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The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at −80 °C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. In addition, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate. [ABSTRACT FROM AUTHOR]

Optical microscopy videos enable experts to analyze the motion of several biological elements. Particularly in blood samples infected with Trypanosoma cruzi (T. cruzi), microscopy videos reveal a dynamic scenario where the parasites' motions are conspicuous. While parasites have self-motion, cells are inert and may assume some displacement under dynamic events, such as fluids and microscope focus adjustments. This paper analyzes the trajectory of T. cruzi and blood cells to discriminate between these elements by identifying the following motion patterns: collateral, fluctuating, and pan–tilt–zoom (PTZ). We consider two approaches: i) classification experiments for discrimination between parasites and cells; and ii) clustering experiments to identify the cell motion. We propose the trajectory step dispersion (TSD) descriptor based on standard deviation to characterize these elements, outperforming state-of-the-art descriptors. Our results confirm motion is valuable in discriminating T. cruzi of the cells. Since the parasites perform the collateral motion, their trajectory steps tend to randomness. The cells may assume fluctuating motion following a homogeneous and directional path or PTZ motion with trajectory steps in a restricted area. Thus, our findings may contribute to developing new computational tools focused on trajectory analysis, which can advance the study and medical diagnosis of Chagas disease. [ABSTRACT FROM AUTHOR]

Hyperspectral imaging has emerged as a novel imaging modality in the medical field, offering the ability to acquire images of biological tissues while simultaneously providing biochemical insights for in-depth tissue analysis. This approach facilitates early disease diagnosis, presenting advantages over traditional medical imaging techniques. Addressing challenges such as the computational burden of existing convolutional neural networks (CNNs) and imbalances in sample data, this paper introduces a lightweight GhostMRNet for the classification of microscopic hyperspectral images of human blood cells. The proposed model employs Ghost Modules to replace conventional convolutional layers and a cascading approach with small convolutional kernels for multiscale feature extraction, aiming to enhance feature extraction capabilities while reducing computational complexity. Additionally, an SE (Squeeze-and-Excitation) module is introduced to selectively allocate weights to features in each channel, emphasizing informative features and efficiently achieving spatial–spectral feature extraction in microscopic hyperspectral imaging. We evaluated the performance of the proposed GhostMRNet and compared it with other state-of-the-art models using two real medical hyperspectral image datasets. The experimental results demonstrate that GhostMRNet exhibits a superior performance, with an overall accuracy (OA), average accuracy (AA), and Kappa coefficient reaching 99.965%, 99.565%, and 0.9925, respectively. In conclusion, the proposed GhostMRNet achieves a superior classification performance at a smaller computational cost, thereby providing a novel approach for blood cell detection. [ABSTRACT FROM AUTHOR]

Simple Summary: Oncolytic herpes simplex viruses (oHSVs) employ as natural entry receptors nectin1 or HVEM. In contrast, retargeted oHSVs (ReHVs) employ as an entry receptor a tumor-associated antigen that no longer interacts with nectin1/HVEM and, thus, spares normal cells. Prior to entry, both oHSVs and ReHVs attach to cells by interaction of gC and gB glycoproteins with heparan sulphates and chondroitin sulphates (glycosaminoglycans–GAGs). The systemic delivery of oncolytic viruses would be the ideal route for hard-to-reach or metastatic cancers that constitute unmet clinical needs. In an accompanying paper, we reported that the blood complement and the antiviral neutralizing antibodies represent the major blood factors that inactivate systemically administered ReHVs. Here, we asked whether ReHV adsorption to GAGs acts as a sink and subtracts the virus from circulation. We report that a genetic modification in gC, which reduced its interaction with GAGs, resulted in a longer half-life of circulating ReHV and a higher anticancer efficacy of systemically (but not intratumorally) administered ReHV. The role of the interaction with cell-surface glycosaminoglycans (GAGs) during in vivo HSV infection is currently unknown. The rationale of the current investigation was to improve the anticancer efficacy of systemically administered retargeted oHSVs (ReHVs) by decreasing their binding to GAGs, including those of endothelial cells, blood cells, and off-tumor tissues. As a proof-of-principle approach, we deleted seven amino acids critical for interacting with GAGs from the glycoprotein C (gC) of R-337 ReHV. The modification in the resulting R-399 recombinant prolonged the half-life in the blood of systemically administered R-399 and enhanced its biodistribution to tumor-positive lungs and to the tumor-negative liver. Ultimately, it greatly increased the R-399 efficacy against metastatic-like lung tumors upon IV administration but not against subcutaneous tumors upon IT administration. These results provide evidence that the increased efficacy seen upon R-399 systemic administration correlated with the slower clearance from the circulation. To our knowledge, this is the first in vivo evidence that the partial impairment of the gC interaction with GAGs resulted in a prolonged half-life of circulating ReHV, an increase in the amount of ReHV taken up by tissues and tumors, and, ultimately, an enhanced anticancer efficacy of systemically administered ReHV. [ABSTRACT FROM AUTHOR]

