The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

Abstract Background: Natural killer (NK) cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC) restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives: The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB)-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods: In our study, CB-Mononuclear Cells’ (MNCs) CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep) CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs) in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand). The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results: Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05). CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively). Conclusions: Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy. Keywords: Umbilical Cord Blood, Natural Killer Cell, Mesenchymal Stem Cel

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic

Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Understanding the health consequences associated with exposure to risk factors is necessary to inform public health policy and practice. To systematically quantify the contributions of risk factor exposures to specific health outcomes, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 aims to provide comprehensive estimates of exposure levels, relative health risks, and attributable burden of disease for 88 risk factors in 204 countries and territories and 811 subnational locations, from 1990 to 2021. Methods: The GBD 2021 risk factor analysis used data from 54 561 total distinct sources to produce epidemiological estimates for 88 risk factors and their associated health outcomes for a total of 631 risk–outcome pairs. Pairs were included on the basis of data-driven determination of a risk–outcome association. Age-sex-location-year-specific estimates were generated at global, regional, and national levels. Our approach followed the comparative risk assessment framework predicated on a causal web of hierarchically organised, potentially combinative, modifiable risks. Relative risks (RRs) of a given outcome occurring as a function of risk factor exposure were estimated separately for each risk–outcome pair, and summary exposure values (SEVs), representing risk-weighted exposure prevalence, and theoretical minimum risk exposure levels (TMRELs) were estimated for each risk factor. These estimates were used to calculate the population attributable fraction (PAF; ie, the proportional change in health risk that would occur if exposure to a risk factor were reduced to the TMREL). The product of PAFs and disease burden associated with a given outcome, measured in disability-adjusted life-years (DALYs), yielded measures of attributable burden (ie, the proportion of total disease burden attributable to a particular risk factor or combination of risk factors). Adjustments for mediation were applied to account for relationships involving risk factors that act indirectly on outcomes via intermediate risks. Attributable burden estimates were stratified by Socio-demographic Index (SDI) quintile and presented as counts, age-standardised rates, and rankings. To complement estimates of RR and attributable burden, newly developed burden of proof risk function (BPRF) methods were applied to yield supplementary, conservative interpretations of risk–outcome associations based on the consistency of underlying evidence, accounting for unexplained heterogeneity between input data from different studies. Estimates reported represent the mean value across 500 draws from the estimate's distribution, with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values across the draws. Findings: Among the specific risk factors analysed for this study, particulate matter air pollution was the leading contributor to the global disease burden in 2021, contributing 8·0% (95% UI 6·7–9·4) of total DALYs, followed by high systolic blood pressure (SBP; 7·8% [6·4–9·2]), smoking (5·7% [4·7–6·8]), low birthweight and short gestation (5·6% [4·8–6·3]), and high fasting plasma glucose (FPG; 5·4% [4·8–6·0]). For younger demographics (ie, those aged 0–4 years and 5–14 years), risks such as low birthweight and short gestation and unsafe water, sanitation, and handwashing (WaSH) were among the leading risk factors, while for older age groups, metabolic risks such as high SBP, high body-mass index (BMI), high FPG, and high LDL cholesterol had a greater impact. From 2000 to 2021, there was an observable shift in global health challenges, marked by a decline in the number of all-age DALYs broadly attributable to behavioural risks (decrease of 20·7% [13·9–27·7]) and environmental and occupational risks (decrease of 22·0% [15·5–28·8]), coupled with a 49·4% (42·3–56·9) increase in DALYs attributable to metabolic risks, all reflecting ageing populations and changing lifestyles on a global scale. Age-standardised global DALY rates attributable to high BMI and high FPG rose considerably (15·7% [9·9–21·7] for high BMI and 7·9% [3·3–12·9] for high FPG) over this period, with exposure to these risks increasing annually at rates of 1·8% (1·6–1·9) for high BMI and 1·3% (1·1–1·5) for high FPG. By contrast, the global risk-attributable burden and exposure to many other risk factors declined, notably for risks such as child growth failure and unsafe water source, with age-standardised attributable DALYs decreasing by 71·5% (64·4–78·8) for child growth failure and 66·3% (60·2–72·0) for unsafe water source. We separated risk factors into three groups according to trajectory over time: those with a decreasing attributable burden, due largely to declining risk exposure (eg, diet high in trans-fat and household air pollution) but also to proportionally smaller child and youth populations (eg, child and maternal malnutrition); those for which the burden increased moderately in spite of declining risk exposure, due largely to population ageing (eg, smoking); and those for which the burden increased considerably due to both increasing risk exposure and population ageing (eg, ambient particulate matter air pollution, high BMI, high FPG, and high SBP). Interpretation: Substantial progress has been made in reducing the global disease burden attributable to a range of risk factors, particularly those related to maternal and child health, WaSH, and household air pollution. Maintaining efforts to minimise the impact of these risk factors, especially in low SDI locations, is necessary to sustain progress. Successes in moderating the smoking-related burden by reducing risk exposure highlight the need to advance policies that reduce exposure to other leading risk factors such as ambient particulate matter air pollution and high SBP. Troubling increases in high FPG, high BMI, and other risk factors related to obesity and metabolic syndrome indicate an urgent need to identify and implement interventions

