Centenarian hippocampus displays high levels of astrocytic metallothioneins.

The hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life, lower cognitive impairment, and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [≥97 years], old, and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular, several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover, we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall, these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging.

Blood RNA-Seq profiling reveals a set of circular RNAs differentially expressed in frail individuals.

BACKGROUND: Frailty is an intermediate and reversible geriatric syndrome that often precedes dependence. Therefore, its identification is essential to prevent dependence. Several molecules have been proposed as biomarkers of frailty, but none of them have reached clinical practice. Recently, circular RNAs have emerged as new non-coding RNAs. Their regulatory role together with their high stability in biofluids makes them good candidates as biomarkers for various processes, but, to date, no study has characterized the expression of circRNA in frailty. RESULTS: We studied RNA from leukocytes of 35 frails and 35 robust individuals. After RNA-Sequencing, circRNA detection was performed by CIRI2 and Circexplorer2 and differential expression analysis by DESeq2. Validation was performed by Quantitative-PCR. Linear Discriminant Analysis was performed to determine the best circRNA combination to discriminate frail from robust. In addition, CircRNA candidates were studied in 13 additional elder donors before and after a 3-month physical intervention. We found 89 differentially expressed circRNAs (p-value<0.05, FC>|1.5|) with frailty. Upregulation of hsa_circ_0007817, hsa_circ_0101802 and hsa_circ_0060527 in frail individuals was validated. The combination of hsa_circ_0079284, hsa_circ_0007817 and hsa_circ_0075737 levels showed a great biomarker value with a 95.9% probability of correctly classifying frail and robust individuals. Moreover, hsa_circ_0079284 levels decreased after physical intervention in concordance with an improvement in frailty scores. CONCLUSIONS: This work describes for the first time a different expression pattern of circular RNA (circRNAs) between frail and robust individuals. Moreover, the level of some circRNAs is modulated after a physical intervention. These results suggest that they could be used as minimally invasive biomarkers of frailty.

Early biochemical analysis of COVID-19 patients helps severity prediction.

COVID-19 pandemic has put the protocols and the capacity of our Hospitals to the test. The management of severe patients admitted to the Intensive Care Units has been a challenge for all health systems. To assist in this challenge, various models have been proposed to predict mortality and severity, however, there is no clear consensus for their use. In this work, we took advantage of data obtained from routine blood tests performed on all individuals on the first day of hospitalization. These data has been obtained by standardized cost-effective technique available in all the hospitals. We have analyzed the results of 1082 patients with COVID19 and using artificial intelligence we have generated a predictive model based on data from the first days of admission that predicts the risk of developing severe disease with an AUC = 0.78 and an F1-score = 0.69. Our results show the importance of immature granulocytes and their ratio with Lymphocytes in the disease and present an algorithm based on 5 parameters to identify a severe course. This work highlights the importance of studying routine analytical variables in the early stages of hospital admission and the benefits of applying AI to identify patients who may develop severe disease.

Immune cell population and cytokine profiling suggest age dependent differences in the response to SARS-CoV-2 infection.

Aging population is at higher risk of developing severe COVID-19, including hospitalization and death. In this work, to further understand the relationship between host age-related factors, immunosenescence/exhaustion of the immune system and the response to the virus, we characterized immune cell and cytokine responses in 58 COVID-19 patients admitted to the hospital and 40 healthy controls of different age ranges. Lymphocyte populations and inflammatory profiles were studied in blood samples, using different panels of multicolor flow cytometry. As expected, our analysis reveals differences at both the cellular and cytokine level in COVID-19 patients. Interestingly, when the age range analysis was carried out, the immunological response to the infection was found to differ with age, being especially affected in the group of 30-39 years. In this age range, an increased exhausted T cell response and a decrease of naïve T helper lymphocytes was found in patients, as well as a reduced concentration of the proinflammatory TNF, IL-1ß and IL-8 cytokines. Besides, the correlation between age and the study variables was evaluated, and multiple cell types and interleukins were found to correlate with donor age. Notably, the correlations of T helper naïve and effector memory cells, T helper 1-17 cells, TNF, IL-10, IL-1ß, IL-8, among others, showed differences between healthy controls and COVID-19 patients. Our findings, in the context of other previous studies, suggest that aging affects the behavior of the immune system in COVID-19 patients. They suggest that young individuals are able to mount an initial response to SARS-CoV-2, but some of them present an accelerated exhaustion of the cell response and an insufficient inflammatory response, resulting in a moderate to severe COVID-19. On the other hand, in older patients there is a smaller immune cell response to the virus, reflected in fewer differences in immune populations between COVID-19 patients and controls. Nevertheless, old patients show more evidence of an inflammatory phenotype, suggesting that the underlying inflammation associated with their age is exacerbated by the SARS-CoV-2 infection.

Women in the field of multiple sclerosis: How they contributed to paradigm shifts.

History is full of women who made enormous contributions to science. While there is little to no imbalance at the early career stage, a decreasing proportion of women is found as seniority increases. In the multiple sclerosis (MS) field, 44% of first authors and only 35% of senior authors were female. So, in this review, we highlight ground-breaking research done by women in the field of MS, focusing mostly on their work as principal investigators. MS is an autoimmune disorder of the central nervous system (CNS), with evident paradigm shifts in the understating of its pathophysiology. It is known that the immune system becomes overactivated and attacks myelin sheath surrounding axons. The resulting demyelination disrupts the communication signals to and from the CNS, which causes unpredictable symptoms, depending on the neurons that are affected. Classically, MS was reported to cause mostly physical and motor disabilities. However, it is now recognized that cognitive impairment affects more than 50% of the MS patients. Another shifting paradigm was the involvement of gray matter in MS pathology, formerly considered to be a white matter disease. Additionally, the identification of different T cell immune subsets and the mechanisms underlying the involvement of B cells and peripheral macrophages provided a better understanding of the immunopathophysiological processes present in MS. Relevantly, the gut-brain axis, recognized as a bi-directional communication system between the CNS and the gut, was found to be crucial in MS. Indeed, gut microbiota influences not only different susceptibilities to MS pathology, but it can also be modulated in order to positively act in MS course. Also, after the identification of the first microRNA in 1993, the role of microRNAs has been investigated in MS, either as potential biomarkers or therapeutic agents. Finally, concerning MS therapeutical approaches, remyelination-based studies have arisen on the spotlight aiming to repair myelin loss/neuronal connectivity. Altogether, here we emphasize the new insights of remarkable women that have voiced the impact of cognitive impairment, white and gray matter pathology, immune response, and that of the CNS-peripheral interplay on MS diagnosis, progression, and/or therapy efficacy, leading to huge breakthroughs in the MS field.

Microbial dysbiosis and lack of SCFA production in a Spanish cohort of patients with multiple sclerosis.

Background: Multiple sclerosis (MS) is a chronic, demyelinating, and immune-mediated disease of the central nervous system caused by a combination of genetic, epigenetic, and environmental factors. The incidence of MS has increased in the past several decades, suggesting changes in the environmental risk factors. Much effort has been made in the description of the gut microbiota in MS; however, little is known about the dysbiosis on its function. The microbiota produces thousands of biologically active substances among which are notable the short-chain fatty acid (SCFA) excretion. Objectives: Analyze the interaction between microbiota, SCFAs, diet, and MS. Methods: 16S, nutritional questionnaires, and SCFAS quantification have been recovered from MS patients and controls. Results: Our results revealed an increment in the phylum Proteobacteria, especially the family Enterobacteriaceae, a lack in total SCFA excretion, and an altered profile of SCFAs in a Spanish cohort of MS patients. These alterations are more evident in patients with higher disability. Conclusions: The abundance of Proteobacteria and acetate and the low excretion of total SCFAs, especially butyrate, are common characteristics of MS patients, and besides, both are associated with a worse prognosis of the disease.

