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Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al.1.

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD).New methodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation.ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers.Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

AbstractTo overcome the challenges of a manual leukocyte differential count, we have developed FlowDiff, an 8-colour, single tube flow cytometry panel, and investigated whether it could potentially replace the manual differential in our laboratory. The instrument was set up in accordance with the EuroFlow settings, and the protocol comprised a stain-lyse no wash process, taking approximately 30 min of working time, without the addition of a toxic lysis reagent. We found a very good correlation for all leukocyte populations between FlowDiff and the Sysmex XN analyzer in 80 normal, non-flagged samples. In addition, FlowDiff showed a very good correlation with manual differential in 168 abnormal samples, as well as a high diagnostic accuracy. FlowDiff correctly identified all samples with acute leukemia (N = 13) and differentiated all B-lymphomas (N = 49) in samples with lymphocytosis. Moreover, FlowDiff detected an additional five samples with B-lymphocytosis without any prior hematological malignancy, which turned out to be a B-lymphoma. Our data suggest that FlowDiff, our 8-colour flow cytometry-based differential, is comparable to, and can successfully substitute the manual differential. [ABSTRACT FROM AUTHOR]

Background: Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Methods: Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase–activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. Results: CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. Conclusions: These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Aim Methods Results Conclusions As traditional Chinese medicinal materials, E'jiao and cubilose are rich in various bioactive substances with good antioxidant, anti‐inflammatory, and immune‐regulating effects.To obtain the optimal ratio of synergistic effect between E'jiao and cubilose.The antioxidant capacity of E'jiao and cubilose digestive fluid was evaluated in vitro, as well as the intracellular oxidation balance between HSF cells and 3D whole‐skin model induced by H2O2.E'jiao, cubilose, and their different ratios of composites had better scavenging ability against free radicals such as DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl). Using HSF cells induced by H2O2 as an oxidative damage model, it was found that the combination of E'jiao and cubilose in a ratio of 2:3 significantly enhanced the activities of SOD, GSH‐PX, and CAT enzymes compared to the separate treatments of E'jiao and cubilose, as well as other combination ratios. It effectively reduced the accumulation of reactive oxygen species caused by H2O2 in HSF (Human Skin Fibroblast) cells and protected the integrity of the cells. Further analysis using flow cytometry and a 3D full‐thickness skin model revealed that the combination ratio of 2:3 increased the proportion of H2O2‐treated cells in the S%+G2% phase from 19.1% + 7.4%–22.1% + 28.8%, helping oxidatively damaged cells partially recover their proliferative capacity. It also promoted the expression of collagen I and collagen IV in the 3D full‐thickness skin model, with improvement rates reaching 168.00% and 123.68%, respectively.These findings indicate that the combination of E'jiao and cubilose in a ratio of 2:3 exhibits good synergistic effects, enhancing the ability of cells to resist oxidative damage, promoting cellular renewal and metabolism, and improving skin antiwrinkle capacity. [ABSTRACT FROM AUTHOR]

The unique heterogenous landscape of myelodysplastic syndromes/neoplasms (MDS) has resulted in continuous redefinition of disease sub-entities, in view of the novel translational research data that have clarified several areas of the pathogenesis and the progression of the disease. The new international classifications (WHO 2022, ICC 2022) have incorporated genomic data defining phenotypical alterations, that guide clinical management of specific patient subgroups. On the other hand, for over a decade, multiparameter flow cytometry (MFC) has proven its value as a complementary diagnostic tool for these diseases and although it has never been established as a mandatory test for the baseline evaluation of MDS patients in international guidelines, it is almost universally adopted in everyday clinical practice for the assessment of suspected cytopenias through simplified scoring systems or elaborate analytical strategies for the detection of immunophenotypical dysplastic features in every hematopoietic cell lineage in the bone marrow (BM). In this review, we explore the clinically meaningful interplay of MFC data and genetic profiles of MDS patients, to reveal the currently existing and the potential future role of each methodology for routine clinical practice, and the benefit of the patients. We reviewed the existing knowledge and recent advances in the field and discuss how an integrated approach could lead to patient re-stratification and guide personalized management. [ABSTRACT FROM AUTHOR]

Background: IFN-γ+CD4+ cells (type 1 helper T cells, Th1) represent a critical component of the inflammatory environment in the lungs of chronic obstructive pulmonary disease (COPD). Identifying influencing factors related to COPD-associated Th1 cells will enhance our understanding of the inflammatory mechanisms involved and facilitate the development of targeted interventions. Method: We describe T-cell immunoglobulin and mucin-domain containing-3 (Tim3) as a key gene regulating COPD-associated Th1 cells through single-cell sequencing, flow cytometry and knockout mice. Results: Our findings indicate that Havcr2 expression gradually increases during CD4+ T cell activation in COPD mice, with Tim3 being highly expressed on both CD4+ T cells and Th1 cells. Notably, the knockout of HAVCR2 further promotes the infiltration of CD4+ T cells and the expression of IFN-γ in the lungs, resulting in a more severe emphysema phenotype, although it does not significantly affect TNF-α expression. Additionally, NFIL3, an upstream regulator of Tim3, is also highly expressed in the CD4+ T cells of COPD mice. Mice with NFIL3 knockout exhibit phenotypes similar to those of HAVCR2 knockout mice, along with a significant downregulation of Tim3 expression. In vitro , we simulated the activation process by polarizing primary CD4+ Tn cells from COPD mice and observed that NFIL3/Tim3 expression was significantly upregulated following Th1 polarization. Conclusion: Our study demonstrates that the NFIL3/Tim3 axis plays a role in Th1 imbalance in the lungs of COPD by inhibiting Th1 differentiation. [ABSTRACT FROM AUTHOR]