The paper considers the problem of automating the creation of an annotated dataset for further use in a system for detecting, localizing and classifying blood cells in an image using deep learning. The subject of the research is the processes of digital image processing for object detection and localization. Objective. The aim of this study is to create a pipeline of digital image processing methods that can automatically generate an annotated set of blood smear images. This set will then be used to train and validate deep learning models, significantly reducing the time required by machine learning specialists. Method. The proposed approach for object detection and localization is based on digital image processing methods such as filtering, thresholding, binarization, contour detection, and filling. The pipeline for detection and localization includes the following steps: The given fragment of text describes a process that involves noise reduction, conversion to the HSV color model, defining a mask for white blood cells and platelets, detecting the contours of white blood cells and platelets, determining the coordinates of the upper left and lower right corners of white blood cells and platelets, calculating the area of the region inside the bounding box, saving the obtained data, and determining the most common color in the image; filling the contours of leukocytes and platelets with said color; defining a mask for red blood cells; defining the contours of red blood cells; determining the coordinates of the upper left and lower right corners of red blood cells; calculating the area of the region within the bounding box; entering data about the found objects into the dataframe; saving to a .csv file for future use. With an unlabeled image dataset and a generated .csv file using image processing libraries, any researcher should be able to recreate a labeled dataset. Results. The developed approach was implemented in software for creating an annotated dataset of blood smear images Conclusions. The study proposes and justifies an approach to automatically create a set of annotated data. The pipeline is tested on a set of unlabelled data and a set of labelled data is obtained, consisting of cell images and a .csv file with the attributes “file name”, “type”, “xmin”, “ymin”, “xmax”, “ymax”, “area”, which are the coordinates of the bounding box for each object. The number of correctly, incorrectly, and unrecognised objects is calculated manually, and metrics are calculated to assess the accuracy and quality of object detection and localisation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. Methods: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. Results: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. Conclusion: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance. [ABSTRACT FROM AUTHOR]

Infection of piglets with Glaesserella parasuis (G. parasuis) induces host immunosuppression. However, the mechanism underlying the immunosuppression of piglets remains unclear. Activation of the PD-1/PD-L1 axis has been shown to trigger host immunosuppression. Baicalin possesses anti-inflammatory and immunomodulatory functions. However, whether baicalin inhibits PD-1/PD-L1 activation and thus alleviates host immunosuppression has not been investigated. In this study, the effect of baicalin on the attenuation of piglet immunosuppression induced by G. parasuis was evaluated. Seventy piglets were randomly divided into the control group, infection group, levamisole group, BMS-1 group, 25 mg/kg baicalin group, 50 mg/kg baicalin group and 100 mg/kg baicalin group. Following pretreatment with levamisole, BMS-1 or baicalin, the piglets were challenged with 1 × 108 CFU of G. parasuis. Our results showed that baicalin, levamisole and BMS-1 modified routine blood indicators and biochemical parameters; downregulated IL-1β, IL-10, IL-18, TNF-α and IFN-γ mRNA expression; and upregulated IL-2 and IL-8 mRNA expression in blood. Baicalin, levamisole and BMS-1 increased the proportions of CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells and CD3–CD21+ B cells in the splenocyte population, increased the proportions of CD3+ T cells, CD3+CD4+ T cells and CD3+CD8+ T cells in the blood, and inhibited PD-1/PD-L1 and TIM-3 activation. Baicalin, levamisole and BMS-1 reduced p-PI3K, p-Akt, and p-mTOR expression, the p-MEK1/2/MEK1/2 and p-ERK1/2/ERK1/2 ratios and increased RAS expression. Baicalin, levamisole and BMS-1 provided substantial protection against G. parasuis challenge and relieved tissue histopathological damage. Our findings might provide new strategies for controlling G. parasuis infection and other immunosuppressive diseases. [ABSTRACT FROM AUTHOR]

Platelet transfusions are routine procedures in clinical treatment aimed at preventing bleeding in critically ill patients, including those with cancer, undergoing surgery, or experiencing trauma. However, platelets are susceptible blood cells that require specific storage conditions. The availability of platelet concentrates is limited to five days due to various factors, including the risk of bacterial contamination and the occurrence of physical and functional changes known as platelet storage lesions. In this article, the problems related to platelet storage lesions are categorized into four groups depending on research areas: storage conditions, additive solutions, new testing methods for platelets (proteomic and metabolomic analysis), and extensive data modeling of platelet production (mathematical modeling, statistical analysis, and artificial intelligence). This article provides extensive information on the challenges, potential improvements, and novel perspectives regarding platelet storage. [ABSTRACT FROM AUTHOR]

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders. The ability to maintain blood stem cells (HSCs) in vitro would allow us to provide better therapies for blood diseases. Here, the authors report that polymer-organised extracellular proteins, coupled to soft environments mimicking bone marrow stiffness, allow stromal cells to maintain HSCs in vitro. [ABSTRACT FROM AUTHOR]

Nanoparticles (NP) play a crucial role in nanomedicine, serving as carriers for localized therapeutics to allow for precise drug delivery to specific disease sites and conditions. When injected systemically, NP can directly interact with various blood cell types, most critically with circulating platelets. Hence, the potential activation/inhibition of platelets following NP exposure must be evaluated a priori due to possible debilitating outcomes. In recent years, various studies have helped resolve the physicochemical parameters that influence platelet‐NP interactions, and either emphasize nanoparticles' therapeutic role such as to augment hemostasis or to inhibit thrombus formation, or conversely map their potential undesired side effects upon injection. In the present review, we discuss some of the main effects of several key NP types including polymeric, ceramic, silica, dendrimers and metallic NPs on platelets, with a focus on the physicochemical parameters that can dictate these effects and modulate the therapeutic potential of the NP. Despite the scientific and clinical significance of understanding Platelet‐NP interactions, there is a significant knowledge gap in the field and a critical need for further investigation. Moreover, improved guidelines and research methodologies need to be developed and implemented. Our outlook includes the use of biomimetic in vitro models to investigate these complex interactions under both healthy physiological and disease conditions. [ABSTRACT FROM AUTHOR]