The innovative animal monitoring device for experimental autoimmune encephalomyelitis ("I AM D EAE"): A more detailed evaluation for improved results.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is the most widely used animal model for multiple sclerosis (MS). It is a rapid model, commonly induced in rodents. Even if EAE does not replicate all MS characteristics, it is appropriate to investigate the development of the disease, including the immune and neuroinflammatory aspects. Besides, EAE has also been shown to be a relevant model for pre-clinical studies, as several drugs effective in the model are beneficial for MS patients. However, despite its widespread use, there is no consensus on the clinical assessment of animals. Most researchers perform a daily evaluation and classify them on a 5-point scale, but many authors also use in-between scores or apply other systems. Besides, among the 5-point scale, different score definitions are used, and most of them do not recapitulate the signs or symptoms each animal can show. Thus, based on our experience with EAE, the aim of the present work was to develop a new scoring system. METHODS: We designed the "I AM D EAE" tool that independently evaluates 9 different items - an innovative and detailed scoring system, yet simple for non-experts to use. The new scale was tested in EAE-induced mice at three experiments, and different evaluators assessed the animals blindly. RESULTS: The "I AM D EAE" scoring system highly correlates to the commonly used 5-point scale and, importantly, it enables a more detailed evaluation. CONCLUSIONS: Considering its high reproducibility and inter-rater reliability, "I AM D EAE" is a useful tool for EAE monitoring.

Profiling of Plasma Extracellular Vesicle Transcriptome Reveals That circRNAs Are Prevalent and Differ between Multiple Sclerosis Patients and Healthy Controls.

(1) Background: Extracellular vesicles (EVs) are released by most cell types and are implicated in several biological and pathological processes, including multiple sclerosis (MS). Differences in the number and cargo of plasma-derived EVs have been described in MS. In this work, we have characterised the EV RNA cargo of MS patients, with particular attention to circular RNAs (circRNAs), which have attracted increasing attention for their roles in physiology and disease and their biomarker potential. (2) Methods: Plasma-derived EVs were isolated by differential centrifugation (20 patients, 8 controls), and RNA-Sequencing was used to identify differentially expressed linear and circRNAs. (3) Results: We found differences in the RNA type distribution, circRNAs being enriched in EVs vs. leucocytes. We found a number of (corrected p-value < 0.05) circRNA significantly DE between the groups. Nevertheless, highly structured circRNAs are preferentially retained in leukocytes. Differential expression analysis reports significant differences in circRNA and linear RNA expression between MS patients and controls, as well as between different MS types. (4) Conclusions: Plasma derived EV RNA cargo is not a representation of leukocytes' cytoplasm but a message worth studying. Moreover, our results reveal the interest of circRNAs as part of this message, highlighting the importance of further understanding RNA regulation in MS.

Extracellular Vesicles in Blood: Sources, Effects, and Applications.

Extracellular vesicles (EVs) are important players for intercellular communication. EVs are secreted by almost all cell types; they can transfer information between nearby or distant cells, and they are highly abundant in body fluids. In this review, we describe the general characteristics of EVs, as well as isolation and characterization approaches. Then, we focus on one of the most relevant sources of EVs: the blood. Indeed, apart from EVs secreted by blood cells, EVs of diverse origins travel in the bloodstream. We present the numerous types of EVs that have been found in circulation. Besides, the implications of blood-derived EVs in both physiological and pathological processes are summarized, highlighting their potential as biomarkers for the diagnosis, treatment monitoring, and prognosis of several diseases, and also as indicators of physiological modifications. Finally, the applications of EVs introduced in the circulatory system are discussed. We describe the use of EVs from distinct origins, naturally produced or engineered, autologous, allogeneic, or even from different species and the effects they have when introduced in circulation. Therefore, the present work provides a comprehensive overview of the components, effects, and applications of EVs in blood.

