Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses

Ricin Toxicokinetics and Its Sensitive Detection in Mouse Sera or Feces Using Immuno-PCR

Ricin (also called RCA-II or RCA(60)), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Several in vitro methods have been designed for ricin detection in complex food matrices in the event of intentional contamination. Recently, a novel Immuno-PCR (IPCR) assay was developed with a limit of detection of 10 fg/ml in a buffer matrix and about 10-1000-fold greater sensitivity than other methods in various food matrices.In order to devise a better diagnostic test for ricin, the IPCR assay was adapted for the detection of ricin in biological samples collected from mice after intoxication. The limit of detection in both mouse sera and feces was as low as 1 pg/ml. Using the mouse intravenous (iv) model for ricin intoxication, a biphasic half-life of ricin, with a rapid t(1/2)α of 4 min and a slower t(1/2)β of 86 min were observed. The molecular biodistribution time for ricin following oral ingestion was estimated using an antibody neutralization assay. Ricin was detected in the blood stream starting at approximately 6-7 h post- oral intoxication. Whole animal histopathological analysis was performed on mice treated orally or systemically with ricin. Severe lesions were observed in the pancreas, spleen and intestinal mesenteric lymph nodes, but no severe pathology in other major organs was observed.The determination of in vivo toxicokinetics and pathological effects of ricin following systemic and oral intoxication provide a better understanding of the etiology of intoxication and will help in the future design of more effective diagnostic and therapeutic methods

A Dominant-Negative Approach That Prevents Diphthamide Formation Confers Resistance to Pseudomonas Exotoxin A and Diphtheria Toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments

The endothelial glycocalyx: composition, functions, and visualization

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging

Vascular permeability, vascular hyperpermeability and angiogenesis

The vascular system has the critical function of supplying tissues with nutrients and clearing waste products. To accomplish these goals, the vasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. Physiologists and many vascular biologists differ as to the definition of vascular permeability and the proper methodology for its measurement. We review these conflicting views, finding that both provide useful but complementary information. Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). We demonstrate that three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, and the anatomic pathways by which molecules of different size cross-vascular endothelium. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. Finally, we list the numerous (at least 25) gene products that different authors have found to affect vascular permeability in variously engineered mice and classify them with respect to their participation, as far as possible, in BVP, AVH and CVH. Further work will be required to elucidate the signaling pathways by which each of these molecules, and others likely to be discovered, mediate the different types of vascular permeability

Observation of Two New Excited Ξb0 States Decaying to Λb0 K-π+

Two narrow resonant states are observed in the Λb0K-π+ mass spectrum using a data sample of proton-proton collisions at a center-of-mass energy of 13 TeV, collected by the LHCb experiment and corresponding to an integrated luminosity of 6 fb-1. The minimal quark content of the Λb0K-π+ system indicates that these are excited Ξb0 baryons. The masses of the Ξb(6327)0 and Ξb(6333)0 states are m[Ξb(6327)0]=6327.28-0.21+0.23±0.12±0.24 and m[Ξb(6333)0]=6332.69-0.18+0.17±0.03±0.22 MeV, respectively, with a mass splitting of Δm=5.41-0.27+0.26±0.12 MeV, where the uncertainties are statistical, systematic, and due to the Λb0 mass measurement. The measured natural widths of these states are consistent with zero, with upper limits of Γ[Ξb(6327)0]<2.20(2.56) and Γ[Ξb(6333)0]<1.60(1.92) MeV at a 90% (95%) credibility level. The significance of the two-peak hypothesis is larger than nine (five) Gaussian standard deviations compared to the no-peak (one-peak) hypothesis. The masses, widths, and resonant structure of the new states are in good agreement with the expectations for a doublet of 1D Ξb0 resonances

Study of the ψ2(3823)ψ_2(3823) and χc1(3872)χ_{c1}(3872) states in B+→(Jψπ+π−)K+B^+ \rightarrow \left( Jψπ^+π^-\right)K^+ decays

The decays $B^+\rightarrow J/\psi \pi^+ \pi^- K^+$ are studied using a data set corresponding to an integrated luminosity of 9fb$^{-1}$ collected with the LHCb detector in proton-proton collisions between 2011 and 2018. Precise measurements of the ratios of branching fractions with the intermediate $\psi_2(3823)$, $\chi_{c1}(3872)$ and $\psi(2S)$ states are reported. The decay of $B^+\rightarrow \psi_2(3823)K^+$ with $\psi_2(3823)\rightarrow J\psi\pi^+\pi^-$ is observed for the first time with a significance of 5.1 standard deviations. The mass differences between the $\psi_2(3823)$, $\chi_{c1}(3872)$ and $\psi(2S)$ states are measured to be $$\begin{array}{rcl} m_{\chi_{c1(3872)}} - m_{\psi_2(3823)} &= & 47.50 \pm 0.53 \pm 0.13\,\mathrm{MeV/}c^2\,, \\ m_{\psi_2(3823)} - m_{\psi(2S)} &= & 137.98 \pm 0.53 \pm 0.14\,\mathrm{MeV/}c^2\,, \\ m_{\chi_{c1}(3872)} - m_{\psi(2S)} &= & 185.49 \pm 0.06 \pm 0.03\,\mathrm{MeV/}c^2\,, \end{array}$$ resulting in the most precise determination of the $\chi_{c1}(3782)$ mass. The width of the $\psi_2(3823)$ state is found to be below 5.2MeV at 90\% confidence level. The Breit-Wigner width of the $\chi_{c1}(3872)$ state is measured to be $$\Gamma^{\mathrm{BW}}_{\chi_{c1}(3872)} = 0.96^{+0.19}_{-0.18}\pm0.21 \mathrm{MeV},$$ which is inconsistent with zero by 5.5 standard deviations

Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells

To investigate the dependence of individual immunological processes on DC, a transgenic mouse system (CD11c-DTR/GFP mice) has been developed that allows conditional depletion of CD11c+ DC in vivo through administration of diphtheria toxin. We have performed careful histological analysis of CD11c-DTR/GFP mice at different time points after diphtheria toxin injection and confirmed the transient depletion of CD11c+ cells from lymph nodes and spleen. Unexpectedly, the injection of diphtheria toxin completely depleted marginal zone and metallophilic M(Phi) from the spleen and their sinusoidal counterparts from the lymph nodes. This finding limits the use of CD11c-DTR/GFP mice for the analysis of the role of DC to models and read outs that are proven to be independent of marginal zone and sinusoidal M(Phi)