Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression

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X-ray-based virtual slicing of TB-infected lungs

Hollow organs such as the lungs pose a considerable challenge for post-mortem imaging in preclinical research owing to their extremely low contrast and high structural complexity. The aim of our study was to enhance the contrast of tuberculosis lesions for their stratification by 3D x-ray&-based virtual slicing. Organ samples were taken from five control and five tuberculosis-infected mice. Micro-Computed Tomography (CT) scans of the subjects were acquired in vivo (without contrast agent) and post-mortem (with contrast agent). The proposed contrast-enhancing technique consists of x-ray contrast agent uptake (silver nitrate and iodine) by immersion. To create the histology ground-truth, the CT scan of the paraffin block guided the sectioning towards specific planes of interest. The digitalized histological slides reveal the presence, extent, and appearance of the contrast agents in lung structures and organized aggregates of immune cells. These findings correlate with the contrast-enhanced micro-CT slice. The abnormal densities in the lungs due to tuberculosis disease are concentrated in the right tail of the lung intensity histograms. The increase in the width of the right tail (~376%) indicates a contrast enhancement of the details of the abnormal densities. Postmortem contrast agents enhance the x-ray attenuation in tuberculosis lesions to allow 3D visualization by polychromatic x-ray CT, providing an advantageous tool for virtual slicing of whole lungs. The proposed contrast-enhancing technique combined with computational methods and the diverse micro-CT modalities will open the doors to the stratification of lesion types associated with infectious diseases.The research leading to these results received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement no. 115337, whose resources comprise funding from the European Union Seventh Framework Programme (FP7/2007–2013) and EFPIA companies in kind contribution. This work was partially funded by projects RTC-2015-3772-1, TEC2015-73064-EXP and TEC2016-78052-R from the Spanish Ministry of Economy, TOPUS S2013/MIT-3024 project from the regional government of Madrid and by the Department of Health, UK. This study (was supported by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013–2016) and co-financed by the European Social Fund (ESF) “ESF investing in your future”. The authors thank Dr.Guembe from CIMA-Universidad de Navarra for preparing and staining the tissue sections, and to Dr. Guerrero-Aspizua and Prof. Conti of the Department of Bioengineering, Universidad Carlos III de Madrid for the pathology evaluation

First-time-in-human study and prediction of early bactericidal activity for GSK3036656, a potent leucyl-tRNA synthetase inhibitor for tuberculosis treatment

This first-time-in-human (FTIH) study evaluated the safety, tolerability, pharmacokinetics, and food effect of single and repeat oral doses of GSK3036656, a leucyl-tRNA synthetase inhibitor. In part A, GSK3036656 single doses of 5 mg (fed and fasted), 15 mg, and 25 mg and placebo were administered. In part B, repeat doses of 5 and 15 mg and placebo were administered for 14 days once daily. GSK3036656 showed dose-proportional increase following single-dose administration and after dosing for 14 days. The maximum concentration of drug in serum (Cmax) and area under the concentration-time curve from 0 h to the end of the dosing period (AUC0–τ) showed accumulation with repeated administration of approximately 2- to 3-fold. Pharmacokinetic parameters were not altered in the presence of food. Unchanged GSK3036656 was the only drug-related component detected in plasma and accounted for approximately 90% of drug-related material in urine. Based on total drug-related material detected in urine, the minimum absorbed doses after single (25 mg) and repeat (15 mg) dosing were 50 and 78%, respectively. Unchanged GSK3036656 represented at least 44% and 71% of the 25- and 15-mg doses, respectively. Clinical trial simulations were performed to guide dose escalation during the FTIH study and to predict the GSK3036656 dose range that produces the highest possible early bactericidal activity (EBA0–14) in the prospective phase II trial, with consideration of the predefined exposure limit. GSK3036656 was well tolerated after single and multiple doses, with no reports of serious adverse events. (This study has been registered at ClinicalTrials.gov under identifier NCT03075410.