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Investigation of signaling cross-talk between platelets and neutrophils

Summary Platelet activation is known to be associated with the release of a vast array of chemokines and proinflammatory lipids which induce pleiotropic effects on a wide variety of tissues and cells, including neutrophils. Protein ectodomain shedding and microparticle formation are also important processes occurring during platelet activation, although their physiological functions have not been fully defined. During thrombosis, the recruitment of neutrophils to activated platelets is considered an important step linking thrombosis to inflammatory responses. This phenomenon is highly regulated and influenced by the cross-talk between platelets and neutrophils. This cross-talk involves a variety of mediators including adhesion molecules, chemokines, shed proteins and various proinflammatory lipids. The initial studies presented in this thesis have examined the impact of platelet shed proteins on neutrophil adhesion and activation. These studies demonstrated that the most extensive surface protein shedding was observed in the procoagulant phenotype of platelets (PS positive) so-called SCIP (Sustained Calcium-Induced Platelet morphology) suggesting that ectodomain shedding is influenced by different platelet activation states. I have found that these shed proteins can negatively regulate the adhesive function of platelets, by reducing integrin alphaIIb beta3 engagement of immobilized fibrinogen, leading to decreased platelet adhesion and spreading. However, this platelet inhibitory activity was spread out over at least 4 distinct chromatographic peaks, making purification of the biological active molecules challenging. Notably, shed material from the surface of platelets appeared to have limited capacity to regulate neutrophil adhesion or activation, with most of the neutrophil activating activity residing within the granule component of activated platelets. Studies presented in chapter 4 have focused on the identification and characterization of the major platelet released product(s) regulating neutrophil adhesive function. Initial studies excluded a major contribution for released small molecules (<5kDa) in inducing neutrophil activation. Through the use of different chromatographic procedures I was able to identify the alpha-granule protein NAP-2 (Neutrophil Activating Protein-2) as the principal component released by platelets regulating neutrophil adhesive function. This was somewhat unexpected, given previous findings for an important role for soluble P-selectin and shed CD40L in regulating neutrophil activation. Subsequent studies in chapter 5 investigated the distribution of neutrophil activating activity in various platelet fractions and revealed that the majority of activity resided on/within intact platelets (~70%), with ~20% found in the microparticle (MP)-free releasate and ~10% in MP. In addition to the unexpected activity distribution, the activation status of platelets also had a major influence on their ability to modulate neutrophil reactivity. Platelet degranulation and P-selectin expression was critical for inducing heterotypic platelet-neutrophil adhesion and for an increase in Mac-1 expression/activation, a process enhanced by platelet conversion to SCIPs. Analysis of the key proinflammatory mediators promoting Mac-1 activation revealed an important co-operative role for PAF, leukotrienes and NAP-2 in promoting neutrophil activation. Taken together, these studies suggest an important role for transcellular lipid metabolism between platelets and neutrophils in enhancing subsequent neutrophil activation. The studies presented in chapter 6 reveal an important feedback activation mechanism operating between platelet and neutrophils, that serves to enhance the procoagulant function of platelets. These studies reveal that the aggregation of neutrophils with TRAP-stimulated platelets not only enhances Mac-1 activation, but also feeds back on platelets to enhance cytosolic calcium flux. This feedback function of neutrophils was dependent on Mac-1 ligand engagement, P-selectin and PAF-generation and was associated with the conversion of platelets from a PS negative to a PS positive phenotype, leading to enhanced platelet procoagulant function. The exact molecular mechanism by which neutrophils induce platelet procoagulant formation remains to be fully delineated, however given the importance of these cells in promoting innate immune and inflammatory functions, such mechanisms may have pathophysiological importance. In summary, the results from my PhD thesis have provided new insights into the complex regulation of neutrophil activation by various components of activated platelets. The finding that neutrophils can provide feedback regulation of platelets to promote procoagulant function may provide new insight into the role of transcellular activation mechanisms in promoting inflammatory and thrombotic responses in a variety of human diseases

Investigation of signaling cross-talk between platelets and neutrophils

Summary Platelet activation is known to be associated with the release of a vast array of chemokines and proinflammatory lipids which induce pleiotropic effects on a wide variety of tissues and cells, including neutrophils. Protein ectodomain shedding and microparticle formation are also important processes occurring during platelet activation, although their physiological functions have not been fully defined. During thrombosis, the recruitment of neutrophils to activated platelets is considered an important step linking thrombosis to inflammatory responses. This phenomenon is highly regulated and influenced by the cross-talk between platelets and neutrophils. This cross-talk involves a variety of mediators including adhesion molecules, chemokines, shed proteins and various proinflammatory lipids. The initial studies presented in this thesis have examined the impact of platelet shed proteins on neutrophil adhesion and activation. These studies demonstrated that the most extensive surface protein shedding was observed in the procoagulant phenotype of platelets (PS positive) so-called SCIP (Sustained Calcium-Induced Platelet morphology) suggesting that ectodomain shedding is influenced by different platelet activation states. I have found that these shed proteins can negatively regulate the adhesive function of platelets, by reducing integrin alphaIIb beta3 engagement of immobilized fibrinogen, leading to decreased platelet adhesion and spreading. However, this platelet inhibitory activity was spread out over at least 4 distinct chromatographic peaks, making purification of the biological active molecules challenging. Notably, shed material from the surface of platelets appeared to have limited capacity to regulate neutrophil adhesion or activation, with most of the neutrophil activating activity residing within the granule component of activated platelets. Studies presented in chapter 4 have focused on the identification and characterization of the major platelet released product(s) regulating neutrophil adhesive function. Initial studies excluded a major contribution for released small molecules (<5kDa) in inducing neutrophil activation. Through the use of different chromatographic procedures I was able to identify the alpha-granule protein NAP-2 (Neutrophil Activating Protein-2) as the principal component released by platelets regulating neutrophil adhesive function. This was somewhat unexpected, given previous findings for an important role for soluble P-selectin and shed CD40L in regulating neutrophil activation. Subsequent studies in chapter 5 investigated the distribution of neutrophil activating activity in various platelet fractions and revealed that the majority of activity resided on/within intact platelets (~70%), with ~20% found in the microparticle (MP)-free releasate and ~10% in MP. In addition to the unexpected activity distribution, the activation status of platelets also had a major influence on their ability to modulate neutrophil reactivity. Platelet degranulation and P-selectin expression was critical for inducing heterotypic platelet-neutrophil adhesion and for an increase in Mac-1 expression/activation, a process enhanced by platelet conversion to SCIPs. Analysis of the key proinflammatory mediators promoting Mac-1 activation revealed an important co-operative role for PAF, leukotrienes and NAP-2 in promoting neutrophil activation. Taken together, these studies suggest an important role for transcellular lipid metabolism between platelets and neutrophils in enhancing subsequent neutrophil activation. The studies presented in chapter 6 reveal an important feedback activation mechanism operating between platelet and neutrophils, that serves to enhance the procoagulant function of platelets. These studies reveal that the aggregation of neutrophils with TRAP-stimulated platelets not only enhances Mac-1 activation, but also feeds back on platelets to enhance cytosolic calcium flux. This feedback function of neutrophils was dependent on Mac-1 ligand engagement, P-selectin and PAF-generation and was associated with the conversion of platelets from a PS negative to a PS positive phenotype, leading to enhanced platelet procoagulant function. The exact molecular mechanism by which neutrophils induce platelet procoagulant formation remains to be fully delineated, however given the importance of these cells in promoting innate immune and inflammatory functions, such mechanisms may have pathophysiological importance. In summary, the results from my PhD thesis have provided new insights into the complex regulation of neutrophil activation by various components of activated platelets. The finding that neutrophils can provide feedback regulation of platelets to promote procoagulant function may provide new insight into the role of transcellular activation mechanisms in promoting inflammatory and thrombotic responses in a variety of human diseases

