Alterations in the immune system persist after one year of convalescence in severe COVID-19 patients.

Introduction: Severe COVID-19 originates a myriad of alterations in the immune system during active disease, especially in the T and NK cell compartments, but several studies in the last year have unveiled some alterations that persist in convalescence. Although most of the studies follow the participants for a short recovery time, studies following patients up to three or six months still find alterations. We aimed at evaluating changes in the NK, T and B cell compartments after severe COVID-19 in participants with a median recovery time of eleven months. Methods: Eighteen convalescent of severe COVID-19 (CSC), 14 convalescent of mild COVID-19 (CMC) and nine controls were recruited. NKG2A, NKG2C, NKG2D and the activating receptor NKp44 were evaluated in NKbright, NKdim and NKT subpopulations. In addition, CD3 and CD19 were measured and a basic biochemistry with IL-6 levels was obtained. Results: CSC participants showed lower NKbright/NKdim ratio, higher NKp44 expression in NKbright subpopulations, higher levels of serum IL-6, lower levels of NKG2A+ T lymphocytes and a trend to a lower expression of CD19 in B lymphocytes compared to controls. CMC participants showed no significant alterations in the immune system compared to controls. Conclusions: These results are concordant with previous studies, which find alterations in CSC weeks or months after resolution of the symptoms, and point to the possibility of these alterations lasting one year or more after COVID-19 resolution.

Antibacterial effect of 3D printed mesoporous bioactive glass scaffolds doped with metallic silver nanoparticles.

The development of new biomaterials for bone tissue regeneration with high bioactivity abilities and antibacterial properties is being intensively investigated. We have synthesized nanocomposites formed by mesoporous bioactive glasses (MBGs) in the ternary SiO2, CaO and P2O5 system doped with metallic silver nanoparticles (AgNPs) that were homogenously embedded in the MBG matrices. Ag/MBG nanocomposites have been directly synthesized and silver species were spontaneously reduced to metallic AgNPs by high temperatures (700 °C) obtained of last MBG synthesis step. Three-dimensional silver-containing mesoporous bioactive glass scaffolds were fabricated showing uniformly interconnected ultrapores, macropores and mesopores. The manufacture method consisted of a combination of a single-step sol-gel route in the mesostructure directing agent (P123) presence and a biomacromolecular polymer such as (hydroxypropyl)methyl cellulose (HPMC) as the macrostructure template, followed by rapid prototyping (RP) technique. Biological properties of Ag/MBG nanocomposites were evaluated by MC3T3-E1 preosteoblastic cells culture tests and bacterial (E. coli and S. aureus) assays. The results showed that the MC3T3-E1 cells morphology was not affected while preosteoblastic proliferation decreased when the presence of silver increased. Antimicrobial assays indicated that bacterial growth inhibition and biofilm destruction were directly proportional to the increased presence of AgNPs in the MBG matrices. Furthermore, in vitro co-culture of MC3T3-E1 cells and S. aureus bacteria confirmed that AgNPs presence was necessary for antibacterial activity, and AgNPs slightly affected cell proliferation parameters. Therefore, 3D printed scaffolds with hierarchical pore structure and high antimicrobial capacity have potential applications in bone tissue regeneration. STATEMENT OF SIGNIFICANCE: This study combines three key scientific aspects for bone tissue engineering: (i) materials with high bioactivity to repair and regenerate bone tissue that (ii) contain antibacterial agents to reduce the infection risk (iii) in the form of three-dimensional scaffolds with hierarchical porosity. Innovative methodology is described here: sol-gel method, which is employed to obtain mesoporous bioactive glass matrices doped with metallic silver nanoparticles where different polymer templates facilitate the different size scales presence, and rapid prototyping technique that provides ultra-large macroporosity according to computer-aided design. The dual scaffolds obtained are biocompatible and deliver active doses of silver capable of combating bone infections, which represent one of the most serious complications associated to surgical treatments of bone diseases and fractures.

Polymorphisms in ARNTL/BMAL1 and CLOCK Are Not Associated with Multiple Sclerosis in Spanish Population.

