Quantitative Analysis of Phagocytosis in Whole Blood Using Double Staining and Visualization.

Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the ability to phagocytize is associated with many diseases and aging of the immune system. Assessment of phagocytosis dynamics requires quantification of bacteria inside and outside the phagocyte. Although flow cytometry is the most common method for assessing phagocytosis, it does not include visualization and direct quantification of location of bacteria. Here, we used double-labeled Escherichia coli cells to evaluate phagocytosis by flow cytometry (cell sorting) and confocal microscopy, as well as employed image cytometry to provide high-throughput quantitative and spatial recognition of the double-labeled E. coli associated with the phagocytes. Retention of pathogens on the surface of myeloid and lymphoid cells without their internalization was suggested to be an auxiliary function of innate immunity in the fight against infections. The developed method of bacterial labeling significantly increased the accuracy of spatial and quantitative measurement of phagocytosis in whole blood and can be recommended as a tool for phagocytosis assessment by image cytometry.

Deep Learning for Cell Migration in Nonwoven Materials and Evaluating Gene Transfer Effects following AAV6-ND4 Transduction.

Studying cell settlement in the three-dimensional structure of synthetic biomaterials over time is of great interest in research and clinical translation for the development of artificial tissues and organs. Tracking cells as physical objects improves our understanding of the processes of migration, homing, and cell division during colonisation of the artificial environment. In this study, the 3D environment had a direct effect on the behaviour of biological objects. Recently, deep learning-based algorithms have shown significant benefits for cell segmentation tasks and, furthermore, for biomaterial design optimisation. We analysed the primary LHON fibroblasts in an artificial 3D environment after adeno-associated virus transduction. Application of these tools to model cell homing in biomaterials and to monitor cell morphology, migration and proliferation indirectly demonstrated restoration of the normal cell phenotype after gene manipulation by AAV transduction. Following the 3Rs principles of reducing the use of living organisms in research, modeling the formation of tissues and organs by reconstructing the behaviour of different cell types on artificial materials facilitates drug testing, the study of inherited and inflammatory diseases, and wound healing. These studies on the composition and algorithms for creating biomaterials to model the formation of cell layers were inspired by the principles of biomimicry.

T-cell immunity against senescence: potential role and perspectives.

The development of age-associated diseases is related to the accumulation of senescent cells in the body. These are old non-functional cells with impaired metabolism, which are unable to divide. Such cells are also resistant to programmed cell death and prone to spontaneous production of some inflammatory factors. The accumulation of senescent cells is related to the age-associated dysfunction of organs and tissues as well as chronic inflammation that enhances with age. In the young organism, senescent cells are removed with the innate immunity system. However, the efficiency of this process decreases with age. Nowadays, more and more evidences are accumulating to support the involvement of specific immunity and T-lymphocytes in the fight against senescent cells. It has great physiological importance since the efficient elimination of senescent cells requires a high diversity of antigen-recognizing receptors to cover the entire spectrum of senescent-associated antigens with high precision and specificity. Developing the approaches of T-cell immunity stimulation to generate or amplify a physiological immune response against senescent cells can provide new perspectives to extend active longevity. In this mini-review, the authors summarize the current understanding of the role of T-cell immunity in the fight against senescent cells and discuss the prospects of stimulating adaptive immunity for combating the accumulation of senescent cells that occurs with age.

Reliable Hallmarks and Biomarkers of Senescent Lymphocytes.

The phenomenon of accumulation of senescent adaptive immunity cells in the elderly is attracting attention due to the increasing risk of global epidemics and aging of the global population. Elderly people are predisposed to various infectious and age-related diseases and are at higher risk of vaccination failure. The accumulation of senescent cells increases age-related background inflammation, "Inflammaging", causing lymphocyte exhaustion and cardiovascular, neurodegenerative, autoimmune and cancer diseases. Here, we present a comprehensive contemporary review of the mechanisms and phenotype of senescence in the adaptive immune system. Although modern research has not yet identified specific markers of aging lymphocytes, several sets of markers facilitate the separation of the aging population based on normal memory and exhausted cells for further genetic and functional analysis. The reasons for the higher predisposition of CD8+ T-lymphocytes to senescence compared to the CD4+ population are also discussed. We point out approaches for senescent-lymphocyte-targeting markers using small molecules (senolytics), antibodies and immunization against senescent cells. The suppression of immune senescence is the most relevant area of research aimed at developing anti-aging and anti-cancer therapy for prolonging the lifespan of the global population.

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Molecular Markers of Blood Cell Populations Can Help Estimate Aging of the Immune System.

