Enantioselective Approach for Expanding the Three-Dimensional Space of Tetrahydroquinoline to Develop BET Bromodomain Inhibitors.

The pharmaceutical industry has a pervasive need for chiral specific molecules with optimal affinity for their biological targets. However, the mass production of such compounds is currently limited by conventional chemical routes, that are costly and have an environmental impact. Here, we propose an easy access to obtain new tetrahydroquinolines, a motif found in many bioactive compounds, that is rapid and cost effective. Starting from simple raw materials, the procedure uses a proline-catalyzed Mannich reaction followed by the addition of BF3 ⋅ OEt2 , which generates a highly electrophilic aza-ortho-quinone methide intermediate capable of reacting with different nucleophiles to form the diversely functionalized tetrahydroquinoline. Moreover, this enantioselective one-pot process provides access for the first time to tetrahydroquinolines with a cis-2,3 and trans-3,4 configuration. As proof of concept, we demonstrate that a three-step reaction sequence, from simple and inexpensive starting compounds and catalysts, can generate a BD2-selective BET bromodomain inhibitor with anti-inflammatory effect.

Effect of Novel AKT Inhibitor Vevorisertib as Single Agent and in Combination with Sorafenib on Hepatocellular Carcinoma in a Cirrhotic Rat Model.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. The AKT pathway is often activated in HCC cases, and a longer exposure to tyrosine kinase inhibitors such as sorafenib may lead to over-activation of the AKT pathway, leading to HCC resistance. Here, we studied the efficacy of a new generation of allosteric AKT inhibitor, vevorisertib, alone or in combination with sorafenib. To identify specific adverse effects related to the background of cirrhosis, we used a diethylnitrosamine (DEN)-induced cirrhotic rat model. Vevorisertib was tested in vitro on Hep3B, HepG2, HuH7 and PLC/PRF cell lines. Rats were treated weekly with intra-peritoneal injections of DEN for 14 weeks to obtain cirrhosis with fully developed HCC. After that, rats were randomized into four groups (n = 7/group): control, sorafenib, vevorisertib and the combination of vevorisertib + sorafenib, and treated for 6 weeks. Tumor progression was followed by MRI. We demonstrated that the vevorisertib is a highly potent treatment, blocking the phosphorylation of AKT. The tumor progression in the rat liver was significantly reduced by treatment with vevorisertib + sorafenib (49.4%) compared to the control group (158.8%, p < 0.0001). Tumor size, tumor number and tumor cell proliferation were significantly reduced in both the vevorisertib group and vevorisertib + sorafenib groups compared to the control group. Sirius red staining showed an improvement in liver fibrosis by vevorisertib and the combination treatment. Moreover, vevorisertib + sorafenib treatment was associated with a normalization in the liver vasculature. Altogether, vevorisertib as a single agent and its combination with sorafenib exerted a strong suppression of tumor progression and improved liver fibrosis. Thus, results provide a rationale for testing vevorisertib in clinical settings and confirm the importance of targeting AKT in HCC.

Targeting Akt in Hepatocellular Carcinoma and Its Tumor Microenvironment.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide, and its incidence is rising. HCC develops almost exclusively on the background of chronic liver inflammation, which can be caused by chronic alcohol consumption, viral hepatitis, or an unhealthy diet. The key role of chronic inflammation in the process of hepatocarcinogenesis, including in the deregulation of innate and adaptive immune responses, has been demonstrated. The inhibition of Akt (also known as Protein Kinase B) directly affects cancer cells, but this therapeutic strategy also exhibits indirect anti-tumor activity mediated by the modulation of the tumor microenvironment, as demonstrated by using Akt inhibitors AZD5363, MK-2206, or ARQ 092. Moreover, the isoforms of Akt converge and diverge in their designated roles, but the currently available Akt inhibitors fail to display an isoform specificity. Thus, selective Akt inhibition needs to be better explored in the context of HCC and its possible combination with immunotherapy. This review presents a compact overview of the current knowledge concerning the role of Akt in HCC and the effect of Akt inhibition on the HCC and liver tumor microenvironment.

A proliferation-inducing ligand-mediated anti-inflammatory response of astrocytes in multiple sclerosis.