A Murine Model of falciparum-Malaria by In Vivo Selection of Competent Strains in Non-Myelodepleted Mice Engrafted with Human Erythrocytes

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NODscid/β2m−/− mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NODscid/β2m−/− mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D70087/N9 as a reference strain for model development. Pf3D70087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid

Análisis de las interacciones moleculares y del tráfico entre el citosol y el núcleo del factor de transcripción NF-kB(p50/p65) y de su inhibidor IkBa. Mecanismo de acción de corticodes y prostratina sobre la activación de NF-kB y la replicación del VIH

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-09-2004This work studies a fundamental problem in the management of HIV infection: the search of new strategies to eliminate viral resewoirs. that represents a rnajor obstacle to erradicate HIV from the patient. It has been addresed in two parts. In the first part, we analize molecular mechanisms implicated in latency and reactivation of HIV in lymphocytes. The trafic of NF-KB and its inhibitor, Ida, between nucleus and cytoplasm has been analized in two models, human PBMC and T cell blasts denved from them. We have found that these proteins shuttle between nucleus and cytoplasm of resting human lymphocytes. When we culture these cells with leptomycin B, an nuclear export inhibitor. p65 and IKBa accumulates into the nucleus. In these circumstances, we have dernonstrated an physical interaction between them by immunoprecipitation. When we activate these cells with PMA, we see also l~Baaccumulationin to the nucleus, but it does not bind to NF-kB. When we add also leptomycin B. we can see a more evident accumulation of lkBa in the nucleus, but the binding to DNA does not dissapear by gel shiR assay. In transfecting experiments with luciferase gene under the control of LTR, we have obsewed an inhibition of leptomycin B of its transactivation with PMA. We have also demonstrated an increment in lKBu levels in human lymphocytes cultured with dexamethasone that wrrelated with an inhibition of NF-kB binding to DNA and HIV propagation in culture. In the sewnd part, we have analized the effecto of Prostratin in HIV replication. Prostratin is a non-tumorogenic phorbol ester that delays HIV replication in vitro. but paradoxically reactivates HIV in latently-infected cells. To get a better insight into the mechanisms of action of prostratin we have analysed the effect of prostratin on HIV reactivation and wreceptor expression in human lymphocytes. LTR vectors, KB- and SP-1- driven luciferase wnstructs transfected in human PBMC were transactivated by prostratin. In another set of experiments PBMCs were transfected with full-length infectious viral clones. Prostratin induced HIV transcription and viral expression as detected by luciferase activity in cellular extracts and p24 levels in culture supematants. respectively. Expression of the HIV wreceptors CCR5 and CXCR4 was inhibited by prostratin and wnwmitantly. prostratin inhibited the infection of PBMC with R5 and X4 strains. However, infection with a pseudotyped viral clone that enter into the cells independently of HIV receptors was not inhibited by prostratin. These results contribute to explain the paradoxical effects of prostratin. On one hand, through the induction of NF-KB and Spl, prostratin induces HIV reactivation in latenly-infected cells. On the other hand, strong and persistent down-regulation of HIV receptors decreases reinfection of new targets and delays HIV propagation. These data support the potential use of prostratin to reactivate HIV from latency and purga viral resewoir

TRAF Family Proteins Link PKR with NF-κB Activation

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-2

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-5

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>together with CMV-IκBα or pcDNA3.1 as negative control, and then activated with anti-CDand IL-2, PHA and IL-2, or maintained in the absence of activation. Viral replication was determined by quantification of HIV p24-gag antigen in culture supernatants (a) after 5 days of transfection or (b) after 7 days of transfection. Numbers on the top of the bars represent fold HIV-replication relative to unstimulated T cells transfected with pcDNA3.1. Differences in p24-gag production were significant for resting and anti-CD-activated T cells (p < 0.05) and a trend towards statistical significance was found in PHA-activated T cells (p = 0.081)

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-8

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB