Rebeca Alonso-Arias’s research while affiliated with University of Oviedo and other places

Background Memory responses to the antigens that an individual encounters throughout life may vary with the intensity and duration of antigen contacts or even with changes in immune status over time. This work aims to characterise specific responses to latent CMV, seasonal influenza and novel SARS-CoV-2 infections in immunocompetent individuals over 60 years of age. Specific cellular and humoral responses were identified by IFN-γ and granzyme B release by ELISpot and antibody level measurement. T lymphocyte subpopulation phenotypes were characterised by flow cytometry. Results Cellular and humoral responses to these viruses were detected in almost all patients. Influenza and SARS-CoV-2 cellular responses were positively correlated. There was no significant correlation between CMV and influenza or SARS-CoV-2 responses although both were consistently lower in CMV-seropositive patients. CMV responses were negatively correlated with the levels of the least differentiated subsets of T lymphocytes, and positively correlated with the most differentiated ones, contrary to what happened with the influenza responses. Nevertheless, SARS-CoV-2 cellular responses were negatively correlated with the most differentiated CD8⁺ T lymphocytes, while humoral responses were negatively correlated with the least differentiated T lymphocytes. Responses to the three viruses were correlated with a Th1/Th2/Th17 balance in favour of Th1. Conclusions The results indicate that memory responses differ depending on the durability of the antigen stimulus. Cellular responses to novel pathogens resemble those generated by seasonal but not CMV infection. Subpopulation distribution and the level of specific T lymphocytes against previous pathogens could be used as immunocompetent status biomarkers in older adults reflecting their ability to generate memory responses to new pathogens.

Background Memory responses to the antigens that an individual encounters throughout life may vary with the intensity and duration of antigen contacts or even changes in immune status over time. This work aims to characterise specific responses to chronic CMV, seasonal influenza and novel SARS-CoV-2 infections in immunocompetent individuals over 60 years of age. Specific cellular and humoral responses were identified by IFN-γ and granzyme-B released by ELISpot and antibody level measurement. T lymphocyte subpopulation phenotypes were characterized by flow cytometry. Results Cellular and humoral responses to these viruses were detected in almost all patients. Influenza and SARS-CoV-2 cellular responses were positively correlated. There was no significant correlation of CMV with influenza or SARS-CoV-2 responses although both were consistently lower in CMV-seropositive patients. CMV responses were negatively correlated with the levels of the least differentiated subsets of T lymphocytes, and positively correlated with the most differentiated ones, contrary to what happened with the influenza responses. Nevertheless, SARS-CoV-2 cellular responses were negatively correlated with the most differentiated CD8⁺ T lymphocytes, while humoral responses were negatively correlated with the least differentiated T lymphocytes. Responses to the three viruses were correlated with a Th1/Th2/Th17 balance in favour of Th1. Conclusions Results indicate that memory responses differ depending on the durability of the antigen stimulus. Cellular responses to novel pathogens resemble those generated by seasonal but not chronic antigens. Subpopulation distribution and the level of specific T lymphocytes against previous pathogens could be used as immunocompetent status biomarkers in older adults reflecting their ability to generate memory responses to new pathogens.

Expansion of CD4 ⁺ CD28 null T‐lymphocytes is common in chronic heart failure (CHF) patients. Its ability to produce high levels of proinflammatory cytokines is probably the key role of these cells in CHF. IL‐10 is a candidate for limiting CD4 ⁺ CD28 null T‐lymphocyte responses, whereas tumour necrosis factor (TNF) is the cytokine most closely involved in the loss of CD28 expression. Serum levels of TNF and IL‐10 were measured in 65 CHF patients (mean age, 65.2 ± 13.84 years). Patients with an IL‐10/TNF ratio ≥1 had significantly lower levels of CD4 ⁺ CD28 null T‐lymphocytes than those with a ratio <1. In vitro, IL‐10 reduced the frequency of proliferative CD4 ⁺ CD28 null T‐lymphocytes stimulated with anti‐CD3. Pre‐treatment with IL‐10 before anti‐CD3 stimulation was required for the cytokine to inhibit TNF production by CD4 ⁺ CD28 null T‐lymphocytes. In addition to the previously described effect of IL‐10 on HLA‐DR and ICAM‐1 expression, LFA‐3 protein and mRNA levels were reduced in the presence of the cytokine in monocytes. IL‐10 inhibition on CD4 ⁺ CD28 null T‐lymphocytes may be mediated by a reduction in HLA class II and LFA‐3 expression because blocking interactions with these costimulators has similar effects to those of IL‐10 treatment. Moreover, costimulation through CD2/LFA‐3 interaction is enough to induce proliferation and cytokine production in CD4 ⁺ CD28 null T‐lymphocytes.