The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

Abstract Background: Natural killer (NK) cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC) restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives: The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB)-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods: In our study, CB-Mononuclear Cells’ (MNCs) CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep) CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs) in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand). The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results: Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05). CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively). Conclusions: Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy. Keywords: Umbilical Cord Blood, Natural Killer Cell, Mesenchymal Stem Cel

Induction of apoptosis in human tumor cell lines by platelets

Introduction: It has been reported that platelets can eradicate tumor cells in vitro, although the mechanism of this effect has not been determined. The effect of platelets on the induction of apoptosis in tumor cells is largely unknown. Materials and methods: To investigate this effect, two human hematologic cell lines, K562 and Daudi, were independently faced with unstimulated and thrombin-activated platelets. After the elapsed co-culture time, the levels of caspase-3 and CD95 were evaluated as a sign of cell death and apoptosis. In addition, immortalized cells were evaluated using trypan blue, 7-AAD and WST-1 methods. Results: CD95 and caspase-3 levels were significantly increased in both cell lines compared with the control cells (P<0.05). Beside, considerably lower number of living cells were shown by trypan blue, 7-AAD and WST-1 in the treatment groups compared with the control group (P<0.05). Conclusion: This study demonstrated the role of apoptosis in tumor inhibition and implied the ability of platelets to induce apoptosis in tumor cell lines

Irradiation of platelets in transfusion medicine: risk and benefit judgments

Irradiation of platelet products is generally used to prevent transfusion-associated graft-versus-host disease (TA-GvHD) as well as transfusion-transmitted infections. As an essential prerequisite, gamma-irradiation of blood products prior to transfusion is required in patients who may develop TA-GVHD. Most studies suggest that gamma irradiation has no significant effect on the quality of platelet products; however, more recent studies have shown that the oxidative effects of gamma irradiation can lead to the induction of platelet storage lesion (PSL) and to some extent reduce the efficiency of transfused platelets. As the second widely used irradiation technique, UV-illumination was primarily introduced to reduce the growth of infectious agents during platelet storage, with the advantage that this method can also prevent TA-GvHD. However, the induction of oxidative conditions and platelet pre-activation that lead to PSL is more pronounced after UV-based methods of pathogen reduction. Since these lesions are large enough to clearly affect the post-transfusion platelet recovery and survival, more studies are needed to improve the safety and effectiveness of pathogen reduction technologies (PRTs). Therefore, pointing to other benefits of PRTs, such as preventing TA-GvHD or prolonging the shelf life of products by eliminating the possibility of pathogen growth during storage, does not yet seem to justify their widespread use due to above-mentioned effects. Even for gamma-irradiated platelets, some researchers have suggested that due to decreased 1-hour post-transfusion increments and increased risk of platelet refractoriness, their use should be limited to the patients who may develop TA-GVHD. It is noteworthy that due to the effect of X-rays in preventing TA-GvHD, some recent studies are underway to examine its effects on the quality and effectiveness of platelet products and determine whether X-rays can be used as a more appropriate and cost-effective alternative to gamma radiation. The review presented here provides a detailed description about irradiation-based technologies for platelet products, including their applications, mechanistic features, advantages, and disadvantages

The impact of HLA-E polymorphisms in graft-versus-host disease following HLA-E matched allogeneic hematopoietic stem cell transplantation

The non-classical MHC class-I mainly involves in the regulation of innate immune responses where HLA-E plays a significant role in the cell identification by natural killer cells. HLA-E is a main regulatory ligand for natural killer cells and given the importance of these effector cells in hematopoietic stem cell transplantation, we investigated the effect of HLA-E polymorphisms on post-hematopoietic stem cell transplantation outcomes. The study group included 56 donor-patient pairs with underlying malignant hematological disorders undergoing HLA-E matched allogeneic hematopoietic stem cell transplantation. They were genotyped for HLA-E locus using a sequence specific primer-polymerase chain reaction. The median follow-up was 20.6 months (range 0.2-114.8) and the parameters assessed were acute and chronic graft-versus-host disease and overall survival. We showed a lower frequency of acute graft-versus-host disease (grade II or more; p=0.02) and chronic graft-versus-host disease (extensive; p=0.04) in the patients with HLA-E*0103/0103 genotype compared to other genotypes of HLA-E. There was also an association between HLA-E*0103/0103 and improved overall survival (p=0.001)