Background: Guided bone regeneration (GBR) has become a necessary practice in implantology. Absorbable membranes have shown advantages over non-absorbable membranes, such as blood support of bone tissue. This study aimed to evaluate five collagen membranes in rat calvaria critical-size defects through a histomorphometric analysis of the inflammatory profile during the initial phase of bone repair. Materials and methods: A total of 72 Albinus Wistar rats were used for the study, divided into six groups, with 12 animals per group, and two experimental periods, 7 and 15 days. The groups were as follows: the CG (clot), BG (Bio-Gide®), JS (Jason®), CS (Collprotect®), GD (GemDerm®), and GDF (GemDerm Flex®). Results: Data showed that the BG group demonstrated an inflammatory profile with an ideal number of inflammatory cells and blood vessels, indicating a statistically significant difference between the JS and CS groups and the BG group in terms of the number of inflammatory cells and a statistically significant difference between the JS and CS groups and the GD group in terms of angiogenesis (p < 0.05). Conclusions: We conclude that different origins and ways of obtaining them, as well as the thickness of the membrane, can interfere with the biological response of the material. [ABSTRACT FROM AUTHOR]

Nanomaterials have been extensively used in the biomedical field due to their unique physical and chemical properties. They promise wide applications in the diagnosis, prevention, and treatment of diseases. Nanodrugs are generally transported to target tissues or organs by coupling targeting molecules or enhanced permeability and retention effect (EPR) passively. As intravenous injection is the most common means of administration of nanomedicine, the transport process inevitably involves the interactions between nanoparticles (NPs) and blood cells. Platelets are known to not only play a critical role in normal coagulation by performing adhesion, aggregation, release, and contraction functions, but also be associated with pathological thrombosis, tumor metastasis, inflammation, and immune reactions, making it necessary to investigate the effects of NPs on platelet function during transport, particularly the way in which their physical and chemical properties determine their interaction with platelets and the underlying mechanisms by which they activate and induce platelet aggregation. However, such data are lacking. This review is intended to summarize the effects of NPs on platelet activation, aggregation, release, and apoptosis, as well as their effects on membrane proteins and morphology in order to shed light on such key issues as how to reduce their adverse reactions in the blood system, which should be taken into consideration in NP engineering. [ABSTRACT FROM AUTHOR]

The search for the molecular markers of osteoporosis (OP), based on the analysis of differential deoxyribonucleic acid (DNA) methylation in bone cells and peripheral blood cells, is promising for developments in the field of the early diagnosis and targeted therapy of the disease. The Runt-related transcription factor 2 (RUNX2) gene is one of the key genes of bone metabolism, which is of interest in the search for epigenetic signatures and aberrations associated with the risk of developing OP. Based on pyrosequencing, the analysis of the RUNX2 methylation profile from a pool of peripheral blood cells in men and women over 50 years of age of Russian ethnicity from the Volga-Ural region of Russia was carried out. The level of DNA methylation in three CpG sites of the RUNX2 gene was assessed and statistically significant hypomethylation was revealed in all three studied CpG sites in men (U = 746.5, p = 0.004; U = 784, p = 0.01; U = 788.5, p = 0.01, respectively) and in one CpG site in women (U = 537, p = 0.03) with primary OP compared with control. In the general sample, associations were preserved for the first CpG site (U = 2561, p = 0.0001766). The results were obtained for the first time and indicate the existence of potentially new epigenetic signatures of RUNX2 in individuals with OP. [ABSTRACT FROM AUTHOR]

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single‐cell resolution. Interestingly, we detected a few progenitor cells and non‐erythroid cells from both origins. In PSC‐derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN‐derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic‐related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony‐forming unit‐erythroid cells. Inhibition of CD99 reduced the proliferation of PSC‐derived cells and facilitated erythroid maturation. Furthermore, CD99–CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation. [ABSTRACT FROM AUTHOR]

C-mannosylation is a post-translational modification that occurs intracellularly in the endoplasmic reticulum. In humans, biosynthesis of C-mannosylation in proteins containing thrombospondin type 1 repeat is catalyzed by the DPY19 family; nonetheless, biological functions of protein C-mannosylation are not yet fully understood, especially in tumor progression. Vasculogenic mimicry (VM) is the formation of fluid-conducting channels by highly invasive and genetically deregulated tumor cells, enabling the tumors to form matrix-embedded vasculogenic structures, containing plasma and blood cells to meet the metabolic demands of rapidly growing tumors. In this study, we focused on DPY19L3, a C-mannosyltransferase, and aimed to unravel its role in VM. Knockout of DPY19L3 inhibited the formation of VM in HT1080 human fibrosarcoma cells. Re-expression of wild-type DPY19L3 recovered VM formation; however, DPY19L3 isoform2, an enzymatic activity-defect mutant, did not restore it, suggesting that the C-mannosyltransferase activity of DPY19L3 is crucial to its function. Furthermore, the knockdown of DPY19L3 in MDA-MB-231 breast cancer cells hindered its network formation ability. Altogether, our findings suggest that DPY19L3 is required for VM formation and stipulate the relevance of C-mannosylation in oncogenesis. [ABSTRACT FROM AUTHOR]

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells that represent the interface between blood cells on one side and hepatocytes on the other side. LSECs not only form a barrier within the hepatic sinus, but also play important physiological functions such as regulating hepatic vascular pressure, anti-inflammatory and anti-fibrotic. Pathologically, pathogenic factors can induce LSECs capillarization, that is, loss of fenestra and dysfunction, which are conducive to early steatosis, lay the foundation for the progression of metabolic dysfunction-associated fatty liver disease (MAFLD), and accelerate metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. The unique localization, phenotype, and function of LSECs make them potential candidates for reducing liver injury, inflammation, and preventing or reversing fibrosis in the future. [ABSTRACT FROM AUTHOR]