Disrupted circadian cycle has been reported in multiple sclerosis (MS). Previous genome-wide association studies (GWAS) singled out over 230 variants associated with MS. A study performed in a Slavic population identified two new single nucleotide polymorphisms (SNPs), rs6811520 (CLOCK) and rs3789327 (ARNTL/BMAL1), associated with MS risk. However, these regions that codify the capital regulators of circadian rhythm had not been linked to the disease before, so replication in independent populations is warranted to ascertain possible geographical differences. Our aim was to replicate the associations reported in the ARNTL/BMAL1 and CLOCK genes in a Spanish cohort with a maximum of 974 MS patients and 626 controls. In this study, 956 MS patients and 612 controls were successfully genotyped for rs6811520 and 943 MS patients and 598 controls for rs3789327.Clinical variables (age at disease onset, EDSS, or relapses) were collected in a maximum of 549 patients. No statistically significant differences were found between cases and controls for the analyzed SNPs, even after stratifications by sex, clinical form, or HLA-DRB1*15:01 status. No influence of the SNPs was found on age at disease onset, EDSS, or annual relapse rate at 5 years after onset. In conclusion, our study does not replicate the associations observed in the previously investigated Slavic population.

Unraveling the Influence of HHEX Risk Polymorphism rs7923837 on Multiple Sclerosis Pathogenesis.

One of the multiple sclerosis (MS) risk polymorphisms, rs7923837, maps near the HHEX (hematopoietically-expressed homeobox) gene. This variant has also been associated with type 2 diabetes susceptibility and with triglyceride levels, suggesting its metabolic involvement. HHEX plays a relevant role as a negative regulator of inflammatory genes in microglia. A reciprocal repression was reported between HHEX and BCL6, another putative risk factor in MS. The present study evidenced statistically significant lower HHEX mRNA levels in lymphocytes of MS patients compared to those of controls, showing a similar trend in MS patients to the already described eQTL effect in blood from healthy individuals. Even though no differences were found in protein expression according to HHEX genotypes, statistically significant divergent subcellular distributions of HHEX appeared in patients and controls. The epistatic interaction detected between BCL6 and HHEX MS-risk variants in healthy individuals was absent in patients, indicative of a perturbed reciprocal regulation in the latter. Lymphocytes from MS carriers of the homozygous mutant genotype exhibited a distinctive, more energetic profile, both in resting and activated conditions, and significantly increased glycolytic rates in resting conditions when compared to controls sharing the HHEX genotype. In contrast, significantly higher mitochondrial mass was evidenced in homozygous mutant controls.

Impact of Multiple Sclerosis Risk Polymorphism rs7665090 on MANBA Activity, Lysosomal Endocytosis, and Lymphocyte Activation.

Deficiencies in Mannosidase ß (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3'UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant ß-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS.

Genetic variation in NDFIP1 modifies the metabolic patterns in immune cells of multiple sclerosis patients.

One of the 233 polymorphisms associated with multiple sclerosis (MS) susceptibility lies within the NDFIP1 gene, and it was previously identified as eQTL in healthy controls. NDFIP1 shows interesting immune functions and is involved in the development of the central nervous system. We aimed at studying the NDFIP1 variant on activation and metabolism of immune cells. NDFIP1 mRNA and protein expression were assessed in PBMCs by qPCR and western blot in 87 MS patients and 84 healthy controls genotyped for rs4912622. Immune activation after PHA stimulation was evaluated by CD69 upregulation, and metabolic function of both basal and PHA-activated lymphocytes was studied by Seahorse Xfp-Analyzer. In minor-allele homozygous controls but not in patients, we found higher NDFIP1 expression, significantly reduced protein levels, and CD69 upregulation in B- and T-cells. PBMCs from minor-allele homozygous controls showed significantly higher basal mitochondrial respiration and ATP production compared to major-allele carriers, while minor-allele homozygous patients showed significantly lower metabolic activity than carriers of the major allele. In conclusion, we describe associations in minor-allele homozygous controls with lower levels of NDFIP1 protein, CD69 upregulation, and raised mitochondrial activity, which are not replicated in MS patients, suggesting a NDFIP1 differential effect in health and disease.