Aging of the immune system involves functional changes in individual cell populations, in hematopoietic tissues and at the systemic level. They are mediated by factors produced by circulating cells, niche cells, and at the systemic level. Age-related alterations in the microenvironment of the bone marrow and thymus cause a decrease in the production of naive immune cells and functional immunodeficiencies. Another result of aging and reduced tissue immune surveillance is the accumulation of senescent cells. Some viral infections deplete adaptive immune cells, increasing the risk of autoimmune and immunodeficiency conditions, leading to a general degradation in the specificity and effectiveness of the immune system in old age. During the COVID-19 pandemic, the state-of-the-art application of mass spectrometry, multichannel flow cytometry, and single-cell genetic analysis have provided vast data on the mechanisms of aging of the immune system. These data require systematic analysis and functional verification. In addition, the prediction of age-related complications is a priority task of modern medicine in the context of the increase in the aged population and the risk of premature death during epidemics. In this review, based on the latest data, we discuss the mechanisms of immune aging and highlight some cellular markers as indicators of age-related immune disbalance that increase the risk of senile diseases and infectious complications.

Hematopoietic Stem Cells and the Immune System in Development and Aging.

Hematopoietic stem cells (HSCs) support haematopoiesis throughout life and give rise to the whole variety of cells of the immune system. Developing in the early embryo, passing through the precursor stage, and maturing into the first HSCs, they undergo a fairly large number of divisions while maintaining a high regenerative potential due to high repair activity. This potential is greatly reduced in adult HSCs. They go into a state of dormancy and anaerobic metabolism to maintain their stemness throughout life. However, with age, changes occur in the pool of HSCs that negatively affect haematopoiesis and the effectiveness of immunity. Niche aging and accumulation of mutations with age reduces the ability of HSCs to self-renew and changes their differentiation potential. This is accompanied by a decrease in clonal diversity and a disturbance of lymphopoiesis (decrease in the formation of naive T- and B-cells) and the predominance of myeloid haematopoiesis. Aging also affects mature cells, regardless of HSC, therefore, phagocytic activity and the intensity of the oxidative burst decrease, and the efficiency of processing and presentation of antigens by myeloid cells is impaired. Aging cells of innate and adaptive immunity produce factors that form a chronic inflammatory background. All these processes have a serious negative impact on the protective properties of the immune system, increasing inflammation, the risk of developing autoimmune, oncological, and cardiovascular diseases with age. Understanding the mechanisms of reducing the regenerative potential in a comparative analysis of embryonic and aging HSCs, the features of inflammatory aging will allow us to get closer to deciphering the programs for the development, aging, regeneration and rejuvenation of HSCs and the immune system.

Age-Related Changes in the Clustering of Blood Populations in Cynomolgus Monkeys Depend on Sex and Immune Status.

Non-anthropoid primates cynomolgus monkeys (Macaca fascicularis), also known as crab-eating macaques, are increasingly used in biomedical and preclinical studies due to their evolutionary proximity to humans, sharing similar diets, infectious and senile diseases. Age-related changes and sexual dimorphism of the immune system of C. monkeys have not been sufficiently characterized in literature, though age and sex differences affect the course of diseases and sensitivity to medications. Aging in C. monkeys is accompanied by an increase in CD3+CD4+CD8+ (DP-T) cells, plasma B-cells, and a decrease in platelets. Erythromyeloid bias has also been noticed in older animals. There was an increase in eosinophils, haematocrit (HCT) and haemoglobin concentration (HGB). Senile decline in the function of the immune system had sex differences. An increase in the number of monocytes, cytotoxic lymphocytes (CTL) and a decrease in the T-helper population were more pronounced in older females. A significant reduction in the number of B-cells and activated T-cells was detected in males only. A moderate correlation with the regression model of aging was established for DP-T, HCT and HGB. The reduction in the B cells count in males and the increase in CTL level in females are moderately correlated with age. Other blood cell populations did not show significant correlations in the regression models due to their high sample variability. The novel cell population CD3-CD20loCD16/CD56+, presumably NK-cells subset, was revealed. This cell population demonstrated an increase trend with age in both sexes. Population-statistical age norms for different sexes for young and very old macaques were established. The blood population clusters associated with sex and immune status in older animals were also identified.

Biodiversity: the overlooked source of human health.

Biodiversity is the measure of the variation of lifeforms in a given ecological system. Biodiversity provides ecosystems with the robustness, stability, and resilience that sustains them. This is ultimately essential for our survival because we depend on the services that natural ecosystems provide (food, fresh water, air, climate, and medicine). Despite this, human activity is driving an unprecedented rate of biodiversity decline, which may jeopardize the life-support systems of the planet if no urgent action is taken. In this article we show why biodiversity is essential for human health. We raise our case and focus on the biomedicine services that are enabled by biodiversity, and we present known and novel approaches to promote biodiversity conservation.

Use of Personal Resources May Influence the Rate of Biological Aging Depending on Individual Typology.

Individual hobbies and interests, the ways of spending leisure time develop personal resources influencing health and wellbeing. The literature analysis helped selecting thirteen personal resources that also affect the rate of aging: sports, order, creativity, intellect, handwork, kindness, Humor, spirituality, risk, nature, achievements, optimism, communication. In 1632 people, (840 women and 792 men) personal resources were assessed using a questionnaire developed in-house. Biological age was determined by health indicators. The personal typology was determined by testing functional asymmetry, physique, interaction style, emotionality, profession, marital status, gender, age, and place of residence. The data were processed by correlation and cluster analysis and methods of automatic artificial neural networks (ANN). Personal resources were used as input continuous variables. Personality types were used as input categorical variables. The index of relative biological aging (RBA) was applied as an output continuous variable. We also calculated the correlation between the RBA index and the applied personal resources in different types of personalities. For most female types including investigative occupations, psychomotor emotionality, living in urban areas, asthenic physique, negative correlations were found between most personal resources and the aging index. In men, resources that slow down aging are found only for certain types: enterprising and conventional professions, ambidexter and left-handed, intellectual emotionality, athletic physique. In conclusion, with the help of the trained ANN, we selected personal resources that slow down aging. For women of all types, there are common resources reducing RBA index including nature, intellect, and achievements. For men, ANN was unable to find common resources that slow down aging. However, with an individual selection of resources, a trained neural network gives a favorable forecast of the ability to slow down the biological aging of a particular man by changing his hobbies and interests and ways of spending free time.

Religiosity, Spirituality and Biopsychological Age of Professionals in Russia.

The challenges of modern civilization resulted in the premature biological and psychological aging of professionals of working age. This phenomenon raises both medical and psychological problems associated with personality factors that affect psychobiological maturity and the rate of aging. The influence of religiosity and spirituality on biopsychological age remains the least studied area of psychology. Progress in this area will help to identify the components of religiosity-predictors of the aging rate of professionals. The sample included 295 people (148 women) aged 24 to 54 years (average age 31.7 years) and consisted of Christians (67.12%), Muslims (5.76%), Buddhists, deists, Shintoists, etc., (7.79%) and atheists (17.29%). The average work experience was 9 years. Using correlation analysis and methods of multivariate linear regression and t-test for independent samples, we found that the religiosity of professionals increases with natural aging and deterioration of their physical condition and does not depend on gender. Religiosity to a greater extent affects psychological age, the indicator of the psychobiological maturity of a professional and, to a lesser extent, biological age. Most of the indicators of religiosity are inherent in a person who is more mature in psychobiological terms. The biological age of professionals increases due to asthenic experiences, while gaining faith in God, unusual religious experiences and the existential meaning of life can reduce it. An increase in the spirituality of professionals is associated with a slowdown in the rate of biological aging.

Acceleration of Biological Aging and Underestimation of Subjective Age Are Risk Factors for Severe COVID-19.

In an epidemic, it is important to have methods for reliable and rapid assessment of risk groups for severe forms of the disease for their priority vaccination and for the application of preventive lockdown measures. The aim of this study was to investigate risk factors for severe forms of COVID-19 in adults using indicators of biological and subjective aging. Longitudinal studies evaluated the severity of the disease and the number of cases. Respondents (447) were divided into "working group" and "risk group" (retirees with chronic diseases). During the lockdown period (in mid-2020), accelerated aging was observed in the group of workers (by 3.9-8 years for men and an increase at the tendency level for women). However, the respondents began to feel subjectively younger (by 3.3-7.2 years). In the risk group, there were no deviations from the expected biopsychological aging. The number of cases at the end of 2020 was 31% in workers and 0% in the risk group. Reasonably, the risk group followed the quarantine rules more strictly by 1.5 times. In working men, indicators of relative biological and relative subjective aging (measured in both 2019 and mid-2020) significantly influenced the incidence at the end of 2020. In women, only the indicators obtained in mid-2020 had a significant impact. The relative biological aging of an individual tested in the middle of 2020 had a direct impact on the risk of infection (p < 0.05) and on the probability of death (p < 0.0001). On the contrary, an increase in the relative subjective (psychological) aging index reduced the risk of infection (at the tendency level, p = 0.06) and the risk of death (p < 0.0001). Both the risk of infection and the risk of death increased with calendar age at the tendency level. Conclusions: Indicators of individual relative biological and subjective aging affect the probability of getting COVID-19 and its severity. The combination of high indicators of biological aging and underestimated indicators of subjective aging is associated with increased chances of developing severe forms of the disease.

Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System.

Apelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance.

Can Blood-Circulating Factors Unveil and Delay Your Biological Aging?

According to the World Health Organization, the population of over 60 will double in the next 30 years in the developed countries, which will enforce a further raise of the retirement age and increase the burden on the healthcare system. Therefore, there is an acute issue of maintaining health and prolonging active working longevity, as well as implementation of early monitoring and prevention of premature aging and age-related disorders to avoid early disability. Traditional indicators of biological age are not always informative and often require extensive and expensive analysis. The study of blood factors is a simple and easily accessible way to assess individual health and supplement the traditional indicators of a person's biological age with new objective criteria. With age, the processes of growth and development, tissue regeneration and repair decline; they are gradually replaced by enhanced catabolism, inflammatory cell activity, and insulin resistance. The number of senescent cells supporting the inflammatory loop rises; cellular clearance by autophagy and mitophagy slows down, resulting in mitochondrial and cellular damage and dysfunction. Monitoring of circulated blood factors not only reflects these processes, but also allows suggesting medical intervention to prevent or decelerate the development of age-related diseases. We review the age-related blood factors discussed in recent publications, as well as approaches to slowing aging for healthy and active longevity.

Multi-layered Spatial Transcriptomics Identify Secretory Factors Promoting Human Hematopoietic Stem Cell Development.

Hematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro.

Vast Self-Renewal Potential of Human AGM Region HSCs Dramatically Declines in the Umbilical Cord Blood.

Human hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region during Carnegie stages (CS) 14-17. Although we previously reported that these HSCs can generate no less than 300 daughter HSCs, their actual number has never been established. Here, we show that a single human AGM region HSC can generate 600-1,600 functional daughter HSCs. The presence of HSCs in the CS 17 liver in one case gave us a unique opportunity to describe a reduction of HSC self-renewal potential after liver colonization. From a clinical perspective, the efficacy of long-term hematopoietic regeneration depends on HSC self-renewal capacity. We quantitatively show that this capacity dramatically declines in the umbilical cord blood compared with HSCs in the AGM region. A full appreciation of the vast regenerative potential of the first human embryo-derived HSCs sets a new bar for generation of clinically useful HSCs from pluripotent stem cells.

Comparative Dynamics of Individual Ageing among the Investigative Type of Professionals Living in Russia and Russian Migrants to the EU Countries.

The goal of this study was to uncover the influence of professional activity, migration, and gender on dynamics of subjective age and ageing biomarkers. We examined the representatives of investigative types of professions (ITP), 30-75 years old in Russia, (101/62 women), and Russian migrants to the European Union, (101/56 women). ITPs appeared to be ageing slower than statistical standards; men age faster than women; the pre-retirement group (51-65 years old) showed acceleration of relative biological ageing in the Russian sample (women +4.5 years, men +10.7 years) against the EU sample, suggesting a boost of pre-retirement stress in Russia; subjectively, Russian people (51-65 years old) feel close to their chronological age, while EU people perceive themselves far below their calendar age (men-lower by 20.4, women-lower by 10.9 years). The subjective ageing depends on the country of residence, while biological ageing depends on occupation, gender, and negative expectations of retirement.

Analysis of the Spatiotemporal Development of Hematopoietic Stem and Progenitor Cells in the Early Human Embryo.

Definitive hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region in both mice and humans. An ex vivo culture approach has enabled recapitulation and analysis of murine HSC development. Knowledge of early human HSC development is hampered by scarcity of tissue: analysis of both CFU-C and HSC development in the human embryo is limited. Here, we characterized the spatial distribution and temporal kinetics of CFU-C development within early human embryonic tissues. We then sought to adapt the murine ex vivo culture system to recapitulate human HSC development. We show robust expansion of CFU-Cs and maintenance, but no significant expansion, of human HSCs in culture. Furthermore, we demonstrate that HSCs emerge predominantly in the middle section of the dorsal aorta in our culture system. We conclude that there are important differences between early mouse and human hematopoiesis, which currently hinder the quest to recapitulate human HSC development ex vivo.

Molecular Mechanisms Governing the Stem Cell's Fate in Brain Cancer: Factors of Stemness and Quiescence.

Cellular quiescence is a reversible, non-cycling state controlled by epigenetic, transcriptional and niche-associated molecular factors. Quiescence is a condition where molecular signaling pathways maintain the poised cell-cycle state whilst enabling rapid cell cycle re-entry. To achieve therapeutic breakthroughs in oncology it is crucial to decipher these molecular mechanisms employed by the cancerous milieu to control, maintain and gear stem cells towards re-activation. Cancer stem-like cells (CSCs) have been extensively studied in most malignancies, including glioma. Here, the aberrant niche activities skew the quiescence/activation equilibrium, leading to rapid tumor relapse after surgery and/or chemotherapy. Unraveling quiescence mechanisms promises to afford prevention of (often multiple) relapses, a key problem in current glioma treatment. This review article covers the current knowledge regarding normal and aberrant cellular quiescence control whilst also exploring how different molecular mechanisms and properties of the neighboring cells can influence the molecular processes behind glioma stem cell quiescence.

Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach.

Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.