) from gestation days 26 to 65 after synchronisation. In Trial 2, the remaining goats were randomly and equally divided into two treatments: CG and RG from days 95 to 125 of gestation. Placental traits, fetal weight, serum parameters, nitric oxide (NO), angiogenesis gene expression and cotyledon proteome were measured at the end of each trial. In early pregnancy, the total and relative weights of placenta, uterine caruncle and cotyledon, as well as fetus, were increased (P<0·05) in RG. The NO content in maternal serum was also increased (P<0·05) in RG. In all, fifty differentially expressed proteins were identified in cotyledon. The up-regulated proteins are related to proliferation and fission of trophoblast cell and the placenta angiogenesis. During the late pregnancy trial, placental weight was increased (P<0·05) in RG, but weight of the fetus was decreased (P<0·05). The capillary density in the cotyledon was also decreased (P<0·01). A total of fifty-eight proteins were differentially expressed in cotyledon. The up-regulated proteins in RG are related to placenta formation, blood flow regulation and embryonic development. These results indicated that feed intake restriction during gestation influenced the placental and fetal development in a stage-dependent manner. These findings have important implications for developing novel nutrient management strategies in goat production.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

Quantitative profiling of tyrosine phosphorylation revealed changes in the activity of the T cell receptor signaling pathway upon cisplatin-induced apoptosis.

In order to better understand the cellular responses to the chemotherapeutic drug cisplatin and the mechanisms leading to apoptosis and potential side effects, we performed a SILAC-based quantitative phosphotyrosine analysis of Jurkat T cells exposed to cisplatin. Signaling molecules in the T cell receptor (TCR) pathway were enriched among proteins displaying reduced phosphorylation levels. The results were verified by immunoblotting and/or phospho-flow cytometry for a selected set of proteins, including the tyrosine kinases Lck and Zap70, and downstream targets Itk, Plcγ1 and Erk. In contrast to the effects on the T cell signaling pathways, the dually phosphorylated form of p38α MAPK was increased in treated cells, and activation of this signaling pathway was verified by immunoblot analysis of phosphorylation levels of p38α MAPK and the downstream targets Atf2 and MAPKAPK2. Activation of the p38α MAPK signaling pathway has been suggested to be one of the main mechanisms by which cisplatin induces apoptosis. Our results indicate that cisplatin may reduce the activity of proteins involved in the TCR signaling pathway, which has an important role in regulating proliferation of T cells, and may contribute to explain previous observations where cisplatin has been reported to inhibit proliferation of T cells. BIOLOGICAL SIGNIFICANCE: In this study, a quantitative phosphotyrosine analysis was performed to identify changes of the phosphoproteome during exposure of Jurkat T cells by cisplatin. The results of the phosphoproteome analysis were complemented with immunoblotting and temporal phospho-flow analysis. An initial activation of the p38α MAPK signaling pathway was detected at early time points of cisplatin treatment, a response previously suggested to be part of the mechanism by which cisplatin induces apoptosis. Furthermore, reduced phosphorylation levels of proteins involved in signaling downstream of the TCR during apoptosis were found by the phosphotyrosine proteome analysis. Our study can support to elucidate the mechanism behind the previously observed immunosuppressive effect of cisplatin.