Evidence supports that hyaluronic acid (HA) can promote tissue regeneration and reduce inflammation. This study aimed to assess the effects of a bilayered cellulose-coated HA scaffold on oral wound healing. A film-type 3% HA scaffold with bilayer cellulose coating was prepared and compared with an HA scaffold without coating. To evaluate cytocompatibility, human gingival fibroblasts were exposed to both scaffolds, and cell viability, flow cytometry, and scratch wound assays were performed. In addition, in vivo and ex vivo wound-healing assays were performed. Cytocompatibility tests showed no cytotoxicity for either HA scaffold. The scratch wound assay revealed a significant reduction in the open wound area in both HA scaffolds compared with that in the control (p < 0.05); however, no differences were observed between the scaffolds with and without cellulose coating. In vivo wound healing analysis showed significantly higher healing rates on day 3 in the HA scaffolds than in the control (p < 0.05), with no significant differences between the scaffolds. HA scaffolds with coating showed lower CD68 and higher vimentin expression than the control (p < 0.05), whereas HA scaffolds without coating did not. Ex vivo wound healing analysis revealed significantly higher re-epithelialization rates in both scaffolds than in the control (p < 0.05). Within the limits of this study, the HA scaffold with coating showed enhanced wound healing efficacy, indicating its potential for oral wound healing applications. [ABSTRACT FROM AUTHOR]

Research on central nervous system tumors (CNSTs) has a significant impact on the diagnosis and prognosis of patients. Currently, CNSTs are classified according to the schema proposed by the World Health Organization (WHO), which considers clinical, histopathological, and molecular characteristics, highlighting the importance of tumor biology for accurate diagnosis and optimal treatment approaches. Despite these advances, assessing DNA ploidy—a marker of tumor aggressiveness—remains complex in CNSTs. This review investigates the utility of DNA index (DNAi) and DNA ploidy analysis by flow cytometry in diagnosing CNSTs and prognosing their outcomes. We systematically reviewed studies in the PubMed database from 1990 to the present using the keywords "DNA Index", "Brain", "Flow cytometry", and "Ploidy". We identified 151 studies, 36 of which met our inclusion criteria. We found considerable variation in sample sizes and methodological variation across the studies. Discrepancies between the reported DNAi and ploidy values were observed. Aneuploidy is generally associated with more aggressive tumors, although exceptions exist. Higher DNAi levels correlate with increased malignancy, notably in glioblastomas, astrocytomas, and meningiomas, whereas diploid astrocytomas and oligodendrogliomas are associated with shorter survival rates. DNA ploidy assessment via flow cytometry could predict CNST behavior, yet methodological issues with tissue selection, adequate control samples, and technique variability remain. DNAi and ploidy assessments show promise as prognostic markers in CNSTs. However, the standardization of flow cytometry protocols and alignment with the current WHO classification schema are essential steps to integrate ploidy analysis in routine CNST assessment. [ABSTRACT FROM AUTHOR]

Gaultheria Kalm ex L. (Ericaceae), a type of evergreen shrub, known as a natural source of methyl salicylate, possesses rich germplasm resources, strong habitat adaptability, significant ornamental value, and noteworthy pharmacological activities. However, due to the paucity of whole genomic information, genetically deep research in these areas remains limited. Consequently, we intend to obtain genome data through high-throughput sequencing, gene annotation, flow cytometry, transcription factors prediction and genetic marker analysis for a representative species of this genus, with Gaultheria prostrata selected for our study. In this study, we preliminarily obtained the genome of G. prostrata through next-generation sequencing methods. Utilizing 47.94 Gb of high-quality sequence data (108.95× coverage), assembled into 114,436 scaffolds, with an N50 length of 33,667 bp. The genome size assembled by SOAPdenovo, approximately 417 Mb, corresponded closely to predictions by flow cytometry (440 Mb) and k -mer analysis (447 Mb). The genome integrity was evaluated using BUSCO with 91%. The heterozygosity ratio was 0.159%, the GC content was 38.85%, and the repetitive regions encompassed over 34.6% of the genome. A total of 26,497 protein-coding genes have been predicted and annotated across Nr, Swissprot, GO, KEGG, and Pfam databases. Among these, 14,377 and 2,387 genes received functional annotation in Nr and Swissprot, respectively; 21,895, 24,424, and 22,330 genes were similarly annotated in GO, KEGG, and Pfam. Moreover, A total of 279,785 SSRs were identified and 345,270 primers for these SSRs were designed. Within the various nucleotide types of SSRs, AG/CT and AAG/CTT constituted the predominant dinucleotide and trinucleotide repeat types in G. prostrata. In addition, 1,395 transcription factors (TFs) from 75 TF families, 462 transcription regulators (TRs) from 33 TR families and 840 protein kinase (PKs) from 118 PK families were identified in this genome. We also performed phylogenetic analyses of G. prostrata and related species, including estimation of divergence times and expansion and contraction analyses, followed by positive selection analyses of orthologous gene pairs of G. prostrata and its close relative Vaccinium corymbosum. These results provide a reference for in-depth study of genus Gaultheria , contributing to future functional and comparative genomics analyses and providing supporting data for the development of molecular markers. [ABSTRACT FROM AUTHOR]

