Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits IκBα and enhances HIV-1 replication in human T lymphocytes

<p>Abstract</p> <p>Background</p> <p>Degradation of p65/RelA has been involved in both the inhibition of NF-κB-dependent activity and the onset of apoptosis. However, the mechanisms of NF-κB degradation are unclear and can vary depending on the cell type. Cleavage of p65/RelA can produce an amino-terminal fragment that was shown to act as a dominant-negative inhibitor of NF-κB, thereby promoting apoptosis. However, the opposite situation has also been described and the production of a carboxy-terminal fragment that contains two potent transactivation domains has also been related to the onset of apoptosis. In this context, a carboxy-terminal fragment of p65/RelA (ΔNH₂p65), detected in non-apoptotic human T lymphocytes upon activation, has been studied. T cells constitute one of the long-lived cellular reservoirs of the human immunodeficiency virus type 1 (HIV-1). Because NF-κB is the most important inducible element involved in initiation of HIV-1 transcription, an adequate control of NF-κB response is of paramount importance for both T cell survival and viral spread. Its major inhibitor IκBα constitutes a master terminator of NF-κB response that is complemented by degradation of p65/RelA.</p> <p>Results and conclusions</p> <p>In this study, the function of a caspase-3-mediated carboxy-terminal fragment of p65/RelA, which was detected in activated human peripheral blood lymphocytes (PBLs), was analyzed. Cells producing this truncated p65/RelA did not undergo apoptosis but showed a high viability, in spite of caspase-3 activation. ΔNH₂p65 lacked most of DNA-binding domain but retained the dimerization domain, NLS and transactivation domains. Consequently, it could translocate to the nucleus, associate with NF-κB1/p50 and IκBα, but could not bind -κB consensus sites. However, although ΔNH₂p65 lacked transcriptional activity by itself, it could increase NF-κB activity in a dose-dependent manner by hijacking IκBα. Thus, its expression resulted in a persistent transactivation activity of wild-type p65/RelA, as well as an improvement of HIV-1 replication in PBLs. Moreover, ΔNH₂p65 was increased in the nuclei of PMA-, PHA-, and TNFα-activated T cells, proving this phenomenon was related to cell activation. These data suggest the existence of a novel mechanism for maintaining NF-κB activity in human T cells through the binding of the carboxy-terminal fragment of p65/RelA to IκBα in order to protect wild-type p65/RelA from IκBα inhibition.</p

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

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Modifications in host cell structure and functions mediated by Tat intracellular expression are greatly dependent on the second exon

1 page.-- Poster presentation.Peer reviewe

An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination

THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcriptiondependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcriptiondependent recombination.Ministerio de Educación y Ciencia BMC2000-0409 SAF2003-00204Junta de Andalucía CVI10

Selective miRNA inhibition in CD8+ cytotoxic T lymphocytes enhances HIV-1 specific cytotoxic responses

miRNAs dictate relevant virus-host interactions, offering new avenues for interventions to achieve an HIV remission. We aimed to enhance HIV-specific cytotoxic responses—a hallmark of natural HIV control— by miRNA modulation in T cells. We recruited 12 participants six elite controllers and six patients with chronic HIV infection on long-term antiretroviral therapy ("progressors"). Elite controllers exhibited stronger HIV-specific cytotoxic responses than the progressors, and their CD8+T cells showed a miRNA (hsa-miR-10a-5p) significantly downregulated. When we transfected ex vivo CD8(+) T cells from progressors with a synthetic miR-10a-5p inhibitor, miR-10a-5p levels decreased in 4 out of 6 progressors, correlating with an increase in HIV-specific cytotoxic responses. The effects of miR-10a-5p inhibition on HIV-specific CTL responses were modest, short-lived, and occurred before day seven after modulation. IL-4 and TNF-α levels strongly correlated with HIV-specific cytotoxic capacity. Thus, inhibition of miR-10a-5p enhanced HIV-specific CD8(+) T cell capacity in progressors. Our pilot study proves the concept that miRNA modulation is a feasible strategy to combat HIV persistence by enhancing specific cytotoxic immune responses, which will inform new approaches for achieving an antiretroviral therapy-free HIV remission

Systemic and Pulmonary Vascular Remodelling in Chronic Obstructive Pulmonary Disease

Background: Chronic Obstructive Pulmonary Disease (COPD) is associated with subclinical systemic atherosclerosis and pulmonary vascular remodelling characterized by intimal hyperplasia and luminal narrowing. We aimed to determine differences in the intimal thickening of systemic and pulmonary arteries in COPD subjects and smokers. Secondary aims include comparisons with a non-smokers group; determining the clinical variables associated with systemic and pulmonary intimal thickening, and the correlations between systemic and pulmonary remodelling changes. Methods: All consecutive subjects undergoing lung resection were included and divided into 3 groups: 1) COPD, 2) smokers, and 3) non-smokers. Sections of the 5th intercostal artery and muscular pulmonary arteries were measured by histo-morphometry. Four parameters of intimal thickening were evaluated: 1) percentage of intimal area (%IA), 2) percentage of luminal narrowing, 3) intimal thickness index, and 4) intima-to-media ratio. Results: In the adjusted analysis, the systemic arteries of COPD subjects showed greater intimal thickening (%IA) than those of smokers (15.6 +/- 1.5% vs. 14.2 +/- 1.6%, p = 0.038). In the pulmonary arteries, significant differences were observed for % IA between the 2 groups (37.3 +/- 2.2% vs. 29.3 +/- 2.3%, p = 0.016). Among clinical factors, metabolic syndrome, gender and COPD status were associated with the systemic intimal thickening, while only COPD status was associated with pulmonary intimal thickening. A correlation between the % IA of the systemic and pulmonary arteries was observed (Spearman's rho = 0.46, p = 0.008). Conclusions: Greater intimal thickening in systemic and pulmonary arteries is observed in COPD patients than in smokers. There is a correlation between systemic and pulmonary vascular remodelling in the overall population

Selective miRNA Modulation Fails to Activate HIV Replication in In Vitro Latency Models

HIV remains incurable because of viral persistence in latent reservoirs that are inaccessible to antiretroviral therapy. A potential curative strategy is to reactivate viral gene expression in latently infected cells. However, no drug so far has proven to be successful in vivo in reducing the reservoir, and therefore new anti-latency compounds are needed. We explored the role of microRNAs (miRNAs) in latency maintenance and their modulation as a potential anti-latency strategy. Latency models based on treating resting CD4 T cells with chemokine (C-C motif) ligand 19 (CCL19) or interleukin-7 (IL7) before HIV infection and next-generation sequencing were used to identify the miRNAs involved in HIV latency. We detected four upregulated miRNAs (miRNA-98, miRNA-4516, miRNA-4488, and miRNA-7974). Individual or combined inhibition of these miRNAs was performed by transfection into cells latently infected with HIV. Viral replication, assessed 72 h after transfection, did not increase after miRNA modulation, despite miRNA inhibition and lack of toxicity. Furthermore, the combined modulation of five miRNAs previously associated with HIV latency was not effective in these models. Our results do not support the modulation of miRNAs as a useful strategy for the reversal of HIV latency. As shown with other drugs, the potential of miRNA modulation as an HIV reactivation strategy could be dependent on the latency model usedThis work wasfunded by the Spanish Ministry of Economy and Competitiveness(PIE 13/00040) and the Spanish AIDS Research Network (RIS)(RD16/0025/0001) as part of the Plan Estatal I+D+I and co-financedby Instituto de Salud Carlos III (ISCIII)-Subdirección General deEvaluación y Fomento de la Investigación and Fondo Europeo deDesarrollo Regional (FEDER) (European Regional DevelopmentFund). M.R.L-H. was supported by the Spanish Ministry of Economyand Competitiveness with ISCIII-FEDER funding (PIE 13/00040 and RD12/0017/0017). N.M.E. and C.G. were supported bythe Spanish AIDS Research Network (RD12/0017/0017 and RD16/0025/001

A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.This work was supported by the Consejería de Conocimiento, Innovación y Universidades de la Junta de Andalucía (FQM-206, FQM-274, FQM-135 and P20–01234); VI Plan Propio Universidad de Sevilla (PP2019/00000748), and the European Union (Feder Funds).Peer reviewe

Supplementary material. A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells

S1. Experimental Section: S1.1. Synthesis and Characterization Data. S1.2. Fluorescence Measurements. S1.3. Zeta Potential Measurements. S1.4. Dynamic Light Scattering Measurements. S1.5. Circular Dichroism Spectra. S1.6. Agarose Gel Electrophoresis. S1.7. Atomic Force Microscopy (AFM). S1.8. In Vitro Assays S1.9. Transfection Assays. S1.10. Chlamydomonas reinhardtii Nuclear Transformation. S2. Results and Discussion: S2.1. Characterization of Monomers and Polyurethanes. S2.2. Formation of the polyplexes PUMan/ctDNA. Figures: Fig. S1. FTIR spectra of PUMan and (MBocCis)DTDI. Fig. S2. SEC chromatogram of (MAL)DTDI. Fig. S3. SEC chromatogram of (MBocCis)DTDI. Fig. S4. SEC chromatogram of PUMan. Fig. S5. 1H NMR of (MAL)DTDI. Fig. S6. 1H NMR of (MBocCis)DTDI. Fig. S7. 1H NMR of PUMan. Fig. S8. TGA curve of PUMan. Fig. S9. Plot of EB emission intensities at different N/P values, circular dichroism spectra, zeta potential and hydrodynamic diameters of the PUMan-based polyplexes. Fig.S10. Electrophoresis of polyplexes PUMan/digested pEGFP-C1 and PUMan/digested Phyco69 at different N/P ratios. Fig.S11. Percentage of GFP positive cells after transfection with 3 μg of the plasmid pEGFP-C1 with the indicated reagents. The molar ratio PUMan:FuGENE and PUMan:DOPE was 1:1. Tables: Table S1. Thermal properties of PUMan and its precursors.Peer reviewe

Changes in the immune response against SARS-CoV-2 in individuals with severe COVID-19 treated with high dose of vitamin D

Main cause of severe illness and death in COVID-19 patients appears to be an excessive but ineffectual inflammatory immune response that may cause severe acute respiratory distress syndrome (ARDS). Vitamin D may favour an anti-inflammatory environment and improve cytotoxic response against some infectious diseases. A multicenter, single-blind, prospective, randomized clinical trial was approved in patients with COVID-19 pneumonia and levels of 25-hydroxyvitamin D (25(OH)D) of 14.8 ng/ml (SD: 6.18) to test antiviral efficacy, tolerance and safety of 10,000 IU/day of cholecalciferol (vitamin D3) for 14 days, in comparison with 2000 IU/day. After supplementation, mean serum 25(OH)D levels increased to 19 ng/ml on average in 2000 IU/day versus 29 ng/ml in 10,000 IU/day group (p < 0.0001). Although levels of inflammatory cytokines were not modified by treatment with 10,000 IU/day, there was an increase of anti-inflammatory cytokine IL-10 and higher levels of CD4+ T cells, with predominance of T central memory subpopulation. Cytotoxic response against pseudotyped SARS-CoV-2 infected cells was increased more than 4-fold in patients who received 10,000 IU/day. Moreover, levels of IFNγ were significantly higher in this group. Beneficial effect of supplementation with 10,000 IU/day was also observed in participants who developed ARDS and stayed at the hospital for 8.0 days, whereas those who received 2000 IU/day stayed for 29.2 days (p = 0.0381). Administration of high doses of vitamin D3 as adjuvant of the standard care treatment during hospitalization for COVID-19 may improve the inflammatory environment and cytotoxic response against pseudotyped SARS-CoV-2 infected cells, shortening the hospital stay and, possibly, improving the prognosis.We greatly appreciate all the patients for their participation in this study. We thank the excellent secretarial assistance of Mrs Olga Palao at the Centro Nacional de Microbiología (CNM, Instituto de Salud Carlos III). The authors also acknowledge María C. de la Cruz at Unidad Central de Apoyo a la Investigación Clínica y Ensayos Clínicos (Instituto de Investigación Sanitaria Gregorio Marañon; IiSGM) for her advice and assistance related to the clinical research with medicines. This work was supported by the Coordinated Research Activities at CNM (Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); the Spanish Ministry of Science and Innovation (PID2019–110275RB-I00); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016–2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF) and Fundación Universidad Alfonso X el Sabio (FUAX, Madrid, Spain; Reference 1012010). The work of Montserrat Torres is financed by the Coordinated Research Activities at the CNM (Instituto de Salud Carlos III) (COV20_00679). The work of María Rosa López-Huertas and Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Fernando Ramos Martín is financed by the Spanish Ministry of Science and Innovation (PID2019–110275RB-I00). Drug Cholecalciferol (vitamin D) used in the study was donated by Italfarmaco Group (Cholecalciferol 25,000IU/2,5 ml oral solution). Italfarmaco Group had no role in the design and conduct of the study, in the collection, management, analysis, and interpretation of the data, or the preparation, review, or approval of the manuscript.S