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii. [ABSTRACT FROM AUTHOR]

Interactions between osteolineage cells and myeloid cells play important roles in maintaining skeletal homeostasis. Herein, we find that osteolineage cells transfer mitochondria to myeloid cells. Impairment of the transfer of mitochondria by deleting MIRO1 in osteolineage cells leads to increased myeloid cell commitment toward osteoclastic lineage cells and promotes bone resorption. In detail, impaired mitochondrial transfer from osteolineage cells alters glutathione metabolism and protects osteoclastic lineage cells from ferroptosis, thus promoting osteoclast activities. Furthermore, mitochondrial transfer from osteolineage cells to myeloid cells is involved in the regulation of glucocorticoid-induced osteoporosis, and glutathione depletion alleviates the progression of glucocorticoid-induced osteoporosis. These findings reveal an unappreciated mechanism underlying the interaction between osteolineage cells and myeloid cells to regulate skeletal metabolic homeostasis and provide insights into glucocorticoid-induced osteoporosis progression. Bone and blood lineage cells communicate with each other to maintain skeletal homeostasis. Here, the authors show that MIRO1-mediated mitochondrial transfer from osteolineage to myeloid lineage cells regulates bone resorption by altering glutathione metabolism. [ABSTRACT FROM AUTHOR]

Human type 1 diabetes (T1D) is caused by autoimmune attack on the insulin-producing pancreatic beta cells by islet antigen-reactive T cells. How human islet antigen-reactive (IAR) CD4+ memory T cells from peripheral blood affect T1D progression in the pancreas is poorly understood. Here, we aim to determine if IAR T cells in blood could be detected in pancreas. We identify paired αβ (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new-onset, and established T1D donors, and measured sequence overlap with TCRs in pancreata from healthy, at risk and T1D organ donors. We report extensive TRA junction sharing between IAR T cells and pancreas-infiltrating T cells (PIT), with perfect-match or single-mismatch TRA junction amino acid sequences comprising ~29% total unique IAR TRA junctions (942/3,264). PIT-matched TRA junctions were largely public and enriched for TRAV41 usage, showing significant nucleotide sequence convergence, increased use of germline-encoded versus non-templated residues in epitope engagement, and a potential for cross-reactivity. Our findings thus link T cells with distinctive germline-like TRA chains in the peripheral blood with T cells in the pancreas. Most efforts to examine islet antigen-reactive T cells in type-1 diabetes have been limited to peripheral blood. In this study, the authors examine the T cell receptor (TCR) repertoire of insulin antigen-reactive T cells from peripheral blood from patients with type-1 diabetes as well as pancreata from deceased donors. [ABSTRACT FROM AUTHOR]

Background: Immunotherapeutic approaches, including immune checkpoint inhibitor (ICI) therapy, are increasingly recognized for their potential. Despite notable successes, patient responses to these treatments vary significantly. The absence of reliable predictive and prognostic biomarkers hampers the ability to foresee outcomes. This meta-analysis aims to evaluate the predictive significance of circulating myeloid-derived suppressor cells (MDSC) in patients with solid tumors undergoing ICI therapy, focusing on progression-free survival (PFS) and overall survival (OS). Methods: A comprehensive literature search was performed across PubMed and EMBASE from January 2007 to November 2023, utilizing keywords related to MDSC and ICI. We extracted hazard ratios (HRs) and 95% confidence intervals (CIs) directly from the publications or calculated them based on the reported data. A hazard ratio greater than 1 indicated a beneficial effect of low MDSC levels. We assessed heterogeneity and effect size through subgroup analyses. Results: Our search yielded 4,023 articles, of which 17 studies involving 1,035 patients were included. The analysis revealed that patients with lower levels of circulating MDSC experienced significantly improved OS (HR=2.13 [95% CI 1.51-2.99]) and PFS (HR=1.87 [95% CI 1.29-2.72]) in response to ICI therapy. Notably, heterogeneity across these outcomes was primarily attributed to differences in polymorphonuclear MDSC (PMN-MDSC) subpopulations and varying cutoff methodologies used in the studies. The monocytic MDSC (M-MDSC) subpopulation emerged as a consistent and significant prognostic marker across various subgroup analyses, including ethnicity, tumor type, ICI target, sample size, and cutoff methodology. Conclusions: Our findings suggest that standardized assessment of MDSC, particularly M-MDSC, should be integral to ICI therapy strategies. These cells hold the promise of identifying patients at risk of poor response to ICI therapy, enabling tailored treatment approaches. Further research focusing on the standardization of markers and validation of cutoff methods is crucial for integrating MDSC into clinical practice. [ABSTRACT FROM AUTHOR]

Background: Kidney cancer is a prevalent malignancy with an increasing incidence worldwide. Blood cell indices and inflammation-related markers have shown huge potential as biomarkers for predicting cancer incidences, but that is not clear in kidney cancer. Our study aims to investigate the correlations of blood cell indices and inflammation-related markers with kidney cancer risk. Methods: We performed a population-based cohort prospective analysis using data from the UK Biobank. A total of 466,994 participants, free of kidney cancer at baseline, were included in the analysis. The hazard ratios (HRs) and 95% confidence intervals (CIs) for kidney cancer risk were calculated using Cox proportional hazards regression models. Restricted cubic spline models were used to investigate nonlinear longitudinal associations. Stratified analyses were used to identify high-risk populations. The results were validated through sensitivity analyses. Results: During a mean follow-up of 12.4 years, 1,710 of 466,994 participants developed kidney cancer. The Cox regression models showed that 13 blood cell indices and four inflammation-related markers were associated with kidney cancer incidence. The restricted cubic spline models showed non-linear relationships with kidney cancer. Finally, combined with stratified and sensitivity analyses, we found that the mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet distribution width (PDW), systemic immune-inflammation index (SII), and product of platelet count and neutrophil count (PPN) were related to enhanced kidney cancer risk with stable results. Conclusion: Our findings identified that three blood cell indices (MCHC, RDW, and PDW) and two inflammation-related markers (SII and PPN) were independent risk factors for the incidence of kidney cancer. These indexes may serve as potential predictors for kidney cancer and aid in the development of targeted screening strategies for at-risk individuals. [ABSTRACT FROM AUTHOR]

Certain ocular conditions result from the non-physiological presence of intraocular particles, leading to visual impairment and potential long-term damage. This happens when the normally clear aqueous humor becomes less transparent, thus blocking the visual axis and by intraocular pressure elevation due to blockage of the trabecular meshwork, as seen in secondary open-angle glaucoma (SOAG). Some of these "particle-related pathologies" acquire ocular conditions like pigment dispersion syndrome, pseodoexfoliation and uveitis. Others are trauma-related, such as blood cell accumulation in hyphema. While medical and surgical treatments exist for SOAG, there is a notable absence of effective preventive measures. Consequently, the prevailing clinical approach predominantly adopts a "wait and see" strategy, wherein the focus lies on managing secondary complications and offers no treatment options for particulate matter disposal. We developed a new technique utilizing standing acoustic waves to trap and direct intraocular particles. By employing acoustic trapping at nodal regions and controlled movement of the acoustic transducer, we successfully directed these particles to specific locations within the angle. Here, we demonstrate control and movement of polystyrene (PS) particles to specific locations within an in vitro eye model, as well as blood cells in porcine eyes (ex vivo). The removal of particles from certain areas can facilitate the outflow of aqueous humor (AH) and help maintain optimal intraocular pressure (IOP) levels, resulting in a non-invasive tool for preventing secondary glaucoma. Furthermore, by controlling the location of trapped particles we can hasten the clearance of the AH and improve visual acuity and quality more effectively. This study represents a significant step towards the practical application of our technique in clinical use. [ABSTRACT FROM AUTHOR]

Glufosinate-ammonium (GLA), an organophosphate herbicide, is released at high concentrations in the environment, leading to concerns over its potential genotoxic effects. However, few articles are available in the literature reporting the possible cellular and nuclear effects of this compound. We assessed, by in vitro and in vivo micronucleus assays, the genotoxicity of GLA on cultured human lymphocytes and Lymnaea stagnalis hemocytes at six concentrations: 0.010 (the established acceptable daily intake value), 0.020, 0.050, 0.100, 0.200, and 0.500 µg/mL. In human lymphocytes, our results reveal a significant and concentration-dependent increase in micronuclei frequency at concentrations from 0.100 to 0.500 μg/mL, while in L. stagnalis hemocytes, significant differences were found at 0.200 and 0.500 μg/mL. A significant reduction in the proliferation index was observed at all tested concentrations, with the only exception of 0.010 μg/mL, indicating that the exposure to GLA could lead to increased cytotoxic effects. In L. stagnalis, a significant reduction in laid eggs and body growth was also observed at all concentrations. In conclusion, we provided evidence of the genomic and cellular damage induced by GLA on both cultured human lymphocytes and a model organism's hemocytes; in addition, we also demonstrated its effects on cell proliferation and reproductive health in L. stagnalis. [ABSTRACT FROM AUTHOR]

With a high mortality rate worldwide, Chronic Lymphocytic Leukemia (CLL) poses a serious health risk. Radiologists view the ability to detect blood tumor cells as both important and difficult. It is challenging for many of the current studies to properly examine these blood cancer cells. An intellectual system for separating healthy cells from tumor cells is suggested in this research work. This research primarily depends on the effective implementation of computed tomography (CT) samples of both benign and malignant blood samples. In this research paper, novel methodologies called public active contour pixel mapping and Deep Neural Eagle Perch Fuzzy Segmentation (DNEPFS) are proposed, which help in detecting the blood cell boundary of the unaffected cells. It is quite similar to an eagle using the claw searching algorithm. Here, the eagle uses its sharp claws to tear the prey's skin during the search for its food. 99.64% and 99.32% accuracy are obtained during the research work for benign and malignant nodules using MATLAB R2023a. The computational complexity was found to be less, which is 7.13 s. Different segmentation techniques are used for comparative analyses. According to the research results, this proposed flow is superior to all segmentation techniques currently in use for the accurate detection of tumor cells. Based on the expert's annotation on online samples and clinical samples, the methodology has been validated to prove their effectiveness and throw light on the life of affected patients to resume normalcy – live long. The research work was tested in real-time clinical samples which deliver promising and encouraging results in leukemia detection procedures. [ABSTRACT FROM AUTHOR]

Cell morphology analysis is a crucial diagnostic tool for identifying blood diseases, including acute leukemia. However, the traditional analysis process is time-consuming and requires significant investment in labor and expertise from laboratory doctors. In recent years, deep learning-based automatic blood cell classification techniques have gained popularity. But acquiring image data and annotations in the medical field is often challenging and costly. With the increasing use of deep learning techniques in clinical practice, it has become vital to ensure both accuracy and high-quality annotations. To address these challenges, this paper proposes a blood cell classification method based on Masked Autoencoder (MAE) and active learning (AL), namely MAE4AL. This method utilizes the self-supervised loss of MAE and sample uncertainty to select the most valuable samples for labeling. A comprehensive comparison is conducted between our method and the state-of-the-art blood cell classification technique, which employed ResNeXt. Remarkably, our proposed approach achieves comparable classification performance to ResNeXt when utilizing only 20% of the labeled data. When employing half of the labeled data, our method achieves a classification accuracy of 96.36%, surpassing the ResNeXt model trained with 100% labeled data by 0.79%. • A blood cell classification method is proposed based on MAE and active learning. • Data distribution is extracted more comprehensively by MAE. • High-quality samples are selected through active learning strategy. • Superior and comparable classification performance over state-of-the-art method. [ABSTRACT FROM AUTHOR]

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance. [ABSTRACT FROM AUTHOR]

Background: Series of whole-body cryotherapy (WBC) among healthy and physically active individuals can potentially reduce inflammatory response, although exact mechanisms remain unclear. Methods: The impact of whole-body cryotherapy on inflammation modulators among 28 young males, categorized as non-training (NTR, N = 10), non-training with WBC (NTR-WBC, N = 10), and training with WBC (TR-WBC, N = 8), is investigated in this study. Over a period of eight weeks, NTR-WBC and TR-WBC subjects underwent 24 WBC treatments (−130 °C for 3 min, three times a week), examining changes in mRNA expressions of IL-1A, IL-6, IL-10, IFN-G, SIRT1, SIRT3, SOD2, GSS, and ICAM-1. Results: The received data indicate an acute inflammatory response to initial WBC (increased IL-1A, IL-6, and SIRT), with a greater effect in NTR-WBC. Subsequent sessions showed enhanced expressions of antioxidative genes in both WBC groups, particularly non-trained, suggesting improved oxidative stress adaptation. A notable decrease in ICAM-1 mRNA post-24 WBC treatments in NTR-WBC signifies a potential systemic anti-inflammatory effect. Conclusions: The findings of the study suggest that the combination of regular physical activity with WBC administered three times per week can potentially modulate inflammatory and antioxidant responses. This modulation is evidenced by changes in the expression of genes related to these processes. [ABSTRACT FROM AUTHOR]

In patients with COVID-19, broad panels of immune checkpoint molecules (ICPMs) and the purinergic signaling have not been studied in parallel. We aimed to perform in-depth immunophenotyping of major cell subsets present in human peripheral blood of COVID-19 patients and controls using PD1, TIM3, LAG3, TIGIT, and CD200R, as well as CD39, as markers for the purinergic signaling pathway. We studied 76 COVID-19 patients and 12 healthy controls using peripheral blood mononuclear cells on flow cytometry. Univariable and multivariable statistics were performed. All ICPMs studied were significantly overexpressed on different cell subsets of COVID-19 patients when compared with healthy controls. Elevated lactate dehydrogenase; C-reactive protein; age; and high expression of CD45+, CD39+CD45+, TIM3+CD39+CD4+CD45+, and TIM3+CD39+CD8+CD3+CD4+ cells were significantly associated with severe COVID-19. On multivariable analysis, however, only high expression of CD39+CD45+ (OR 51.4, 95% CI 1.5 to 1763) and TIM3+CD39+CD4+CD3+CD45+ (OR 22.6, 95% CI 1.8 to 277) cells was an independent predictor for severe COVID-19. In conclusion, numerous ICPMs are overexpressed in COVID-19 patients when compared with healthy controls, suggesting a pathophysiological role of these molecules in SARS-CoV-2 infection. However, only TIM3 in co-expression with CD39 remained as a significant independent prognostic ICPM on multivariable analysis. The flow cytometric evaluation of TIM3+CD39+CD4+CD3+CD45+, as well as CD39+CD45+, is a powerful tool for the prognostication of COVID-19 patients on hospital admission. [ABSTRACT FROM AUTHOR]

COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression. This study shows that severity of disease in COVID-19 patients is characterized by the preferential loss of TCF1+ progenitor T-cells needed to self-renew and replenish the immune system. [ABSTRACT FROM AUTHOR]

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic. [ABSTRACT FROM AUTHOR]

Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information from their producer cells to target cells. This communication can in turn affect both normal and pathological processes. Mounting evidence has revealed that dermal wound myofibroblasts (Wmyo) produce MVs, which can transfer biomolecules impacting receptor cells such as human dermal microvascular endothelial cells (HDMECs). While the effects of MVs on HDMECs are generally well described in the literature, little is known about the transport of MVs across the HDMEC barrier, and their potential effect on the barrier integrity remains unknown. Here, we investigated these roles of Wmyo‐derived MVs on two sub‐populations of HDMECs, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Using an in vitro model to mimic the endothelial barrier, we showed that MVs crossed the LEC barrier but not the BEC barrier. In addition, we demonstrated that MVs were able to influence the cell‐cell junctions of HDMECs. Specifically, we observed that after internalization via the predominantly caveolin‐dependent pathway, MVs induced the opening of junctions in BECs. Conversely, in LECs, MVs mainly use the macropinocytosis pathway and induce closure of these junctions. Moreover, proteins in the MV membrane were responsible for this effect, but not specifically those belonging to the VEGF family. Finally, we found that once the LEC barrier permeability was reduced by MV stimuli, MVs ceased to cross the barrier. Conversely, when the BEC barrier was rendered permeable following stimulation with MVs, they were subsequently able to cross the barrier via the paracellular pathway. Taken together, these results suggest that the study of Wmyo‐derived MVs offers valuable insights into their interaction with the HDMEC barrier in the context of wound healing. They highlight the potential significance of these MVs in the overall process. [ABSTRACT FROM AUTHOR]

Background: Progressive decline of allograft function leads to premature graft loss. Forkhead box P3 (FOXP3), a characteristic gene of T-regulatory cells, is known to be essential for auto-antigen tolerance. We assessed the hypothesis that low FOXP3 mRNA splice variant levels in peripheral blood cells early after transplantation are associated with progressive allograft injury. Methods: Blood samples were prospectively collected from 333 incident kidney transplant recipients on the first and 29th postoperative day. We used quantitative polymerase chain reaction to determine transcripts of 3 isotypes of FOXP3 splice variants, including pre-mature FOXP3 and full length FOXP3 (FOXP3fl). We investigated the association between FOXP3 splice variant levels and the declines in estimated glomerular filtration rate (eGFR) of more than 5ml/min/1.73m2 within the first-year post-transplant using logistic regression. Results: We observed lower FOXP3fl levels in recipients with declining eGFR (N = 132) than in recipients with stable eGFR (N = 201), (logarithmic value -4.13 [IQR -4.50 to -3.84] vs -4.00 [4.32 to -3.74], p=0.02). In ad hoc analysis pre-transplant FOXP3fl levels were similar in both groups. The association between FOXP3fl and declining eGFR was confirmed by multivariable analysis adjusted for potential confounding factors (Odds Ratio 0.51, 95% confidence interval 0.28 to 0.91: p=0.02). When stratifying FOXP3fl levels into quartiles, recipients with lower day1 FOXP3fl had the highest rate of declining eGFR (p=0.04). Conclusion: Low FOXP3fl splice variant levels at the first postoperative day in kidney transplant recipients were associated with severe decline of eGFR, a wellknown surrogate for hard endpoints. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis. [ABSTRACT FROM AUTHOR]

Background: The rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called "gating", and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data. Results: We applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations. Conclusions: CITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation. [ABSTRACT FROM AUTHOR]

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors. [ABSTRACT FROM AUTHOR]

Objective: To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. Methods: Participants with chikungunya arthritis were recruited from Colombia from 2019–2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. Results: Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). Conclusion: Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy. [ABSTRACT FROM AUTHOR]

A traumatic hemorrhage is fatal due to the great loss of blood in a short period of time; however, there are a few biomaterials that can stop the bleeding quickly due to the limited water absorption speed. Here, a highly absorbent polymer (HPA), polyacrylate, was prepared as it has the best structure–effectiveness relationship. Within a very short period of time (2 min), HPA continually absorbed water until it swelled up to its 600 times its weight; more importantly, the porous structure comprised the swollen dressing. This instantaneous swelling immediately led to rapid hemostasis in irregular wounds. We optimized the HPA preparation process to obtain a rapidly water-absorbent polymer (i.e., HPA-5). HPA-5 showed favorable adhesion and biocompatibility in vitro. A rat femoral arteriovenous complete shear model and a tail arteriovenous injury model were established. HPA exhibited excellent hemostatic capability with little blood loss and short hemostatic time compared with CeloxTM in both of the models. The hemostatic mechanisms of HPA consist of fast clotting by aggregating blood cells, activating platelets, and accelerating the coagulation pathway via water absorption and electrostatic interaction. HPA is a promising highly water-absorbent hemostatic dressing for rapid and extensive blood clotting after vessel injury. [ABSTRACT FROM AUTHOR]

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods. Rolandelli et al. define how immune cells from the tick Ixodes scapularis are affected by infection and show that this cell population have distinct signatures and functions in immunity, metabolism and proliferation. [ABSTRACT FROM AUTHOR]

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vd2+ T cells, while Vd1+ T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vd1+ and Vd2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-g, TNF-a and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies. [ABSTRACT FROM AUTHOR]

Cellular immunotherapy, particularly CAR-T cells, has shown potential in the improvement of outcomes in patients with refractory and recurrent malignancies of the blood. However, achieving sustainable long-term complete remission for blood cancer remains a challenge, with resistance and relapse being expected outcomes for many patients. Although many studies have attempted to clarify the mechanisms of CAR-T cell therapy failure, the mechanism remains unclear. In this article, we discuss and describe the current state of knowledge regarding these factors, which include elements that influence the CAR-T cell, cancer cells as a whole, and the microenvironment surrounding the tumor. In addition, we propose prospective approaches to overcome these obstacles in an effort to decrease recurrence rates and extend patient survival subsequent to CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

Circulating endothelial cells (CEC) are arising as biomarkers for vascular diseases. However, whether they can be utilized as markers of endothelial cell (EC) senescence in vivo remains unknown. Here, we present a protocol to isolate circulating endothelial cells for a characterization of their senescent signature. Further, we characterize different models of EC senescence induction in vitro and show similar patterns of senescence being upregulated in CECs of aged patients as compared to young volunteers. Replication-(ageing), etoposide-(DNA damage) and angiotensin II-(ROS) induced senescence models showed the expected cell morphology and proliferation-reduction effects. Expression of senescence-associated secretory phenotype markers was specifically upregulated in replication-induced EC senescence. All models showed reduced telomere lengths and induction of the INK4a/ARF locus. Additional p14ARF-p21 pathway activation was observed in replication- and etoposide-induced EC senescence. Next, we established a combined magnetic activated- and fluorescence activated cell sorting (MACS-FACS) based protocol for CEC isolation. Interestingly, CECs isolated from aged volunteers showed similar senescence marker patterns as replication- and etoposide-induced senescence models. Here, we provide first proof of senescence in human blood derived circulating endothelial cells. These results hint towards an exciting future of using CECs as mirror cells for in vivo endothelial cell senescence, of particular interest in the context of endothelial dysfunction and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Background: Neutrophil extracellular traps (NETs) play a key role in thrombus formation in patients with coronavirus disease 2019 (COVID-19). However, the existing detection and observation methods for NETs are limited in their ability to provide quantitative, convenient, and accurate descriptions of in situ NETs. Therefore, establishing a quantitative description of the relationship between NETs and thrombosis remains a challenge. Objective: We employed morphological observations of blood cells and statistical analyses to investigate the correlation between the NETs/neutrophilic segmented granulocyte ratio and mortality risk in patients with COVID-19. Methods: Peripheral blood samples were collected from 117 hospitalized patients with COVID-19 between November 2022 and February 2023, and various blood cell parameters were measured. Two types of smudge cells were observed in the blood and counted: lymphatic and neutral smudge cells. Statistical data analysis was used to establish COVID-19 mortality risk assessment indicators. Results: Morphological observations of neutrophilic smudge cells revealed swelling, eruption, and NETs formation in the neutrophil nuclei. Subsequently, the NETs/neutrophilic segmented granulocyte ratio (NNSR) was calculated. A high concentration of NETs poses a fatal risk for thrombus formation in patients. Statistical analysis indicated that a high NNSR was more suitable for evaluating the risk of death in patients with COVID-19 compared to elevated fibrinogen (FIB) and D-dimer (DD) levels. Conclusion: Observing blood cell morphology is an effective method for the detection of NETs, NNSR are important markers for revealing the mortality risk of patients with COVID-19. [ABSTRACT FROM AUTHOR]

Fluorescent graphitic carbon nitride (g-C3N4) doped with various heteroatoms, such as B, P, and S, named Bg-C3N4, Pg-C3N4, and Sg-C3N4, were synthesized with variable band-gap values as diagnostic materials. Furthermore, they were embedded within hyaluronic acid (HA) microgels as g-C3N4@HA microgel composites. The g-C3N4@HA microgels had a 0.5–20 μm size range that is suitable for intravenous administration. Bare g-C3N4 showed excellent fluorescence ability with 360 nm excitation wavelength and 410–460 emission wavelengths for possible cell imaging application of g-C3N4@HA microgel composites as diagnostic agents. The g-C3N4@HA-based microgels were non-hemolytic, and no clotting effects on blood cells or cell toxicity on fibroblasts were observed at 1000 μg/mL concentration. In addition, approximately 70% cell viability for SKMEL-30 melanoma cells was seen with Sg-C3N4 and its HA microgel composites. The prepared g-C3N4@HA and Sg-C3N4@HA microgels were used in cell imaging because of their excellent penetration capability for healthy fibroblasts. Furthermore, g-C3N4-based materials did not interact with malignant cells, but their HA microgel composites had significant penetration capability linked to the binding function of HA with the cancerous cells. Flow cytometry analysis revealed that g-C3N4 and g-C3N4@HA microgel composites did not interfere with the viability of healthy fibroblast cells and provided fluorescence imaging without any staining while significantly decreasing the viability of cancerous cells. Overall, heteroatom-doped g-C3N4@HA microgel composites, especially Sg-C3N4@HA microgels, can be safely used as multifunctional theragnostic agents for both diagnostic as well as target and treatment purposes in cancer therapy because of their fluorescent nature. [ABSTRACT FROM AUTHOR]

Juvenile primary Sjögren syndrome (pSS) with renal involvement is extremely rare, reported approximately in 50 children, predominantly girls. Here, we present the first reported case of a male child with juvenile pSS with ocular surface disease (previously keratoconjunctivitis sicca), submandibular salivary gland involvement, and tubulointerstitial nephritis. First, two symptoms were clinically apparent at presentation. We illustrate here that kidney involvement in pSS should be actively looked for, as juvenile pSS may be associated with asymptomatic renal involvement. Immunophenotyping of peripheral blood cells using multicolor flow cytometry revealed at the time of diagnosis changes in both adaptive (T memory cells and B memory cells), and innate immunity (an increased activation of natural killer cells, as well as monocytes and neutrophils, and an increased representation of intermediate monocytes). Our case report points to the importance of kidney examination, early diagnosis and therapy in juvenile pSS, as well as highlights international collaboration to obtain more data for this rare disease. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) are somatic stem cells that continuously generate lifelong supply of blood cells through a balance of symmetric and asymmetric divisions. It is well established that the HSC pool increases with age. However, not much is known about the underlying cause for these observed changes. Here, using a novel method combining single-cell ex vivo HSC expansion with mathematical modeling, we quantify HSC division types (stem cell—stem cell (S-S) division, stem cell—progenitor cell (S-P) division, and progenitor cell—progenitor cell (P-P) division) as a function of the aging process. Our time-series experiments reveal how changes in these three modes of division can explain the increase in HSC numbers with age. Contrary to the popular notion that HSCs divide predominantly through S-P divisions, we show that S-S divisions are predominant throughout the lifespan of the animal, thereby expanding the HSC pool. We, therefore, provide a novel mathematical model-based experimental validation for reflecting HSC dynamics in vivo. [ABSTRACT FROM AUTHOR]

The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here, we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. While the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest that both single-lineage and multi- lineage contributions by HSCs collectively drive continuous hematopoiesis, and highlight a close relationship of myeloid and lymphoid development. [ABSTRACT FROM AUTHOR]