The plate counting method is a traditional and widely accepted technique for live cell counting, often employed for Bacillus enumeration and spore forming rate calculations. However, this method requires at least 12 h to generate results, making it unsuitable for real-time monitoring of bacterial growth status and spore transformation rate. Bacillus thuringiensis crystals, produced during sporulation, are widely used as microbial pesticides, with high demand for industrial scale production. Variations in cultivation conditions and harvest timing during large-scale pore production of Bacillus thuringiensis significantly affect spore forming rate, impacting crystallization yield. Nevertheless, there is a lack of real-time monitoring methods for spore conversion rate. Flow cytometry (FCM), a well-established technique for single-cell analysis in eukaryotic cells, has been successfully applied in bacterial detection in environmental and food samples. In this study, we introduced a rapid flow cytometry-based method for determining spore forming rate of Bacillus thuringiensis , with two nucleic acid dyes, SYTO24 and LDS751. The method enables dynamic monitoring of spore, vegetative cell, and viable but non-culturable/dead cell proportions during the whole cultivation process, and spore forming rate could be gained within 30 min. Data of spore forming rate by FCM method is consistent with that by plate counting method, offering a faster and more efficient approach for assessing sporulation status in industrial Bacillus thuringiensis microbial pesticide production. [ABSTRACT FROM AUTHOR]

Introduction: Single-cell RNA sequencing (scRNAseq) and flow cytometry studies in skin are methodologically complex and costly, limiting their accessibility to researchers worldwide. Ideally, RNA and protein-based analyses should be performed on the same lesion to obtain more comprehensive data. However, current protocols generally focus on either scRNAseq or flow cytometry of healthy skin. Methods: We present a novel label-free sample multiplexing strategy, building on the souporcell algorithm, which enables scRNAseq analysis of paired blood and skin samples. Additionally, we provide detailed instructions for simultaneous flow cytometry analysis from the same sample, with necessary adaptations for both healthy and inflamed skin specimens. Results: This tissue multiplexing strategy mitigates technical batch effects and reduces costs by 2-4 times compared to existing protocols. We also demonstrate the effects of varying enzymatic incubation durations (1, 3, and 16 hours, with and without enzyme P) on flow cytometry outcomes. Comprehensive explanations of bioinformatic demultiplexing steps and a detailed step-by-step protocol of the entire experimental procedure are included. Discussion: The protocol outlined in this article will make scRNAseq and flow cytometry analysis of skin samples more accessible to researchers, especially those new to these techniques. [ABSTRACT FROM AUTHOR]

Self-assembling DNA nanoparticles have the potential to significantly advance the targeted delivery of molecular cargo owing to their chemical and architectural flexibility. Recently, it has been demonstrated that the genetic code embedded in DNA nanoparticles produced by the method of DNA origami or related techniques can be recognized and copied by RNA polymerase in vitro. Further, sculpted DNA nanoparticles can serve as a substrate for Cas9-mediated gene modification and gene expression in cell culture. In the present study, we further investigate the ability of DNA origami nanoparticles to be expressed in a human cell line with emphasis on the impact of single-stranded DNA (ssDNA) domains and the contributions of the architectural disposition of genetic control elements, namely promoter and enhancer sequences. Our findings suggest that while cells possess the remarkable capability to express genes within highly folded architectures, the presence and relative density and location of ssDNA domains appears to influence overall levels of gene expression. These results suggest that it may be possible to nuance folded DNA nanoparticle architecture to regulate the rate and/or level of gene expression. Considering the highly malleable architecture and chemistry of self-assembling DNA nanoparticles, these findings motivate further exploration of their potential as an economic nanotechnology platform for targeted gene editing, nucleic acid-based vaccines, and related biotherapeutic applications. [ABSTRACT FROM AUTHOR]

Introduction Methods Results Conclusion Activation‐induced cytidine deaminase (AID) deficiency is a rare autosomal recessive inborn error of immunity (IEI) characterized by increased susceptibility to infections, autoimmunity, and/or autoinflammation. AID plays an important role in immunoglobulin class switching and somatic hypermutation. AID deficiency patients have very low or absent levels of IgG, IgA, and IgE, while IgM level is elevated. The disease is designated as type 2 hyperimmunoglobulin M syndrome (HIGM‐2). To date, around 130 patients with HIGM‐2 have been reported, none from Egypt.Four patients from three different consanguineous families with elevated serum IgM and low IgG and IgA were included in the study. After the exclusion of CD40 and/or CD40L deficiency by flow cytometry, patients’ samples were tested by a panel covering 452 genes (four bases PID‐Pro) on the Illumina Miseq platform.All patients suffered repeated infections since childhood. Patients 1–3 had inflammatory bowel disease‐like (IBD‐like) symptoms, while patient 4 did not have autoimmune manifestations. Patient 1 is the first HIGM‐2 patient to be reported to have renal amyloidosis as part of the autoinflammation. Patients 1–3 had the same pathogenic variant (NM_020661.4 (AID):c.406del, p.Ile136Ter), while patient 4 had another pathogenic variant (NM_020661.4 (AID):c.374G > A, p.Gly125Glu). The variant p.Ile136Ter was not reported before in any of the documented HIGM‐2 patients.HIGM‐2 is a rare IEI that can be overlooked; hence, patients’ diagnosis is delayed. Autoimmune and autoinflammatory manifestations develop later in the disease course leading to significant morbidities. The diagnosis can be suspected after exclusion of CD40/CD40L deficiencies by flow cytometry, and the diagnosis can be confirmed by genetic testing. [ABSTRACT FROM AUTHOR]

Background Study Design and Methods Results Discussion Flow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post‐thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery.Apheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%‐Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%.Fifty‐two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre‐cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%.Cryopreserved samples can be reliably prepared for flow cytometric testing using a step‐wise dilution to preserve cell integrity and robust recoveries. [ABSTRACT FROM AUTHOR]

Heterogeneous particles co-focusing to a single stream is a vital prerequisite for cell counting and enumeration, playing an essential role in flow cytometry and single-cell analysis. Microfluidics-based inertial focusing holds great research prospects due to its simplicity of devices, ease of operation, high throughput, and freedom from external fields. Combining microfluidic channels with two or more different geometries has become a powerful tool for high-efficiency particle focusing. Here, we explored hybrid microfluidic channels for heterogeneous particle co-focusing. Four different annular channels with obstacles distributed on the inner wall were constructed and simulated, obtaining constantly variable secondary flows. Then we used four different fluorescent particles with the size of 10 μm, 12 μm 15 μm, and 20 μm as well as their mixture to perform the inertial focusing experiments of multi-sized particles. Theoretical simulation and experimental results demonstrated a focusing efficiency of >99%. Finally, we further utilized human white blood cells to estimate the co-focusing performance of our hybrid microfluidic channel, resulting in a high focusing efficiency of >92% and a high throughput of ≈8000 cell s−1. The hybrid microfluidic channels, capable of high-precision heterogeneous particle co-focusing, could pave a broad avenue for microfluidic flow cytometry and single-cell analysis. [ABSTRACT FROM AUTHOR]

Background: The objective was to evaluate the level of neutrophil extracellular traps (NETs) in patients with chronic kidney disease (CKD) and periodontitis, and to explore the relationship between NETs and both diseases. Methods: 63 CKD and 40 non-CKD participants were recruited and underwent periodontal examination, among which 35 early CKD patients underwent periodontal therapy. The concentrations of NETs were determined by dsDNA assay in gingival crevicular fluid (GCF) and plasma, and by flow cytometry or immunofluorescence assay in blood and gingival tissues. The correlations between NETs and clinical parameters were analyzed. The influence of periodontal therapy on periodontitis, CKD and NETs concentrations was also evaluated. Results: CKD patients had higher concentrations of NETs in plasma than non-CKD patients, and NETs concentrations were also increased in both GCF and plasma of patients with periodontitis than that of periodontally healthy patients. NETs concentrations were positively correlated with increased clinical parameters of CKD and periodontitis. The positive correlation between CKD and periodontitis was demonstrated. Moreover, periodontal therapy ameliorated periodontitis and CKD, and reduced NETs concentrations in GCF of patients. Conclusions: This study revealed that NETs might be a possible bridge between periodontitis and CKD, and suggested the potential target for therapy. [ABSTRACT FROM AUTHOR]

Objective: Cysteamine, a drug approved to treat cystinosis, has been proposed as a host-directed therapy for M. tuberculosis (Mtb) and SARS-CoV-2. The impact of cysteamine on the immune responses has not been fully investigated. We aimed to in vitro evaluate the immunomodulatory effects of cysteamine on peripheral blood mononuclear cells (PBMCs) using the purified protein derivative (PPD) as a recall antigen, and an unspecific stimulus as staphylococcal enterotoxin B (SEB). Methods: PBMCs isolated from subjects with tuberculosis infection (TBI), those with tuberculosis disease (TB), and healthy controls (HC) were in vitro stimulated with PPD or SEB and treated or not with cysteamine at different concentrations (50 µM–400 µM) for 6 hours (h) and 24 h. We evaluated the T helper1 (Th1) and T cytotoxic1 (Tc1) cell cytokine production by flow cytometry and immune-enzymatic assays. In HC, we also evaluated apoptosis and/or necrosis by flow cytometry. Results: We observed an immunomodulatory effect of cysteamine at 400 µM in PBMCs from TB and TBI subjects. It significantly reduced PPD-specific Th1 responses at 24 h and at 6 h (p=0.0004 and p=0.0009, respectively), and a similar non-significant trend was observed with cysteamine at 200 µM (p=0.06 at 24 h and p=0.14 at 6 h). Moreover, cysteamine at both 400 µM (p<0.0001 and p=0.0187 at 24 h, respectively, and p<0.0001 at 6 h for both) and 200 µM (p=0.0119 and p=0.0028 at 24 h and p=0.0028 and p=0.0003 at 6 h, respectively) significantly reduced SEB-induced Th1 and Tc1 responses. Furthermore, we found that cysteamine induced morphological lymphocyte changes and significantly reduced the lymphocyte percentage in a dose- and time-dependent manner. Cysteamine at 400 µM induced 8% late apoptosis and 1.6% necrosis (p<0.05) at 24 h. In contrast, despite significant differences from untreated conditions (p<0.05), cysteamine at 400 µM for 6 h induced approximately 1% late apoptosis and 0.1% necrosis in the cells. Conclusions: High doses of cysteamine in vitro reduce the percentages of PPD- and SEB-induced Th1 and Tc1 cells and induce late apoptosis and necrosis. Differently, cysteamine at lower doses retains the immunomodulatory effect without affecting cell viability. These findings suggest cysteamine as a potential adjunct to antimicrobial regimens as in the TB or COVID-19 field, for its ability to reduce the inflammatory status. [ABSTRACT FROM AUTHOR]

