Highly Efficient Autologous HIV-1 Isolation by Coculturing Macrophage With Enriched CD4+ T Cells From HIV-1 Patients

We described a novel HIV autologous isolation method based in coculturing macrophages and CD4+T-cell-enriched fractions from peripheral blood collected from antiretroviral-treated (ART) HIV patients. This method allows the isolation of high viral titers of autologous viruses, over 1010HIV RNA copies/ml, and reduces the time required to produce necessary amounts for virus for use as antigens presented by monocyte-derived myeloid cells in HIV therapeutic vaccine approaches. By applying these high titer and autologous virus produced in the patient-derived cells, we intended to elicit a boost of the immunological system response in HIV therapeutic vaccines in clinical trials.This study was partially supported by grants from the Spanish Ministry of Economy (MINECO) (grants: SAF2015-66193-R, SAF-2017-88089-R, RTI2018-096309-B-I00); the Fondo Europeo para el Desarrollo Regional (FEDER); the SPANISH AIDS Research Network RD16/0025/0002 and RD16/0025/0014-ISCIII-FEDER (RIS); the Fondo de Investigación Sanitaria (FIS) PI12/01247 and PI20/00676; and HIVACAT program and the CERCA Programme/Generalitat de Catalunya SGR 615 and SGR 653. The project leading to these results has received funding from “la Caixa” Foundation under agreement. This study was also supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract Nos. HHSN261200800001E and 75N91019D00024.S

Treatment with integrase inhibitors alters SARS-CoV-2 neutralization levels measured with HIV-based pseudotypes in people living with HIV

The presence of neutralizing antibodies (NAbs) is a major correlate of protection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Thus, different in vitro pseudoviruses-based assays have been described to detect NAbs against SARS-CoV-2. However, the determination of NAbs against SARS-CoV-2 in people living with HIV (PLWH) through HIV-based pseudoparticles could be influenced by cross-neutralization activity or treatment, impeding accurate titration of NAbs. Two assays were compared using replication-defective HIV or VSV-based particles pseudotyped with SARS-CoV-2 spike to measure NAbs in COVID-19-recovered and COVID-19-naïve PLWH. The assay based on HIV-pseudoparticles displayed neutralization activity in all COVID-19-recovered PLWH with a median neutralizing titer 50 (NT50) of 1417.0 (interquartile range [IQR]: 450.3-3284.0), but also in 67% of COVID-19-naïve PLWH (NT50: 631.5, IQR: 16.0-1535.0). Regarding VSV-pseudoparticles system, no neutralization was observed in COVID-19-naïve PLWH as expected, whereas in comparison with HIV-pseudoparticles assay lower neutralization titers were measured in 75% COVID-19-recovered PLWH (NT50: 100.5; IQR: 20.5-1353.0). Treatment with integrase inhibitors was associated with inaccurate increase in neutralization titers when HIV-based pseudoparticles were used. IgG purification and consequent elimination of drugs from samples avoided the interference with retroviral cycle and corrected the lack of specificity observed in HIV-pseudotyped assay. This study shows methodological alternatives based on pseudoviruses systems to determine specific SARS-CoV-2 neutralization titers in PLWH

HIV-1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion.

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or to study HIV reservoirs

InnovÁvila: Propuestas de innovación en la Escuela Universitaria de Educación y Turismo de Ávila para la excelencia docente

Memoria ID-038. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2018-2019

Identification of 1,2,3-triazolium salt-based inhibitors of Leishmania infantum trypanothione disulfide reductase with enhanced antileishmanial potency in cellulo and increased selectivity

N-methylation of the triazole moiety present in our recently described triazole-phenyl-thiazole dimerization disruptors of Leishmania infantum trypanothione disulfide reductase (LiTryR) led to a new class of potent in- hibitors that target different binding sites on this enzyme. Subtle structural changes among representative library members modified their mechanism of action, switching from models of classical competitive inhibition to time- dependent mixed noncompetitive inhibition. X-ray crystallography and molecular modeling results provided a rationale for this distinct behavior. The remarkable potency and selectivity improvements, particularly against intracellular amastigotes, of the LiTryR dimerization disruptors 4c and 4d reveal that they could be exploited as leishmanicidal agents. Of note, L. infantum promastigotes treated with 4c significantly reduced their low- molecular-weight thiol content, thus providing additional evidence that LiTryR is the main target of this novel compound.This work has been financially supported by the Spanish MICINN (Projects PID2019-104070RB-C21, PID2019-104070RB-C22 and PID2020-115331 GB-I00), the Spanish Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028), and the Comunidad de Madrid (PLATESA2-CM ref. S-2018/BAA-4370). The Spanish MEC is also acknowledged for FPU grants to A. R. and to J.C.G. P.A.S.M. thanks to the Division of Physio- logical Chemistry and the Otto-Loewi Research Center of the Medical University of Graz for their support with the scienfic cluster where the calculations contained in this work were run. We thank Ricardo Lau- reano-Rodríguez, Juan Antonio Rodríguez-Gutierrez, and Laura Lagar- tera for technical assistance with SPR experiments.Peer reviewe

SARS-CoV-2 viral load in nasopharyngeal swabs is not an independent predictor of unfavorable outcome

The aim was to assess the ability of nasopharyngeal SARS-CoV-2 viral load at first patient’s hospital evaluation to predict unfavorable outcomes. We conducted a prospective cohort study including 321 adult patients with confirmed COVID-19 through RT-PCR in nasopharyngeal swabs. Quantitative Synthetic SARS-CoV-2 RNA cycle threshold values were used to calculate the viral load in log10 copies/mL. Disease severity at the end of follow up was categorized into mild, moderate, and severe. Primary endpoint was a composite of intensive care unit (ICU) admission and/or death (n = 85, 26.4%). Univariable and multivariable logistic regression analyses were performed. Nasopharyngeal SARS-CoV-2 viral load over the second quartile (≥ 7.35 log10 copies/mL, p = 0.003) and second tertile (≥ 8.27 log10 copies/mL, p = 0.01) were associated to unfavorable outcome in the unadjusted logistic regression analysis. However, in the final multivariable analysis, viral load was not independently associated with an unfavorable outcome. Five predictors were independently associated with increased odds of ICU admission and/or death: age ≥ 70 years, SpO2, neutrophils > 7.5 × 103/µL, lactate dehydrogenase ≥ 300 U/L, and C-reactive protein ≥ 100 mg/L. In summary, nasopharyngeal SARS-CoV-2 viral load on admission is generally high in patients with COVID-19, regardless of illness severity, but it cannot be used as an independent predictor of unfavorable clinical outcome

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin β7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19

Diseño, creación y difusión de Objetos de Aprendizaje para el desarrollo de la competencia informacional

Memoria ID-0277. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Aula virtual de Máster en Profesor de Secundaria

Memoria ID-0322. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Drosophila Araucan and Caupolican Integrate Intrinsic and Signalling Inputs for the Acquisition by Muscle Progenitors of the Lateral Transverse Fate

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. Here we identify, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, we show that Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity