NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins.

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

NLRC4-mediated activation of CD1c+ DC contributes to perpetuation of synovitis in rheumatoid arthritis.

The individual contribution of specific myeloid subsets such as CD1c+ conventional DC (cDC) to perpetuation of rheumatoid arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDC in patients with RA and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional, and functional characteristics of CD1c+ and CD141+ cDC and monocytes from the blood and synovial fluid of patients with RA. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC was associated with the presence of this DC subset in the synovial membrane in patients with RA. Moreover, synovial CD1c+ cDC are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFN-γ+IL-17+CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ receptors and NLRC4 as a potential molecular mechanism mediating pathogenic activation, CD64 upregulation, and functional specialization of CD1c+ cDC in response to dsDNA-IgG in patients with RA.

Relevant SARS-CoV-2 viremia is associated with COVID-19 severity: Prospective cohort study and validation cohort.

Early kinetics of SARS-CoV-2 viral load (VL) in plasma determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR) was evaluated as a predictor of poor clinical outcome in a prospective study and assessed in a retrospective validation cohort. Prospective observational single-center study including consecutive adult patients hospitalized with COVID-19 between November 2020 and January 2021. Serial plasma samples were obtained until discharge. Quantitative RT-PCR was performed to assess SARS-CoV-2 VL. The main outcomes were in-hospital mortality, admission to the Intensive Care Unit (ICU), and their combination (Poor Outcome). Relevant viremia (RV), established in the prospective study, was assessed in a retrospective cohort including hospitalized COVID-19 patients from April 2021 to May 2022, in which plasma samples were collected according to clinical criteria. Prospective cohort: 57 patients were included. RV was defined as at least a twofold increase in VL within ≤2 days or a VL > 300 copies/ml, in the first week. Patients with RV (N = 14; 24.6%) were more likely to die than those without RV (35.7% vs. 0%), needed ICU admission (57% vs. 0%) or had Poor Outcome (71.4% vs. 0%), (p < 0.001 for the three variables). Retrospective cohort: 326 patients were included, 18.7% presented RV. Patients with RV compared with patients without RV had higher rates of ICU-admission (odds ratio [OR]: 5.6 [95% confidence interval [CI]: 2.1-15.1); p = 0.001), mortality (OR: 13.5 [95% CI: 6.3-28.7]; p < 0.0001) and Poor Outcome (OR: 11.2 [95% CI: 5.8-22]; p < 0.0001). Relevant SARS-CoV-2 viremia in the first week of hospitalization was associated with higher in-hospital mortality, ICU admission, and Poor Outcome. Findings observed in the prospective cohort were confirmed in a larger validation cohort.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV.

BACKGROUND: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. METHODS: We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. FINDINGS: HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. INTERPRETATION: Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. FUNDING: NIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Fabrication and Characterization of Bioactive Gelatin-Alginate-Bioactive Glass Composite Coatings on Porous Titanium Substrates.

In this research work, the fabrication of biphasic composite implants has been investigated. Porous, commercially available pure Ti (50 vol % porosity and pore distributions of 100-200, 250-355, and 355-500 µm) has been used as a cortical bone replacement, while different composites based on a polymer blend (gelatin and alginate) and bioactive glass (BG) 45S5 have been applied as a soft layer for cartilage tissues. The microstructure, degradation rates, biofunctionality, and wear behavior of the different composites were analyzed to find the best possible coating. Experiments demonstrated the best micromechanical balance for the substrate containing 200-355 µm size range distribution. In addition, although the coating prepared from alginate presented a lower mass loss, the composite containing 50% alginate and 50% gelatin showed a higher elastic recovery, which entails that this type of coating could replicate the functions of the soft tissue in areas of the joints. Therefore, results revealed that the combinations of porous commercially pure Ti and composites prepared from alginate/gelatin/45S5 BG are candidates for the fabrication of biphasic implants not only for the treatment of osteochondral defects but also potentially for any other diseases affecting simultaneously hard and soft tissues.

Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Effect of the Processing Parameters on the Porosity and Mechanical Behavior of Titanium Samples with Bimodal Microstructure Produced via Hot Pressing.

Commercially pure (c.p.) titanium grade IV with a bimodal microstructure is a promising material for biomedical implants. The influence of the processing parameters on the physical, microstructural, and mechanical properties was investigated. The bimodal microstructure was achieved from the blends of powder particles with different sizes, while the porous structure was obtained using the space-holder technique (50 vol.% of ammonium bicarbonate). Mechanically milled powders (10 and 20 h) were mixed in 50 wt.% or 75 wt.% with c.p. titanium. Four different mixtures of powders were precompacted via uniaxial cold pressing at 400 MPa. Then, the specimens were sintered at 750 °C via hot pressing in an argon gas atmosphere. The presence of a bimodal microstructure, comprised of small-grain regions separated by coarse-grain ones, was confirmed by optical and scanning electron microscopies. The samples with a bimodal microstructure exhibited an increase in the porosity compared with the commercially available pure Ti. In addition, the hardness was increased while the Young's modulus was decreased in the specimens with 75 wt.% of the milled powders (20 h).

Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells.

Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells.

Analysis of the Microstructure and Mechanical Properties of Titanium-Based Composites Reinforced by Secondary Phases and B4C Particles Produced via Direct Hot Pressing.

In the last decade, titanium metal matrix composites (TMCs) have received considerable attention thanks to their interesting properties as a consequence of the clear interface between the matrix and the reinforcing phases formed. In this work, TMCs with 30 vol % of B4C are consolidated by hot pressing. This technique is a powder metallurgy rapid process. Incorporation of the intermetallic to the matrix, 20 vol % (Ti-Al), is also evaluated. Here, the reinforcing phases formed by the reaction between the titanium matrix and the ceramic particles, as well as the intermetallic addition, promote substantial variations to the microstructure and to the properties of the fabricated composites. The influences of the starting materials and the consolidation temperature (900 °C and 1000 °C) are investigated. By X-ray diffraction, scanning and transmission electron microscopy analysis, the in-situ-formed phases in the matrix and the residual ceramic particles were studied. Furthermore, mechanical properties are studied through tensile and bending tests in addition to other properties, such as Young's modulus, hardness, and densification of the composites. The results show the significant effect of temperature on the microstructure and on the mechanical properties from the same starting powder. Moreover, the Ti-Al addition causes variation in the interface between the reinforcement and the matrix, thereby affecting the behaviour of the TMCs produced at the same temperature.

Analysis of the Influence of Starting Materials and Processing Conditions on the Properties of W/Cu Alloys.

In this work, a study of the influence of the starting materials and the processing time used to develop W/Cu alloys is carried out. Regarding powder metallurgy as a promising fabrication route, the difficulties in producing W/Cu alloys motivated us to investigate the influential factors on the final properties of the most industrially demanding alloys: 85-W/15-Cu, 80-W/20-Cu, and 75-W/25-Cu alloys. Two different tungsten powders with large variation among their particle size-fine (Wf) and coarse (Wc) powders-were used for the preparation of W/Cu alloys. Three weight ratios of fine and coarse (Wf:Wc) tungsten particles were analyzed. These powders were labelled as "tungsten bimodal powders". The powder blends were consolidated by rapid sinter pressing (RSP) at 900 °C and 150 MPa, and were thus sintered and compacted simultaneously. The elemental powders and W/Cu alloys were studied by optical microscopy (OM) and scanning electron microscopy (SEM). Thermal conductivity, hardness, and densification were measured. Results showed that the synthesis of W/Cu using bimodal tungsten powders significantly affects the final alloy properties. The higher the tungsten content, the more noticeable the effect of the bimodal powder. The best bimodal W powder was the blend with 10 wt % of fine tungsten particles (10-Wf:90-Wc). These specimens present good values of densification and hardness, and higher values of thermal conductivity than other bimodal mixtures.

Dasatinib Reversibly Disrupts Endothelial Vascular Integrity by Increasing Non-Muscle Myosin II Contractility in a ROCK-Dependent Manner.

Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell-cell contacts, altered cell-matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions. Clin Cancer Res; 23(21); 6697-707. ©2017 AACR.

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise.

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Influence of Sintering Temperature on the Microstructure and Mechanical Properties of In Situ Reinforced Titanium Composites by Inductive Hot Pressing.

This research is focused on the influence of processing temperature on titanium matrix composites reinforced through Ti, Al, and B4C reactions. In order to investigate the effect of Ti-Al based intermetallic compounds on the properties of the composites, aluminum powder was incorporated into the starting materials. In this way, in situ TixAly were expected to form as well as TiB and TiC. The specimens were fabricated by the powder metallurgy technique known as inductive hot pressing (iHP), using a temperature range between 900 °C and 1400 °C, at 40 MPa for 5 min. Raising the inductive hot pressing temperature may affect the microstructure and properties of the composites. Consequently, the variations of the reinforcing phases were investigated. X-ray diffraction, microstructural analysis, and mechanical properties (Young's modulus and hardness) of the specimens were carried out to evaluate and determine the significant influence of the processing temperature on the behavior of the composites.

A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity.

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front-back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935-1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B-like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.

Behavioral and molecular changes elicited by acute administration of SR141716 to Delta9-tetrahydrocannabinol-tolerant rats: an experimental model of cannabinoid abstinence.

Whether chronic cannabinoid consumption produces a dependent state comparable to that occurring with other drugs (e.g. the appearance of withdrawal signs when consumption is interrupted), and whether chronic cannabinoid consumption increases the risk of consuming other drugs of greater addictive power, are probably the two questions relating to cannabinoid addiction that provoke the most controversy. The present study was designed to further explore these two questions in laboratory animals. Firstly, we examined the effects of an acute challenge with SR141716 (an antagonist for the cannabinoid CB(1) receptor) in Delta(9)-tetrahydrocannabinol (Delta(9)-THC)-tolerant rats. This antagonist has been reported to precipitate a cannabinoid withdrawal syndrome. Thus, the administration of SR141716 to Delta(9)-THC-tolerant rats reduced inactivity in the open-field test and enhanced responses as tremor, turning and retropulsion-these responses that were only slightly enhanced in control rats. The administration of SR141716 increased the plasma prolactin and the corticosterone concentration in controls, but these increases were much lesser in Delta(9)-THC-tolerant rats. In addition, CRF-mRNA levels in the paraventricular hypothalamic nucleus, while reduced in SR141716-treated controls, were significantly increased in Delta(9)-THC-tolerant rats. The analysis of endocannabinoids also revealed that the administration of SR141716, which was mostly inactive in control rats, was able to reverse the changes in anandamide or 2-arachidonoylglycerol concentrations found in Delta(9)-THC-tolerant rats, in the striatum, limbic forebrain, diencephalon, cerebellum and brainstem, but not in the midbrain and hippocampus. As a second objective, we evaluated whether Delta(9)-THC-tolerant rats were more vulnerable to morphine in a self-administration paradigm. The Delta(9)-THC-tolerant and control rats self-administered morphine to a similar extent, in concordance with the similar values of dopaminergic activity in limbic and motor regions. In summary, our data indicate that Delta(9)-THC-tolerant rats were not more vulnerable to the reinforcing properties of morphine. However, they responded to the blockade of CB(1) receptors by exhibiting slightly but possibly relevant differences in behavioral, endocrine and molecular parameters compared to the response in non-tolerant rats. This is indicative of the existence of a withdrawal syndrome in cannabinoid-tolerant rats that is mild compared with abstinence in opioid-dependent rats.