Background: Breast cancer seriously affects physical and mental health of women. Despite advances in the clinical use of different treatments, breast cancer remains a major cause of mortality. Therefore, it is imperative to identify promising treatment options. In the present study, we investigated the effects of shikonin on 4T1 breast cancer cells and its potential mechanisms of action. Methods: BALB/c-derived mouse breast cancer 4T1 is very close to human breast cancer in growth characteristics and systemic response, so 4T1 cells were selected for further experiments. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial activity, and cellular calreticulin (CRT) exposure were assessed to evaluate the antitumor effects and mechanisms of shikonin in vitro. Orthotopic tumor growth inhibition and splenic immune cell regulation by shikonin were evaluated in 4T1 breast cancer orthotopic mice in vivo. Results: In vitro, shikonin could inhibit cell proliferation, cause apoptosis, disrupt mitochondrial activity, and induce ROS production and CRT exposure. In vivo, shikonin inhibited tumor growth, increased the proportion of CD8+ T cells, and reduced the proportion of regulatory cells (CD25+ Foxp3+ T cells) in the spleen. Conclusions: Shikonin inhibits the growth of 4T1 breast cancer cells by disrupting mitochondrial activity, promoting oxidative stress, and regulating immune function. [ABSTRACT FROM AUTHOR]

MBL is a precursor condition to chronic lymphocytic leukemia (CLL), characterized by monoclonal B-cells in blood. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis that include gains, losses, and copy-neutral loss-of-heterozygosity of large DNA segments. Both MBL and mCAs have been found to increase the risk of CLL and lymphoid malignancies, and the aim of our study was to investigate how mCAs relate to MBL, which is currently unknown. We analyzed genetic, flow cytometric, and hematologic data from 4632 individuals from the Mayo Clinic Biobank and CLL Database. MBL was detected using flow cytometry and classified as high-count (HC) or low-count (LC) MBL based on clone size. mCAs were detected primarily from whole blood DNA using sensitive SNP-array-based analyses. mCAs commonly altered in CLL (deletion of 6q, 11q, 13q, 17p, and trisomy 12) were specific (>99%) to individuals with MBL and CLL. HC-MBL and LC-MBL individuals were 881-fold and 8-fold, respectively, more likely to harbor CLL-associated mCAs than those without MBL. The cell fraction bearing these mCAs typically exceeded the B-cell fraction, suggesting their origin prior to the B-cell lineage. Integrating genetic and blood count data enabled detecting HC-MBL with high specificity in a biobank sample. These results quantify the contribution of mCAs to MBL and could enable large studies of HC-MBL without the need for flow cytometric screening. [ABSTRACT FROM AUTHOR]

