Immunophenotyping of Peripheral Blood Cells in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib.

Chronic lymphocytic leukemia (CLL) is a B-cell-derived hematologic malignancy whose progression depends on active B-cell receptor (BCR) signaling. Despite the spectacular efficacy of Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK), resistance can develop in CLL patients, and alternative therapeutic strategies are therefore required. Cancer immunotherapy has revolutionized cancer care and may be an attractive approach in this context. We speculated that characterizing the immune responses of CLL patients may highlight putative immunotherapeutic targets. Here, we used high-dimensional spectral flow cytometry to compare the distribution and phenotype of non-B-cell immune populations in the circulating blood of CLL patients treated with Ibrutinib displaying a complete response or secondary progression. Although no drastic changes were observed in the composition of their immune subsets, the Ibrutinib-resistant group showed increased cycling of CD8+ T cells, leading to their overabundance at the expense of dendritic cells. In addition, the expression of 11 different surface checkpoints was similar regardless of response status. Together, this suggests that CLL relapse upon Ibrutinib treatment may not lead to major alterations in the peripheral immune response.

Spontaneous and Stimulated Production of Cytokines by Blood Cells Ex Vivo as a Biomarker of Initially High or Low Hypoxia Resistance in Rats.

Spontaneous and stimulated production of cytokines by peripheral blood cells obtained from the caudal vein of male Wistar rats was assessed before testing their resistance to oxygen deficiency in a decompression chamber. To study the spontaneous production of cytokines, heparinized blood cells were incubated in a culture medium (24 h, 5% CO2, 37°C) and the content of proinflammatory cytokines IL-6 and TNFα and anti-inflammatory IL-10 in the culture medium was assessed by ELISA. To stimulate cytokine production, blood cells were incubated for 24 h with LPS, phytohemagglutinin, and concanavalin A at final concentrations of 2, 4, and 4 µg, respectively. Two weeks after blood sampling, individual resistance of the animals to hypoxia in a decompression chamber was determined. In animals with low resistance to hypoxia, the levels of spontaneous production of all three cytokines were significantly higher, while after stimulation, the level of IL-1ß increased by more than 2 times. The animals with spontaneous production of IL-10>50 pg/ml, IL-6>10 pg/ml, and TNFα>10 pg/ml, as well as with the increase in IL-1ß production by more than 2 times upon stimulation were classified as low-resistant. At IL-10<15 pg/ml, IL-6<9 pg/ml, and TNFα<7 pg/ml, as well as the absence of the increase in IL-1ß production upon stimulation, they were classified as high-resistant. The identified features of spontaneous and stimulated production of cytokines can be used as non-invasive biomarkers to determine the resistance to hypoxia without exposure to sublethal hypoxia in a decompression chamber.

Mouse blood cells types and aging prediction using penalized Latent Dirichlet Allocation.

BACKGROUND: Aging is a complex, heterogeneous process that has multiple causes. Knowledge on genomic, epigenomic and transcriptomic changes during the aging process shed light on understanding the aging mechanism. A recent breakthrough in biotechnology, single cell RNAseq, is revolutionizing aging study by providing gene expression profile of the entire transcriptome of individual cells. Many interesting information could be inferred from this new type of data with the help of novel computational methods. RESULTS: In this manuscript a novel statistical method, penalized Latent Dirichlet Allocation (pLDA), is applied to an aging mouse blood scRNA-seq data set. A pipeline is built for cell type and aging prediction. The sequence of models in the pipeline take scRNA-seq expression counts as input, preprocess the data using pLDA and predict the cell type and aging status. CONCLUSIONS: pLDA learns a dimension reduced representation of the expression profile. This representation allows identification of cell types and has predictability of the age of cells.

Revealing differential expression patterns of piRNA in FACS blood cells of SARS-CoV-2 infected patients.

Non-coding RNA expression has shown to have cell type-specificity. The regulatory characteristics of these molecules are impacted by changes in their expression levels. We performed next-generation sequencing and examined small RNA-seq data obtained from 6 different types of blood cells separated by fluorescence-activated cell sorting of severe COVID-19 patients and healthy control donors. In addition to examining the behavior of piRNA in the blood cells of severe SARS-CoV-2 infected patients, our aim was to present a distinct piRNA differential expression portrait for each separate cell type. We observed that depending on the type of cell, different sorted control cells (erythrocytes, monocytes, lymphocytes, eosinophils, basophils, and neutrophils) have altering piRNA expression patterns. After analyzing the expression of piRNAs in each set of sorted cells from patients with severe COVID-19, we observed 3 significantly elevated piRNAs - piR-33,123, piR-34,765, piR-43,768 and 9 downregulated piRNAs in erythrocytes. In lymphocytes, all 19 piRNAs were upregulated. Monocytes were presented with a larger amount of statistically significant piRNA, 5 upregulated (piR-49039 piR-31623, piR-37213, piR-44721, piR-44720) and 35 downregulated. It has been previously shown that piR-31,623 has been associated with respiratory syncytial virus infection, and taking in account the major role of piRNA in transposon silencing, we presume that the differential expression patterns which we observed could be a signal of indirect antiviral activity or a specific antiviral cell state. Additionally, in lymphocytes, all 19 piRNAs were upregulated.

The predictive value of peripheral blood cell mitochondrial gene expression in identifying the prognosis in pediatric sepsis at preschool age.

Background: Sepsis represents a severe manifestation of infection often accompanied by metabolic disorders and mitochondrial dysfunction. Notably, mitochondrial DNA copy number (mtDNA-CN) and the expression of specific mitochondrial genes have emerged as sensitive indicators of mitochondrial function. To investigate the utility of mitochondrial gene expression in peripheral blood cells for distinguishing severe infections and predicting associated outcomes, we conducted a prospective cohort study. Methods: We established a prospective cohort comprising 74 patients with non-sepsis pneumonia and 67 cases of sepsis induced by respiratory infections, aging from 2 to 6 years old. We documented corresponding clinical data and laboratory information and collected blood samples upon initial hospital admission. Peripheral blood cells were promptly isolated, and both total DNA and RNA were extracted. We utilized absolute quantification PCR to assess mtDNA-CN, as well as the expression levels of mt-CO1, mt-ND1, and mt-ATP6. Subsequently, we extended these comparisons to include survivors and non-survivors among patients with sepsis using univariate and multivariate analyses. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic potential. Results: The mtDNA-CN in peripheral blood cells was significantly lower in the sepsis group. Univariate analysis revealed a significant reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 in patients with sepsis. However, multivariate analysis did not support the use of mitochondrial function in peripheral blood cells for sepsis diagnosis. In the comparison between pediatric sepsis survivors and non-survivors, univariate analysis indicated a substantial reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 among non-survivors. Notably, total bilirubin (TB), mt-CO1, mt-ND1, and mt-ATP6 levels were identified as independent risk factors for sepsis-induced mortality. ROC curves were then established for these independent risk factors, revealing areas under the curve (AUCs) of 0.753 for TB (95% CI 0.596-0.910), 0.870 for mt-CO1 (95% CI 0.775-0.965), 0.987 for mt-ND1 (95% CI 0.964-1.000), and 0.877 for mt-ATP6 (95% CI 0.793-0.962). Conclusion: MtDNA-CN and mitochondrial gene expression are closely linked to the severity and clinical outcomes of infectious diseases. Severe infections lead to impaired mitochondrial function in peripheral blood cells. Notably, when compared to other laboratory parameters, the expression levels of mt-CO1, mt-ND1, and mt-ATP6 demonstrate promising potential for assessing the prognosis of pediatric sepsis.

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

A metabolic atlas of blood cells in young and aged mice identifies uridine as a metabolite to rejuvenate aged hematopoietic stem cells.

Aging of hematopoietic stem cells (HSCs) is accompanied by impaired self-renewal ability, myeloid skewing, immunodeficiencies and increased susceptibility to malignancies. Although previous studies highlighted the pivotal roles of individual metabolites in hematopoiesis, comprehensive and high-resolution metabolomic profiles of different hematopoietic cells across ages are still lacking. In this study, we created a metabolome atlas of different blood cells across ages in mice. We reveal here that purine, pyrimidine and retinol metabolism are enriched in young hematopoietic stem and progenitor cells (HSPCs), whereas glutamate and sphingolipid metabolism are concentrated in aged HSPCs. Through metabolic screening, we identified uridine as a potential regulator to rejuvenate aged HSPCs. Mechanistically, uridine treatment upregulates the FoxO signaling pathway and enhances self-renewal while suppressing inflammation in aged HSCs. Finally, we constructed an open-source platform for public easy access and metabolomic analysis in blood cells. Collectively, we provide a resource for metabolic studies in hematopoiesis that can contribute to future anti-aging metabolite screening.