O group is a protective factor for COVID19 in Basque population.

ABO blood groups have recently been related to COVID19 infection. In the present work, we performed this analysis using data from 412 COVID19 patients and 17796 blood donors, all of them from Gipuzkoa, a region in Northern Spain. The results obtained confirmed this relation, in addition to showing a clear importance of group O as a protective factor in COVID19 disease, with an OR = 0.59 (CI95% 0.481-0.7177, p<0.0001) while A, B and AB are risk factors. ABO blood groups are slightly differently distributed in the populations and therefore these results should be replicated in the specific areas with a proper control population.

Inflammaging markers characteristic of advanced age show similar levels with frailty and dependency.

The improvement of life quality and medical advances has resulted in increased life expectancy. Despite this, health status commonly worsens in the last years of life. Frailty is an intermediate and reversible state that often precedes dependency and therefore, its identification may be essential to prevent dependency. However, there is no consensus on the best tools to identify frailty. In this sense, diverse molecules have been proposed as potential biomarkers. Some investigations pointed to an increased chronic inflammation or inflammaging with frailty, while others did not report such differences. In this work, we evaluated the circulating concentration of the inflammaging markers in adults and older adults (aged over 70 years) by ELISA and Luminex techniques. The Barthel Index was applied for the evaluation of dependency and Timed up-and-go, Gait Speed, Short Physical Performance Battery, Tilburg Frailty Indicator and Gerontopole Frailty Screening Tool were used for the identification of frailty. CRP, TNF-α, IL-6 and albumin concentrations were measured, and we found that elevated inflammation is present in older adults, while no differences with frailty and dependency were reported. Our results were consistent for all the evaluated frailty scales, highlighting the need to reconsider increased inflammation as a biomarker of frailty.

Gut Microbiota Changes in Experimental Autoimmune Encephalomyelitis and Cuprizone Mice Models.

Multiple sclerosis (MS) is a chronic and neurodegenerative disease of the central nervous system (CNS) characterized by the immune mediated attack on axons and the subsequent demyelination. There is growing evidence that the gut microbiota of MS patients is altered; however, the connection between demyelination events and changes in the gut microbiota has not been determined. The objective of the current work was to characterize the microbial dysbiosis in two murine demyelinating models and to study the correlation between them. Concurrently, their suitability as predictors of microbial changes in MS patients was assessed. To this purpose, experimental autoimmune encephalomyelitis (EAE) and cuprizone (CPZ) models were induced in C57BL/6 mice that were monitored for 4 and 9 weeks, respectively. Fecal samples were collected during disease progression. Motor skill performance was evaluated by EAE scale measurement in EAE mice and demyelination by magnetic resonance imaging (MRI) in CPZ ones. EAE and CPZ mice revealed drastic microbial changes according to disease progression, adding a new layer of complexity to the understanding of demyelination and remyelination processes. Besides, the reported microbial changes replicate most of the characteristics that define the potential dysbiosis in MS patients. The controlled environment and stable diet that animals have in research centers offer an exceptional scenario to modify animal's microbiota and provide opportunities to study host microbiota interplay with restrained conditions not achievable in human studies. Nevertheless the slight differences from murine model's and patient's microbiota should be considered in the design of studies aiming to modulate the microbiota.

RNA-Seq profiling of leukocytes reveals a sex-dependent global circular RNA upregulation in multiple sclerosis and 6 candidate biomarkers.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, with higher prevalence in women, that leads to neurological disability. The disease course and clinical phenotype are highly variable, and therefore, biomarkers for the diagnosis, classification, monitoring of the disease and treatment assessment are needed. Studies have shown a dysregulation in the coding and non-coding RNAs and proposed some as biomarkers. However, still none of them have reached the clinical practice. Recently, circular RNAs (circRNAs) have emerged as new players in the transcriptome that hold a great potential as biomarkers in several diseases. Leukocytes from 30 MS patients and 20 healthy controls (HCs) were RNA-sequenced to study the linear and circular transcriptome. Differential expression analysis was performed by DESeq, and circRNA candidates were studied in a second cohort (70 MS and 46 HC) by RT-qPCR and in paired samples drawn during the relapse and remission phases (20 patients). Among the differentially expressed circRNAs, 96.1% are upregulated in patients compared with controls, but similar circRNA profiles are found between MS types. The same upregulation trend was observed in females but not in males or in the linear transcriptome. The upregulation of 6 circRNAs was validated, and a change in their expression was found between relapse and remission. The 6 circRNAs showed a good performance to discriminate patients from HC with a combined area under the curve of 0.852. There is global, specific and sex-dependent increase of circRNA expression in MS, and 6 circRNAs are proposed as potential biomarkers.

Relevance of oxidative stress and inflammation in frailty based on human studies and mouse models.

Frailty represents a state of vulnerability and increases the risk of negative health outcomes, which is becoming an important public health problem. Over recent years, multiple independent studies have attempted to identify biomarkers that can predict, diagnose, and monitor frailty at the biological level. Among them, several promising candidates have been associated with frailty status including antioxidants and free radicals, and also inflammatory response biomarkers. In this review, we will summarize the more recent advances in this field. Moreover, the identification of scales and measurements to detect and quantify frailty in aged mice, as well as the generation of mouse models, have started to unravel the underlying biological and molecular mechanisms of frailty. We will discuss them here with an emphasis on murine models with overexpression of glucose-6-phosphate dehydrogenase and loss of function of superoxide dismutase and interleukin 10, which reveal that altered oxidative stress and inflammation pathways are involved in the physiopathology of frailty. In summary, we provide the current available evidence, from both human cohorts and experimental animal models, that highlights oxidative damage and inflammation as relevant biomarkers and drivers of frailty.

T cells and immune functions of plasma extracellular vesicles are differentially modulated from adults to centenarians.

Aging is a universal and complex process that affects all tissues and cells types, including immune cells, in a process known as immunosenescence. However, many aspects of immunosenescence are not completely understood, as the characteristics of the immune cells of nonagenarians and centenarians or the features and implications of extracellular vesicles (EVs). In this study, we analyzed blood samples from 51 individuals aged 20-49 and 70-104 years. We found that senescent CD8 cells accumulate with age, while there is a partial reduction of senescent CD4 cells in nonagenarians and centenarians. Moreover, plasma EVs carry T cell specific markers, but no accumulation of "senescent-like EVs" was found within any of analyzed age groups. Our functional studies of cocultures of peripheral blood mononuclear cells and EVs showed that EVs enhance T cell viability and, under phytohemagglutinin stimulation, they influence cytokine secretion and cell activation in an age-dependent manner. These results underline the importance of EVs on the immune system functioning, and open new perspectives to further study their implication in human aging.

Blood Markers in Healthy-Aged Nonagenarians: A Combination of High Telomere Length and Low Amyloidß Are Strongly Associated With Healthy Aging in the Oldest Old.

Many factors may converge in healthy aging in the oldest old, but their association and predictive power on healthy or functionally impaired aging has yet to be demonstrated. By detecting healthy aging and in turn, poor aging, we could take action to prevent chronic diseases associated with age. We conducted a pilot study comparing results of a set of markers (peripheral blood mononuclear cell or PBMC telomere length, circulating Aß peptides, anti-Aß antibodies, and ApoE status) previously associated with poor aging or cognitive deterioration, and their combinations, in a cohort of "neurologically healthy" (both motor and cognitive) nonagenarians (n = 20) and functionally impaired, institutionalized nonagenarians (n = 38) recruited between 2014 and 2015. We recruited 58 nonagenarians (41 women, 70.7%; mean age: 92.37 years in the neurologically healthy group vs. 94.13 years in the functionally impaired group). Healthy nonagenarians had significantly higher mean PBMC telomere lengths (mean = 7, p = 0.001), this being inversely correlated with functional impairment, and lower circulating Aß40 (total in plasma fraction or TP and free in plasma fraction or FP), Aß42 (TP and FP) and Aß17 (FP) levels (FP40 131.35, p = 0.004; TP40 299.10, p = 0.007; FP42 6.29, p = 0.009; TP42 22.53, p = 0.019; FP17 1.32 p = 0.001; TP17 4.47, p = 0.3), after adjusting by age. Although healthy nonagenarians had higher anti-Aß40 antibody levels (net adsorbed signal or NAS ± SD: 0.211 ± 0.107), the number of participants that pass the threshold (NAS > 3) to be considered as positive did not show such a strong association. There was no association with ApoE status. Additionally, we propose a "Composite Neurologically Healthy Aging Score" combining TP40 and mean PBMC telomere length, the strongest correlation of measured biomarkers with neurologically healthy status in nonagenarians (AUC = 0.904).

Therapeutic Potential of Extracellular Vesicles for Demyelinating Diseases; Challenges and Opportunities.

Multiple Sclerosis is a demyelinating disease of the central nervous system for which no remyelination therapy is available and alternative strategies are being tested. Extracellular vesicles (EVs) have emerged as players in physiological and pathological processes and are being proposed as therapeutic targets and mediators. More concretely, EVs have shown to be involved in myelination related processes such as axon-oligodendrocyte communication or oligodendrocyte precursor cell migration. In addition, EVs have been shown to carry genetic material and small compounds, and to be able to cross the Blood Brain Barrier. This scenario led scientists to test the ability of EVs as myelin regeneration promoters in demyelinating diseases. In this review we will address the use of EVs as remyelination promoters and the challenges and opportunities of this therapy will be discussed.

The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients.

Extracellular vesicles (EVs) are membrane-bound particles involved in intercellular communication. They carry proteins, lipids, and nucleotides such as microRNAs (miRNAs) from the secreting cell that can modulate target cells. We and others have previously described the presence of EVs in peripheral blood of multiple sclerosis (MS) patients and postulated them as novel biomarkers. However, their immune function in MS pathogenesis and the effect during the onset of new immunomodulatory therapies on EVs remain elusive. Here, we isolated plasma EVs from fingolimod-treated MS patients in order to assess whether EVs are affected by the first dose of the treatment. We quantified EVs, analyzed their miRNA cargo, and checked their immune regulatory function. Results showed an elevated EV concentration with a dramatic change in their miRNA cargo 5 h after the first dose of fingolimod. Besides, EVs obtained prior to fingolimod treatment showed an increased immune regulatory activity compared to EVs obtained 5 h post-treatment. This work suggests that EVs are implicated in the mechanism of action of immunomodulatory treatments from the initial hours and opens a new avenue to explore a potential use of EVs for early treatment monitoring.

Progressive changes in non-coding RNA profile in leucocytes with age.

It has been observed that immune cell deterioration occurs in the elderly, as well as a chronic low-grade inflammation called inflammaging. These cellular changes must be driven by numerous changes in gene expression and in fact, both protein-coding and non-coding RNA expression alterations have been observed in peripheral blood mononuclear cells from elder people. In the present work we have studied the expression of small non-coding RNA (microRNA and small nucleolar RNA -snoRNA-) from healthy individuals from 24 to 79 years old. We have observed that the expression of 69 non-coding RNAs (56 microRNAs and 13 snoRNAs) changes progressively with chronological age. According to our results, the age range from 47 to 54 is critical given that it is the period when the expression trend (increasing or decreasing) of age-related small non-coding RNAs is more pronounced. Furthermore, age-related miRNAs regulate genes that are involved in immune, cell cycle and cancer-related processes, which had already been associated to human aging. Therefore, human aging could be studied as a result of progressive molecular changes, and different age ranges should be analysed to cover the whole aging process.

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation.

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.