Background: Epstein-Barr virus (EBV) specific T-cell response measurement can help adjust immunosuppression in transplant patients with persistent infections. We aim to define T-cell responses against EBV in a cohort of pediatric liver-transplant patients. Methods: Thirty-eight immunosuppressed pediatric liver-transplant patients (IP) and 25 EBV-seropositive healthy-adult controls (HC) were included in our cross-sectional study. Based on their EBV serological (S) and viral load (VL) status, patients were categorized into IP-SNEG, IP-SPOSVLNEG and IP-SPOSVLPOS groups. T-cell response was assessed at two timepoints by stimulating cells with EBV peptides (PepTivator®) and performing intracellular-cytokine and activation-induced marker staining. Background subtraction was used to determine EBV-specific T-lymphocyte frequency. Results: Polyfunctional CD8+ T cells indicated previous EBV contact (IP-SNEG 0.00% vs IP-SPOS 0.04% and HC 0.02%; p=0.001 and p=0.01, respectively). Polyfunctional CD8+CD107a+IFNɣ+IL2-TNFα- profile was increased in serology-positive (IP-SNEG 0.01% vs IP-SPOS 0.13% and HC 0.03%; p=0.01 and p=0.50, respectively) and viral-load positive (IP-SPOSVLPOS 0.43% vs IP-SPOSVLNEG 0.07% and HC 0.03%; p=0.03 and p=0.001, respectively) patients. Central-memory cells were increased among serology-positive adults (IP-SNEG 0.00% vs IP-SPOS 0.13% and HC 4.33%; p=0.58 and p=0.002, respectively). At the second timepoint, IP-SNEG patients remained negative (first visit 0.01% vs second visit 0.00%, p=0.44). On the other hand, IP-SPOSVLPOS patients had cleared viral loads and, subsequently, decreased polyfunctional CD8+CD107a+IFNɣ+IL2-TNFα- cells (first visit 0.43% vs second visit 0.10%, p=0.81). Conclusion: Polyfunctional CD8+ EBV-specific T-cell response allows detecting EBV previous contact in liver-transplant children. %CD8+CD107a+IFNɣ+IL2-TNFα- is increased in patients with positive viral loads. Central memory CD4+ T-cell population more effectively determines prior EBV-exposure in adults. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to profile the transcriptional profiles of peripheral blood mononuclear cells (PBMCs) and their immune repertoires affected by anti-synthetase syndrome (ASS) at the single-cell level. Methods: We performed single-cell RNA sequencing (scRNA-seq) analysis of PBMCs and bulk RNA sequencing for patients with ASS (N=3) and patients with anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5+ DM, N=3) along with healthy controls (HCs, N=4). As ASS and MDA5+ DM have similar organ involvements, MDA5+ DM was used as a disease control. The immune repertoire was constructed by reusing the same scRNA-seq datasets. Importantly, flow cytometry was performed to verify the results from the scRNA-seq analysis. Results: After meticulous annotation of PBMCs, we noticed a significant decrease in the proportion of mucosal-associated invariant T (MAIT) cells in ASS patients compared to HCs, while there was a notable increase in the proportion of proliferative NKT cells. Compared with MDA5+ DM patients, in their PBMCs ASS patients presented substantial enrichment of interferon pathways, which were primarily mediated by IFN-II, and displayed a weak immune response. Furthermore, ASS patients exhibited more pronounced metabolic abnormalities, which may in turn affect oxidative phosphorylation pathways. Monocytes from ASS patients appear to play a crucial role as receptive signaling cells for the TNF pathway. Immunophenotyping analysis of PBMCs from ASS patients revealed an increasing trend for the clone type CQQSYSTPWTF. Conclusion: Using single-cell genomic datasets of ASS PBMCs, we revealed a distinctive profile in the immune system of individuals with ASS, compared to that with MDA5+ DM or healthy controls. [ABSTRACT FROM AUTHOR]

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. Key points: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins [ABSTRACT FROM AUTHOR]

Background: In evolutionary biology, identifying and quantifying inter-lineage genome size variation and elucidating the underlying causes of that variation have long been goals. Repetitive elements (REs) have been proposed and confirmed as being among the most important contributors to genome size variation. However, the evolutionary implications of genome size variation and RE dynamics are not well understood. Results: A total of 35 Ensifera insects were collected from different areas in China, including nine species of crickets and 26 species of katydids. The genome sizes of seven species were then determined using flow cytometry. The RepeatExplorer2 pipeline was employed to retrieve the repeated sequences for each species, based on low-coverage (0.1 X) high-throughput Illumina unassembled short reads. The genome sizes of the 35 Ensifera insects exhibited a considerable degree of variation, ranging from 1.00 to 18.34 pg. This variation was more than 18-fold. Similarly, the RE abundances exhibited considerable variation, ranging from 13.66 to 61.16%. In addition, the Tettigonioidea had larger genomes and contained significantly more REs than did the Grylloidea genomes. Analysis of the correlation between RE abundance and the genome size of 35 Ensifera insects revealed that the abundance of REs, transposable elements (TEs), long terminal repeats (LTRs), and long interspersed nuclear elements (LINEs) are significantly correlated with genome size. Notably, there is an inflection point in this correlation, where species with increasingly large genomes (e.g., > 5–10 pg) have repeats that contribute less to genome expansion than expected. Furthermore, this study revealed contrasting evolutionary directions between the Tettigonioidea and Grylloidea clades in terms of the expansion of REs. Tettigonioidea species exhibit a gradual increase in ancestral genome size and RE abundance as they diverge, while Grylloidea species experience sustained genome contraction. Conclusions: This study reveals extensive variation in genome size and RE abundance in Ensifera insects, with distinct evolutionary patterns across two major groups, Tettigonioidea and Grylloidea. This provides valuable insights into the variation in genome size and RE abundance in Ensifera insects, offering a comprehensive understanding of their evolutionary history. [ABSTRACT FROM AUTHOR]