OBJECTIVE: The two related tumor necrosis factor members a proliferation-inducing ligand (APRIL) and B-cell activation factor (BAFF) are currently targeted in autoimmune diseases as B-cell regulators. In multiple sclerosis (MS), combined APRIL/BAFF blockade led to unexpected exacerbated inflammation in the central nervous system (CNS) of patients. Here, we investigate the role of the APRIL/BAFF axis in the CNS. METHODS: APRIL expression was analyzed in MS lesions by immunohistochemistry. The in vivo role of APRIL was assessed in the murine MS model, experimental autoimmune encephalitis (EAE). Functional in vitro studies were performed with human and mouse astrocytes. RESULTS: APRIL was expressed in lesions from EAE. In its absence, the disease was worst. Lesions from MS patients also showed APRIL expression upon infiltration of macrophages. Notably, all the APRIL secreted by these macrophages specifically targeted astrocytes. The upregulation of chondroitin sulfate proteoglycan, sometimes bearing chondroitin sulfate of type E sugar moieties, binding APRIL, in reactive astrocytes explained the latter selectivity. Astrocytes responded to APRIL by producing a sufficient amount of IL-10 to dampen antigen-specific T-cell proliferation and pathogenic cytokine secretion. Finally, an intraspinal delivery of recombinant APRIL before disease onset, shortly reduced EAE symptoms. Repeated intravenous injections of recombinant APRIL before and even at disease onset also had an effect. INTERPRETATION: Our data show that APRIL mediates an anti-inflammatory response from astrocytes in MS lesions. This protective activity is not shared with BAFF. ANN NEUROL 2019;85:406-420.

Modulating the Crosstalk between the Tumor and Its Microenvironment Using RNA Interference: A Treatment Strategy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy with one of the highest mortality rates among solid cancers. It develops almost exclusively in the background of chronic liver inflammation, which can be caused by viral hepatitis, chronic alcohol consumption or an unhealthy diet. Chronic inflammation deregulates the innate and adaptive immune responses that contribute to the proliferation, survival and migration of tumor cells. The continuous communication between the tumor and its microenvironment components serves as the overriding force of the tumor against the body's defenses. The importance of this crosstalk between the tumor microenvironment and immune cells in the process of hepatocarcinogenesis has been shown, and therapeutic strategies modulating this communication have improved the outcomes of patients with liver cancer. To target this communication, an RNA interference (RNAi)-based approach can be used, an innovative and promising strategy that can disrupt the crosstalk at the transcriptomic level. Moreover, RNAi offers the advantage of specificity in comparison to the treatments currently used for HCC in clinics. In this review, we will provide the recent data pertaining to the modulation of a tumor and its microenvironment by using RNAi and its potential for therapeutic intervention in HCC.

Sex Differences in Spontaneous Degranulation Activity of Intrahepatic Natural Killer Cells during Chronic Hepatitis B: Association with Estradiol Levels.

Major sex differences are observed in the prevalence, intensity, and severity of hepatitis B virus (HBV) infection. Here, we investigated degranulation activity of circulating and intrahepatic natural killer (NK) cells from HBV and HCV chronically infected patients before any treatment (n = 125). The frequency of CD107+ NK cells in the female liver was significantly higher compared to that in males during chronic HBV infection (p = 0.002) and correlated with the plasma levels of estradiol (correlation coefficient r = 0.634; p < 0.0001). Our results clearly show sex differences in degranulation activity of intrahepatic NK cells of HBV-infected patients. This probably contributes to the ability of females to better deal with HBV disease.

Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.

The MSRV (multiple sclerosis-associated retrovirus) belongs to the human endogenous retrovirus HERV-W family. The envelope protein originating from the MSRV has been found in most patients with multiple sclerosis (MS). This protein (Env-ms) has pro-inflammatory properties for several types of immune cells and could therefore play a role in MS pathogenesis by promoting the leukocyte diapedesis observed in the central nervous system of patients. Our study aims to analyze the effects of Env-ms on the blood-brain barrier (BBB) at a molecular and functional level. We demonstrate that the recombinant MSRV envelope is able to stimulate several inflammatory parameters in a human BBB in vitro model, the HCMEC/D3 brain endothelial cell line. Indeed, Env-ms induces over-expression of ICAM-1, a major mediator of leukocyte adhesion to endothelial cells, in a dose-dependent manner as well as a strong dose-dependent production of the pro-inflammatory cytokines IL-6 and IL-8. Furthermore, using a silencing approach with siRNAs, we show that Env-ms is recognized via the Toll-like receptor 4 receptor, a pattern recognition receptor of innate immunity present on endothelial cells. We also show, using functional assays, that treatment of brain endothelial cells with Env-ms significantly stimulated the adhesion and the transmigration of activated immune cells through a monolayer of endothelial cells. These findings support the hypothesis that MSRV could be involved in the pathogenesis of MS disease or at least in maintenance of inflammatory conditions, thus fueling the auto-immune disorder. MSRV could also play a role in other chronic inflammatory diseases.

Progression of fibrosis in patients with chronic viral hepatitis is associated with IL-17(+) neutrophils.

BACKGROUND & AIMS: The pro-inflammatory cytokine IL-17 plays a crucial role in liver diseases associated with hepatic fibrosis and increased risk of cancer development. Nevertheless, the cellular source of this cytokine has never been characterized in patients with liver fibrosis. METHODS: In this study, we investigated liver biopsies from 49 patients with chronic viral hepatitis at different stages of liver fibrosis. We monitored IL-17 production by intracellular flow cytometry, immunofluorescence and immunohistochemical in situ stainings, allowing a precise quantification, characterization and localization of IL-17(+) cells. RESULTS: Density of IL-17(+) cells increased with the stage of liver fibrosis specifically in fibrotic septa and portal areas (correlation coefficient r = 0.7373; P < 0.0001). Data clearly show that the frequency of intrahepatic IL-17(+) lymphocytes (including T, NKT and NK cells) was independent on stage of liver fibrosis, and we observed no statistical differences in number of IL-17(+) macrophages during progression of fibrosis. On the other hand, the number of IL-17(+) neutrophils in fibrotic septa and portal areas strongly correlated with the stages of fibrosis (correlation coefficient r = 0.6986; P < 0.0001), contributing significantly to total IL-17 production in liver tissue. CONCLUSIONS: Our data indicate that neutrophils represent an important source of IL-17 in the human liver, especially in late fibrosis stages. Inhibition of this specific harmful subset of neutrophils may offer therapeutic opportunities in fibrotic liver.

Liver-infiltrating CD8(+) lymphocytes as prognostic factor for tumour recurrence in hepatitis C virus-related hepatocellular carcinoma.

BACKGROUND: Chronic liver inflammation and immune/inflammatory response promote hepatocellular carcinoma. The aim of this study was to characterize the immune status of HCV-related cirrhosis in patients with hepatocellular carcinoma (HCV-HCC) as compared to HCV patients without hepatocellular carcinoma. METHOD: Immune markers (CD3, CD4, CD8, CD20, CD56, TCRγδ, FoxP3) and gene expression profiles (CD8α, CD8ß, FoxP3, IL-6, IFN-γ, perforin, RANTES) were analysed in a test cohort by immunohistochemistry and quantitative RT-PCR analysis on serial non-tumorous and tumorous tissues. RESULTS: Immune micro-environment was more inflammatory in HCV-HCC than HCV cirrhotic livers. The number of CD3(+) , CD4(+) , CD8(+) and CD20(+) liver-infiltrating lymphocytes was significantly higher, whereas the number of CD56(+) cells was significantly lower in HCV-HCC compared to HCV cirrhotic parenchyma. These differences were restricted to fibrous septa for CD4(+) and CD20(+) cells and to nodular parenchyma for CD8(+) cells. Gene expressions of CD8α, FoxP3 and RANTES were also significantly higher in HCV-HCC than in HCV cirrhosis. Interestingly, in a large cohort of 63 HCV-HCC patients. The number of CD8(+) cells ≥100/field was associated with significant higher tumour recurrence (P = 0.003) and lower overall survival (P = 0.05) at 5 years. CONCLUSION: High densities of liver-infiltrating lymphocytes in HCV-HCC cirrhotic parenchyma prevail inflammatory conditions and could contribute to tumorigenesis and tumour recurrence. These results could contribute towards better clinical evaluation of patients susceptible for HCC recurrence after curative surgery.

Protein-peptide arrays for detection of specific anti-hepatitis D virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging.