Evaluation of the Role of Potassium Channels in the Proliferation, Migration, and Invasion of Blood Cells.

The potassium channels are one of the key regulators of cell membrane potential and permeability properties of blood cells. The changes in functioning of potassium channels control crucial cell processes such as proliferation, viability, migration, and invasion. The correct estimation of these processes is important for the characterization of physiological and pathophysiological cell states. Here, we present the experimental protocol for evaluation of the role of potassium ion channels in the proliferation, migration, and invasion of blood cells.

Biological functions and biomedical applications of extracellular vesicles derived from blood cells.

There is a growing interest in using extracellular vesicles (EVs) for therapeutic applications. EVs are composed of cytoplasmic proteins and nucleic acids and an external lipid bilayer containing transmembrane proteins on their surfaces. EVs can alter the state of the target cells by interacting with the receptor ligand of the target cell or by being internalised by the target cell. Blood cells are the primary source of EVs, and 1 µL of plasma contains approximately 1.5 × 107 EVs. Owing to their easy acquisition and the avoidance of cell amplification in vitro, using blood cells as a source of therapeutic EVs has promising clinical application prospects. This review summarises the characteristics and biological functions of EVs derived from different blood cell types (platelets, erythrocytes, and leukocytes) and analyses the prospects and challenges of using them for clinical therapeutic applications. In summary, blood cell-derived EVs can regulate different cell types such as immune cells (macrophages, T cells, and dendritic cells), stem cells, and somatic cells, and play a role in intercellular communication, immune regulation, and cell proliferation. Overall, blood cell-derived EVs have the potential for use in vascular diseases, inflammatory diseases, degenerative diseases, and injuries. To promote the clinical translation of blood cell-derived EVs, researchers need to perform further studies on EVs in terms of scalable and reproducible isolation technology, quality control, safety, stability and storage, regulatory issues, cost-effectiveness, and long-term efficacy.

Blood cell parameters and risk of nonalcoholic fatty liver disease: a comprehensive Mendelian randomization study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is on the rise globally, and past research suggests a significant association with various blood cell components. Our goal is to explore the potential correlation between whole blood cell indices and NAFLD risk using Mendelian randomization (MR). METHODS: We analyzed data from 4,198 participants in the 2017-2018 National Health and Nutrition Examination Survey to investigate the link between blood cell indicators and NAFLD. Using various methods like weighted quantile sum and multivariate logistic regression, we assessed the association. Additionally, two-sample Mendelian randomization were employed to infer causality for 36 blood cell indicators and NAFLD. RESULTS: Multivariate logistic regression identified 10 NAFLD risk factors. Weighted quantile sum revealed a positive correlation (p = 6.03e-07) between total blood cell indices and NAFLD, with hemoglobin and lymphocyte counts as key contributors. Restricted cubic spline analysis found five indicators with significant nonlinear correlations to NAFLD. Mendelian randomization showed a notable association between reticulocyte counts and NAFLD using the inverse-variance weighted method. CONCLUSIONS: Hematological markers pose an independent NAFLD risk, with a positive causal link found for reticulocyte count. These results emphasize the importance of monitoring NAFLD and investigating specific underlying mechanisms further.

Numerous Serine/Threonine Kinases Affect Blood Cell Homeostasis in Drosophila melanogaster.

Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.

Epigenetic aging of human blood cells is influenced by the age of the host body.

Allogenic hematopoietic stem cell transplantation is a therapeutic procedure performed over a wide range of donor and recipient age combinations, representing natural experiments of how the age of the recipient affects aging in transplanted donor cells in vivo. We measured DNA methylation and epigenetic aging in donors and recipients and found that biological epigenetic clocks are accelerated in cells transplanted into an older body and decelerated in a younger body. This is the first evidence that the age of the circulating environment influences human epigenetic aging in vivo.

RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks.

ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Methylation of serotonin regulating genes in cord blood cells: association with maternal metabolic parameters and correlation with methylation in peripheral blood cells during childhood and adolescence.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) signaling is involved in neurodevelopment, mood regulation, energy metabolism, and other physiological processes. DNA methylation plays a significant role in modulating the expression of genes responsible for maintaining 5-HT balance, such as 5-HT transporter (SLC6A4), monoamine oxidase A (MAOA), and 5-HT receptor type 2A (HTR2A). Maternal metabolic health can influence long-term outcomes in offspring, with DNA methylation mediating these effects. We investigated associations between maternal metabolic parameters-pre-pregnancy body mass index (pBMI), gestational weight gain (GWG), and glucose tolerance status (GTS), i.e., gestational diabetes mellitus (GDM) versus normal glucose tolerance (NGT)-and cord blood methylation of SLC6A4, MAOA, and HTR2A in participants from our PlaNS birth cohort. CpG sites (15, 9, and 2 in each gene, respectively) were selected based on literature and in silico data. Methylation levels were quantified by bisulfite pyrosequencing. We also examined the stability of methylation patterns in these genes in circulating blood cells from birth to adolescence using longitudinal DNA methylation data from the ARIES database. RESULTS: None of the 203 PlaNS mothers included in this study had preexisting diabetes, 99 were diagnosed with GDM, and 104 had NGT; all neonates were born at full term by planned Cesarean section. Methylation at most CpG sites differed between male and female newborns. SLC6A4 methylation correlated inversely with maternal pBMI and GWG, while methylation at HTR2A site -1665 correlated positively with GWG. None of the maternal metabolic parameters statistically associated with MAOA methylation. DNA methylation data in cord blood and peripheral blood at ages 7 and 15 years were available for 808 participants from the ARIES database; 4 CpG sites (2 in SLC6A4 and 2 in HTR2A) overlapped between the PlaNS and ARIES cohorts. A positive correlation between methylation levels in cord blood and peripheral blood at 7 and 15 years of age was observed for both SLC6A4 and HTR2A CpG sites. CONCLUSIONS: Methylation of 5-HT regulating genes in cord blood cells is influenced by neonatal sex, with maternal metabolism playing an additional role. Inter-individual variations present in circulating blood cells at birth are still pronounced in childhood and adolescence.

Toxicity evaluation of silver nanoparticles synthesized from naringin flavonoid on human promyelocytic leukemic cells and human blood cells.

Increasing applications of silver nanoparticles (AgNPs) in multiple products like cosmetics, medicines, drugs, paints, and other new materials have raised concern for their toxic effects on living beings and the surrounding environment. In the present study, cytotoxicity and genotoxicity of AgNPs synthesized using plant flavonoid (Naringin) as a reducing agent were investigated on human promyelocytic leukemic (HL-60) cells and human blood as an in vitro model. The LC50 of AgNPs was found to be 4.85 µM. Dose-dependent increase in cell death and caspase activity was observed in the presence of AgNPs. The comet assay showed a 60%-70% (p < .05) increase in tail DNA at 0.48 and 0.96 µM AgNPs. CBMN in PBMCs also confirmed the genotoxic potential of AgNPs-induced DNA damage. AgNPs resulted in 1.5-1.54 fold (p < .05) increase in the level of ROS in HL-60 cells after 12 h of exposure. AgNP showed toxicity in human cells through ROS generation and cellular damage through membrane dysfunction, caspase activation, apoptosis, and DNA damage.

Multi-omics integrative analysis revealed characteristic changes in blood cell immunity and amino acid metabolism in a silkworm model of hyperproteinemia.

Hyperproteinemia is a serious metabolic disease of both humans and animals characterized by an abnormally high plasma protein concentration (HPPC). Although hyperproteinemia can cause an imbalance in blood cell homeostasis, the functional changes to blood cells remain unclear. Here, a HPPC silkworm model was used to assess changes to the chromatin accessibility and transcript levels of genes related to blood cell metabolism and immune function. The results showed that HPPC enhanced phagocytosis of blood cells, increased chromatin accessibility and transcript levels of genes involved in cell phagocytosis, proliferation, stress, and programmed death, while genes associated with aromatic amino acid metabolism, and antibacterial peptide synthesis were inhibited in blood cells. Further analysis of the chromatin accessibility of the promoter region found that the high chromatin accessibility of genes sensitive to HPPC, was related to histone modifications, including tri-methylation of lysine residue 4 of histone H3 and acetylation of lysine residue 27 of histone H3. Changes to the chromatin accessibility and transcript levels of genes related to immune function and amino acid metabolism in the blood cells of the HPPC silkworm model provided useful references for future studies of the mechanisms underlying epigenomic regulation mediated by hyperproteinemia.

Single-cell transcriptomics identifies new blood cell populations in Drosophila released at the onset of metamorphosis.

Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, we identified a highly migratory and immune-responsive pupal cell population expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, we present the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.

Effects of bile salt-stimulated lipase on blood cells and associations with disease activity in human inflammatory joint disorders.

The bile salt-stimulated lipase (BSSL) was originally recognized as a lipolytic enzyme expressed by the exocrine pancreas and in some species, notably humans, the lactating mammary gland, being secreted into the duodenum and with the mother's milk, respectively. However, BSSL is also present in the blood and has been assigned additional functions, even beyond the gastrointestinal tract. Conventional BSSL knockout mice are protected from developing disease in animal models of arthritis, and antibodies directed towards BSSL prevent or mitigate disease in similar models. The aim of this study was to investigate the role of BSSL as a newly discovered player in inflammation and specifically in inflammatory joint disorders. As part of mechanism of action, we here show that BSSL is secreted by neutrophils, interacts with monocytes and stimulates their migration in vitro. An anti-BSSL antibody that blocks the human BSSL-monocyte interaction was shown to simultaneously prevent the signaling pathway by which BSSL induce cell migration. Moreover, in this cohort study we show that BSSL levels are significantly higher in blood samples from patients with rheumatoid arthritis and psoriatic arthritis compared to healthy controls. The BSSL levels in patients' blood also correlated with disease activity scores and established inflammatory markers. Hence, although the mode of action is not yet fully clarified, we conclude that BSSL could be considered a proinflammatory component in the innate immune system and thus a possible novel target for treatment of chronic inflammation.

Differences in DNA methylation of HAMP in blood cells predicts the development of type 2 diabetes.

OBJECTIVES: Better disease management can be achieved with earlier detection through robust, sensitive, and easily accessible biomarkers. The aim of the current study was to identify novel epigenetic biomarkers determining the risk of type 2 diabetes (T2D). METHODS: Livers of 10-week-old female New Zealand Obese (NZO) mice, slightly differing in their degree of hyperglycemia and liver fat content and thereby in their diabetes susceptibility were used for expression and methylation profiling. We screened for differences in hepatic expression and DNA methylation in diabetes-prone and -resistant mice, and verified a candidate (HAMP) in human livers and blood cells. Hamp expression was manipulated in primary hepatocytes and insulin-stimulated pAKT was detected. Luciferase reporter assays were conducted in a murine liver cell line to test the impact of DNA methylation on promoter activity. RESULTS: In livers of NZO mice, the overlap of methylome and transcriptome analyses revealed a potential transcriptional dysregulation of 12 hepatokines. The strongest effect with a 52% decreased expression in livers of diabetes-prone mice was detected for the Hamp gene, mediated by elevated DNA methylation of two CpG sites located in the promoter. Hamp encodes the iron-regulatory hormone hepcidin, which had a lower abundance in the livers of mice prone to developing diabetes. Suppression of Hamp reduces the levels of pAKT in insulin-treated hepatocytes. In liver biopsies of obese insulin-resistant women, HAMP expression was significantly downregulated along with increased DNA methylation of a homologous CpG site. In blood cells of incident T2D cases from the prospective EPIC-Potsdam cohort, higher DNA methylation of two CpG sites was related to increased risk of incident diabetes. CONCLUSIONS: We identified epigenetic changes in the HAMP gene which may be used as an early marker preceding T2D.

The Impact of Different Types of Physical Effort on the Expression of Selected Chemokine and Interleukin Receptor Genes in Peripheral Blood Cells.

This study aimed to assess the post-effort transcriptional changes of selected genes encoding receptors for chemokines and interleukins in young, physically active men to better understand the immunomodulatory effect of physical activity. The participants, aged 16-21 years, performed physical exercise tasks of either a maximal multistage 20 m shuttle-run test (beep test) or a repeated speed ability test. The expression of selected genes encoding receptors for chemokines and interleukins in nucleated peripheral blood cells was determined using RT-qPCR. Aerobic endurance activity was a positive stimulant that induced increased expression of CCR1 and CCR2 genes following lactate recovery, while the maximum expression of CCR5 was found immediately post-effort. The increase in the expression of inflammation-related genes encoding chemokine receptors triggered by aerobic effort strengthens the theory that physical effort induces sterile inflammation. Different profiles of studied chemokine receptor gene expression induced by short-term anaerobic effort suggest that not all types of physical effort activate the same immunological pathways. A significant increase in IL17RA gene expression after the beep test confirmed the hypothesis that cells expressing this receptor, including Th17 lymphocyte subsets, can be involved in the creation of an immune response after endurance efforts.