Bay-scale empirical evaluations of how bivalve aquaculture alters plankton composition, and subsequently ecological functioning and higher trophic levels, are lacking. Temporal, inter- and within-bay variation in hydrodynamic, environmental, and aquaculture pressure complicate plankton monitoring design to detect bay-scale changes and inform aquaculture ecosystem interactions. Here, we used flow cytometry to investigate spatio-temporal variations in bacteria and phytoplankton (< 20 μm) composition in four bivalve aquaculture embayments. We observed higher abundances of bacteria and phytoplankton in shallow embayments that experienced greater freshwater and nutrient inputs. Depleted nutrient conditions may have led to the dominance of picophytoplankton cells, which showed strong within-bay variation as a function of riverine vs marine influence and nutrient availability. Although environmental forcings appeared to be a strong driver of spatio-temporal trends, results showed that bivalve aquaculture may reduce near-lease phytoplankton abundance and favor bacterial growth. We discuss confounding environmental factors that must be accounted for when interpreting aquaculture effects such as grazing, benthic-pelagic coupling processes, and microbial biogeochemical cycling. Conclusions provide guidance on sampling considerations using flow cytometry in aquaculture sites based on embayment geomorphology and hydrodynamics. [ABSTRACT FROM AUTHOR]

Background: This study seeks to elucidate the expressions of lncRNA TSIX in Osteoarthritis (OA) and to explore its mechanisms in regulating OA progression. Methods: RT-qPCR was employed to analyze the expression of TSIX in OA patients classified by Kellgren-Lawrence (K-L) grades. Receiver operator characteristic (ROC) was conducted to evaluate the diagnostic value of TSIX. Correlation between TSIX levels and clinical scores such as Lysholm and visual analogue scale (VAS) score was evaluated using Pearson method. IL-1β-induced SW1353 cells served as an in vitro model. The cell function were assessed by flow cytometry and cell counting kit-8 (CCK-8) assay. The relationship between TSIX and miR-320a was verified by luciferase reporting system, while bioinformatics approaches were utilized to predict the downstream target genes of miR-320a. Results: The findings revealed that TSIX level in OA patients was elevated compared to that of the control group, with a notable progressive increase in TSIX expression correlated with higher K-L grades. In OA patients, the Lysholm score showed a negative correlation with TSIX expression, while the VAS score displayed a positive correlation with TSIX levels. Cell studies demonstrated that inhibition of TSIX enhanced cell viability and mitigated IL-1β-induced apoptosis by targeting miR-320a, in addition to promoting Aggrecan and Collagen II secretion. Luciferase reporter assay further validated the targeting interaction among TSIX, miR-320a, and PTEN. Conclusions: This study demonstrated an increased expression of TSIX in OA patients. It suggests that TSIX may play a role in chondrocyte dysfunction during OA by modulating the miR-320a/PTEN axis. [ABSTRACT FROM AUTHOR]

Group 2 Innate Lymphoid Cells (ILC2) are critical drivers of both innate and adaptive type 2 immune responses, known to orchestrate processes involved in tissue restoration and wound healing. In addition, ILC2 have been implicated in chronic inflammatory barrier disorders in type 2 immunopathologies such as allergic rhinitis and asthma. ILC2 in the context of allergen-driven airway inflammation have recently been shown to influence local and systemic metabolism, as well as being rich in lipid-storing organelles called lipid droplets. However, mechanisms of ILC2 lipid anabolism and catabolism remain largely unknown and the impact of these metabolic processes in regulating ILC2 phenotypes and effector functions has not been extensively characterized. ILC2 phenotypes and effector functions are shaped by their metabolic status, and determining the metabolic requirements of ILC2 is critical in understanding their role in type 2 immune responses and their associated pathophysiology. We detail here a novel experimental method of implementing flow cytometry for large scale analysis of fatty acid uptake, storage of neutral lipids, and fatty acid oxidation in primary murine ILC2 with complementary morphological analysis of lipid storage using confocal microscopy. By combining flow cytometry and confocal microscopy, we can identify the metabolic lipid requirements for ILC2 functions as well as characterize the phenotype of lipid storage in ILC2. Linking lipid metabolism pathways to ILC2 phenotypes and effector functions is critical for the assessment of novel pharmaceutical strategies to regulate ILC2 functions in type 2 immunopathologies. [ABSTRACT FROM AUTHOR]

Introduction: Thymus resident B cells were described more than 40 years ago. In early human life, these cells are found predominantly in the medulla and overwhelmingly display an unswitched IgM+ phenotype. The reactivity of thymic IgM B cells, however, is still unclear. Methods: Here, we generated 120 IgM-producing B cell clones from 3 separate thymus specimens obtained from infant, adolescent, and adult donors. Using flow cytometry and a unique high-dimensional ELISA platform, we investigated the clones' reactivity to apoptotic cells as well as to common chemical adducts exposed on modified amino acids and other macromolecules. Results: Regardless of the age, approximately 30-40% of thymic IgM B cells reacted to apoptotic cells. Further, 30-40% displayed reactivity to at least one adduct, including malondialdehyde, Homocysteine, and NEDD 8. Four distinct reactivity patterns were identified through this profiling. Notably, a significant association was observed between reactivity to apoptotic cells, and to one or more adducts, suggesting that the same determinants were recognized in both assays. Additionally, thymic IgM B cells reactive to adducts were more likely to recognize intra-nuclear or intra-cytoplasmic structures in Hep-2 cells as revealed by immunofluorescence staining. Conclusion/Discussion: Collectively, our findings suggest that thymic IgM B cells actively uptake apoptotic bodies and cellular debris in the medulla by binding specific chemical adducts. This mechanism could underpin their antigenpresenting function and further support their role in T-cell negative selection. [ABSTRACT FROM AUTHOR]