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg-anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array.

Lymphocytes degranulation in liver in hepatitis C virus carriers is associated with IFNL4 polymorphisms and ALT levels.

BACKGROUND: The polymorphisms of IFNL4 are strongly associated with both spontaneous hepatitis C virus (HCV) clearance and response to peg-IFN-α/ribavirin treatment. To further establish the biological effects of the IFNL4 and rs1297860 variations, we studied the activity of liver immune cells. METHODS: Fresh liver samples were collected from HCV-infected patients before any treatment and from NASH patients as controls. Degranulation activity of each lymphocyte type was assessed by the surface expression of CD107a. IFNL4 polymorphisms and HCV genotypes were determined. RESULTS: In the liver, frequency of CD107a(+) immune cells was significantly higher in HCV patients compared to NASH patients. Higher degranulation activity was observed in lymphocytes of HCV patients with favorable IFNL4 genotypes compared to patients with unfavorable genotypes. Multivariate regression analyses indicated that serum ALT levels were dependent on both Metavir activity score and frequency of CD107a positive NKT cells. The high level of degranulation activity observed before treatment was associated with a high HCV RNA decline at the early stage of peg-IFN-α/ribavirin treatment in patients with favorable genotypes. CONCLUSIONS: These data underline that intrahepatic lymphocyte degranulation activity in HCV-infected patients is associated with IFNL4 polymorphisms and contributes to the clearance of HCV in patients with favorable genotypes under antiviral therapy.

Spatial resolution in prism-based surface plasmon resonance microscopy.

Several optical surface sensing techniques, such as Surface Plasmon Resonance (SPR), work by imaging the base of a prism by one of its faces. However, such a fundamental optical concern has not been fully analyzed and understood so far, and spatial resolution remains a critical and controversial issue. In SPR, the propagation length L(x) of the surface plasmon waves has been considered as the limiting factor. Here, we demonstrate that for unoptimized systems geometrical aberrations caused by the prism can be more limiting than the propagation length. By combining line-scan imaging mode with optimized prisms, we access the ultimate lateral resolution which is diffraction-limited by the object light diffusion. We describe several optimized configurations in water and discuss the trade-off between L(x) and sensitivity. The improvement of resolution is confirmed by imaging micro-structured PDMS stamps and individual living eukaryote cells and bacteria on field-of-view from 0.1 to 20 mm(2).

A top-down synthesis route to ultrasmall multifunctional Gd-based silica nanoparticles for theranostic applications.

New, ultrasmall nanoparticles with sizes below 5 nm have been obtained. These small rigid platforms (SRP) are composed of a polysiloxane matrix with DOTAGA (1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid)-Gd(3+) chelates on their surface. They have been synthesised by an original top-down process: 1) formation of a gadolinium oxide Gd2O3 core, 2) encapsulation in a polysiloxane shell grafted with DOTAGA ligands, 3) dissolution of the gadolinium oxide core due to chelation of Gd(3+) by DOTAGA ligands and 4) polysiloxane fragmentation. These nanoparticles have been fully characterised using photon correlation spectroscopy (PCS), transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID) and electron paramagnetic resonance (EPR) to demonstrate the dissolution of the oxide core and by inductively coupled plasma mass spectrometry (ICP-MS), mass spectrometry, fluorescence spectroscopy, (29)Si solid-state NMR, (1)H NMR and diffusion ordered spectroscopy (DOSY) to determine the nanoparticle composition. Relaxivity measurements gave a longitudinal relaxivity r1 of 11.9 s(-1) mM(-1) per Gd at 60 MHz. Finally, potentiometric titrations showed that Gd(3+) is strongly chelated to DOTAGA (complexation constant logß110 =24.78) and cellular tests confirmed the that nanoconstructs had a very low toxicity. Moreover, SRPs are excreted from the body by renal clearance. Their efficiency as contrast agents for MRI has been proved and they are promising candidates as sensitising agents for image-guided radiotherapy.

Predictive toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data.

BACKGROUND: Cobalt-ferrite nanoparticles (Co-Fe NPs) are attractive for nanotechnology-based therapies. Thus, exploring their effect on viability of seven different cell lines representing different organs of the human body is highly important. METHODS: The toxicological effects of Co-Fe NPs were studied by in-vitro exposure of A549 and NCIH441 cell-lines (lung), precision-cut lung slices from rat, HepG2 cell-line (liver), MDCK cell-line (kidney), Caco-2 TC7 cell-line (intestine), TK6 (lymphoblasts) and primary mouse dendritic-cells. Toxicity was examined following exposure to Co-Fe NPs in the concentration range of 0.05 -1.2 mM for 24 and 72 h, using Alamar blue, MTT and neutral red assays. Changes in oxidative stress were determined by a dichlorodihydrofluorescein diacetate based assay. Data analysis and predictive modeling of the obtained data sets were executed by employing methods of Knowledge Discovery from Data with emphasis on a decision tree model (J48). RESULTS: Different dose-response curves of cell viability were obtained for each of the seven cell lines upon exposure to Co-Fe NPs. Increase of oxidative stress was induced by Co-Fe NPs and found to be dependent on the cell type. A high linear correlation (R2=0.97) was found between the toxicity of Co-Fe NPs and the extent of ROS generation following their exposure to Co-Fe NPs. The algorithm we applied to model the observed toxicity belongs to a type of supervised classifier. The decision tree model yielded the following order with decrease of the ranking parameter: NP concentrations (as the most influencing parameter), cell type (possessing the following hierarchy of cell sensitivity towards viability decrease: TK6 > Lung slices > NCIH441 > Caco-2 = MDCK > A549 > HepG2 = Dendritic) and time of exposure, where the highest-ranking parameter (NP concentration) provides the highest information gain with respect to toxicity. The validity of the chosen decision tree model J48 was established by yielding a higher accuracy than that of the well-known "naive bayes" classifier. CONCLUSIONS: The observed correlation between the oxidative stress, caused by the presence of the Co-Fe NPs, with the hierarchy of sensitivity of the different cell types towards toxicity, suggests that oxidative stress is one possible mechanism for the toxicity of Co-Fe NPs.

Immune checkpoints on T and NK cells in the context of HBV infection: Landscape, pathophysiology and therapeutic exploitation.

In hepatitis B virus (HBV) infection, the interplay between the virus and the host immune system is crucial in determining the pathogenesis of the disease. Patients who fail to mount a sufficient and sustained anti-viral immune response develop chronic hepatitis B (CHB). T cells and natural killer (NK) cells play decisive role in viral clearance, but they are defective in chronic HBV infection. The activation of immune cells is tightly controlled by a combination of activating and inhibitory receptors, called immune checkpoints (ICs), allowing the maintenance of immune homeostasis. Chronic exposure to viral antigens and the subsequent dysregulation of ICs actively contribute to the exhaustion of effector cells and viral persistence. The present review aims to summarize the function of various ICs and their expression in T lymphocytes and NK cells in the course of HBV infection as well as the use of immunotherapeutic strategies targeting ICs in chronic HBV infection.

Intracellular accumulation and immunological response of NIR-II polymeric nanoparticles.

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

Hepatitis C virus (HCV) evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines.

Understanding how hepatitis C virus (HCV) induces and circumvents the host's natural killer (NK) cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC) molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4) promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFß which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFß effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

Enhanced susceptibility of T lymphocytes to oxidative stress in the absence of the cellular prion protein.

The cellular prion glycoprotein (PrP(C)) is ubiquitously expressed but its physiologic functions remain enigmatic, particularly in the immune system. Here, we demonstrate in vitro and in vivo that PrP(C) is involved in T lymphocytes response to oxidative stress. By monitoring the intracellular level of reduced glutathione, we show that PrP(-/-) thymocytes display a higher susceptibility to H(2)O(2) exposure than PrP(+/+) cells. Furthermore, we find that in mice fed with a restricted diet, a regimen known to increase the intracellular level of ROS, PrP(-/-) thymocytes are more sensitive to oxidative stress. PrP(C) function appears to be specific for oxidative stress, since no significant differences are observed between PrP(-/-) and PrP(+/+) mice exposed to other kinds of stress. We also show a marked evolution of the redox status of T cells throughout differentiation in the thymus. Taken together, our results clearly ascribe to PrP(C) a protective function in thymocytes against oxidative stress.

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. METHODS: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. RESULTS: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. CONCLUSIONS: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.