Background: We previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in ~ 80% of cases. Methods: We report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded. Results: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P = 1.1 × 10-4) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR = 3.70[95%CI 1.3-8.2], P = 2.1 × 10-4). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR = 19.65[95%CI 2.1-2635.4], P = 3.4 × 10-3), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR = 4.40[9%CI 2.3-8.4], P = 7.7 × 10-8). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD] = 43.3 [20.3] years) than the other patients (56.0 [17.3] years; P = 1.68 × 10-5). Conclusions: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.

Background: Diabetic men are less likely to suffer prostate cancer, and insulin signalling through insulin receptors has been long considered. However, the role of insulin-dependent glucose transporters has yet to be elucidated. The unique metabolic properties of prostate cancer are attributed to the central role of androgens. Androgen-sensitive tumour cells have higher mitochondrial activity, while castration-resistant cells exhibit aerobic glycolysis. In addition, to glycolysis, one of the hallmarks of cancer metabolism is increased glucose uptake. However, the prostate's oncogenic value of glucose transporters (GLUTs) needs to be better characterized. This research aims to discover the relevance of insulin-dependent glucose transporters to cancer progression and their importance in the protective role of diabetes in prostate cancer. Methods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used in vitro. Castration-resistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were established from LNCaP cell line. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed. Results: We found that androgens stimulate insulin-independent glucose transporters, while androgen independence is associated with GLUT4 overexpression. The ectopic overexpression of GLUT4 promotes the characteristics of a castration-resistant phenotype. Metabolomics confirmed that hormone-resistant prostate cancer cells show an oxidative metabolism with a clear enrichment in amino acid metabolism. Diabetic TRAMP mice showed total tumour regression, while insulin administration restored proliferation and recovered GLUT4 levels. The levels of GLUT4 increase along with tumour progression in TRAMP mice, and it is reduced by castration and streptozotocin-induced diabetes. Finally, the levels of GLUT4 accumulation in tumour tissues compared to normal epithelial in patients' samples showed a clear co-location with nuclear AR. Conclusion: Here it is confirmed the relevance of insulin-mediated glucose uptake through GLUT4 with prostate cancer progression and its relation to the reduced occurrence of prostate cancer in diabetic men.

Faecalibacterium represents one of the most abundant bacterial groups in the human intestinal microbiota of healthy adults and can represent more than 10% of the total bacterial population, Faecalibacterium prausnitzii being the only recognized species up to the past year. Reduction in the abundance of F. prausnitzii in the human gut has been linked to several human disorders, such as Crohn’s disease. In this study, we developed a strategy to modify the relative abundance of F. prausnitzii in fecal microbiotas as a means of evaluating its contribution to the immunomodulatory effect of intestinal microbiotas with different F. prausnitzii contents using a peripheral blood mononuclear cell (PBMC) model. We used a polyclonal antibody against the surface of F. prausnitzii M21 to capture the bacterium from synthetic and human fecal microbiotas using immunoseparation techniques. As a proof-of-principle study, the levels of immunomodulation exerted by microbiotas of healthy donors (HDs) with different relative abundances of F. prausnitzii, achieved with the above-mentioned immunoseparation technique, were evaluated in a PBMC model. For this purpose, PBMCs were cocultivated with the modified microbiotas or a pure culture of F. prausnitzii and, subsequently, the microbiota of Crohn’s donors was added to the coculture. The cytokine concentration was determined, showing that our experimental model supports the anti-inflammatory effects of this bacterium. IMPORTANCE There is increasing interest in deciphering the contribution of gut microbiota species to health and disease amelioration. The approach proposed herein provides a novel and affordable strategy to probe deeply into microbiota-host interactions by strategically modifying the relative abundance of specific gut microbes, hence facilitating the study of their contribution to a given trait of the microbiota.

Background We previously reported inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity in 1-5% of unvaccinated patients with life-threatening COVID-19, and auto-antibodies against type I IFN in another 15-20% of cases. Methods We report here a genome-wide rare variant burden association analysis in 3,269 unvaccinated patients with life-threatening COVID-19 (1,301 previously reported and 1,968 new patients), and 1,373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. A quarter of the patients tested had antibodies against type I IFN (234 of 928) and were excluded from the analysis. Results No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7 , with an OR of 27.68 (95%CI:1.5-528.7, P= 1.1×10 ⁻⁴ ), in analyses restricted to biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70 [95%CI:1.3-8.2], P= 2.1×10 ⁻⁴ ). Adding the recently reported TYK2 COVID-19 locus strengthened this enrichment, particularly under a recessive model (OR=19.65 [95%CI:2.1-2635.4]; P= 3.4×10 ⁻³ ). When these 14 loci and TLR7 were considered, all individuals hemizygous ( n =20) or homozygous ( n =5) for pLOF or bLOF variants were patients (OR=39.19 [95%CI:5.2-5037.0], P =4.7×10 ⁻⁷ ), who also showed an enrichment in heterozygous variants (OR=2.36 [95%CI:1.0-5.9], P =0.02). Finally, the patients with pLOF or bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P= 1.68×10 ⁻⁵ ). Conclusions Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.