The incidence of primary hepatocellular carcinoma (HCC) has recently ranked fifth in the world, and the incidence rate is increasing year by year worldwide. Therefore, early diagnosis is the highest priority in the treatment of HCC. In this paper, four anti-HCC aptamers were obtained using magnetic bead SELEX technology. Among them, Apt-1 had the smallest Kd value(5.9 nM) and the highest affinity. Flow cytometry results showed that the FITC-aptamers only specifically recognized HCC serum. Circular dichronism (CD) spectral characterization showed a positive peak near 275 nm and a negative peak near 250 nm for all aptamers, elucidating that the secondary structure formed by the candidate aptamers was a stem-loop B-DNA structure. In addition, molecular docking simulations showed that the binding of the HCC target to the candidate aptamer sequences was mainly dominated by hydrogen bonding. The results of the aptamer sensing performance analysis showed that under the optimized assay conditions, a linear relationship (ranging from 1 nM to 1 µM) was achieved, with a limit of detection (LOD) down to 0.75 nM and a LOQ of 2.32 nM. This was further validated in clinical samples, with a positive detection rate of more than 90%. Furthermore, aptamer-mediated in vivo delivery of luciferase mRNA showed that Apt-1-luciferase mRNA could be targeted to the liver and hepatic luciferase expression was significantly increased. These results demonstrate that the aptamer paves the way for clinical application, evidencing significant potential to offer reference information for early diagnosis. [ABSTRACT FROM AUTHOR]

Objectives Large granular lymphocytic leukemia (LGLL) represents a rare neoplasm of mature T cells or natural killer (NK) cells, with an indolent clinical course. Diagnosing LGLL can be challenging because of overlapping features with reactive processes and other mimickers. Methods By presenting 2 challenging cases, we elucidate the differentiation of LGLL from its mimics and highlight potential diagnostic pitfalls. A comprehensive review of the clinicopathologic features of LGLL was conducted. Results Large granular lymphocytic leukemia displays a diverse spectrum of clinical presentations, morphologies, flow cytometric immunophenotypes, and molecular profiles. These features are also encountered in reactive conditions, T-cell clones of uncertain significance, and NK cell clones of uncertain significance. Conclusions In light of the intricate diagnostic landscape, LGLL workup must encompass clinical, morphologic, immunophenotypic, clonal, and molecular findings. Meeting major and minor diagnostic criteria is imperative for the accurate diagnosis of LGLL. [ABSTRACT FROM AUTHOR]

Objectives To determine whether the information provided by short tandem repeat (STR) testing and bone marrow (BM) biopsy specimens following hematopoietic stem cell transplant (HSCT) provides redundant information, leading to test overutilization, without additional clinical benefit. Methods Cases with synchronous STR and flow cytometric immunophenotyping (FCI) testing, as part of the BM evaluation, were assessed for STR/FCI concordance. Results Of 1199 cases (410 patients), we found the overall concordance between STR and FCI was 93%, with most cases (1063) classified as STR–/FCI–. Of all discordant cases, 75 (6%) were STR+/FCI–, with only 5 (6.7%) cases best explained as identification of disease relapse. Eight cases were STR–/FCI+, representing relapsed/residual disease. Analysis of cases 1 year or more from transplant (54% of all cases) indicated only 9 (1.5%) were STR+/FCI–, and none uniquely identified relapse. Conclusions These data suggest that STR analysis performed 1 year or more post-HSCT does not identify unknown cases of relapse. Furthermore, while STR testing is critical for identifying graft failure/rejection within the first year posttransplant, FCI appears superior to STR at detecting late relapses with low-level disease. Therefore, STR testing from patients 1 year or more post-HSCT may be unnecessary, as BM biopsy evaluation is sufficient to identify disease relapse. [ABSTRACT FROM AUTHOR]

Objectives We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. Methods Sixty-one cases of AML with CBFB rearrangement were evaluated. Results The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non–type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. Conclusions Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts. [ABSTRACT FROM AUTHOR]

Objective This study aimed to investigate the utility of neutrophil-related cell population data obtained by automated hematology analyzers in assessing myelodysplastic syndrome cases with decreased granules in neutrophils. Methods A total of 108 subjects were classified into normal granule (n = 35), hypogranulation (n = 37), or hypergranulation (n = 36) groups. Neutrophil cell area and granule area were measured by ImageJ. All samples were analyzed on the XR-1000 and UniCel DxH 800, and neutrophil-related parameters were compared among the 3 groups. Results Neutrophil cell area and the ratio of the granular area showed significant differences among the 3 groups; they were the highest in the hypergranulation group and lowest in the hypogranulation group. XR-1000 data showed significant differences in NE-SFL and NE-FSC among the 3 groups (P <.0001). NE-SFL and NE-FSC discriminated most accurately hypogranulation group against other groups. UniCel DxH 800 data showed significant differences in MN-V-NE, MN-MALS-N, MN-UMALS-NE, SD-UMALS-NE (P <.01), MN-LMALS-NE, and SD-LMALS-NE (P <.05) among the 3 groups. The combination of SD-V-NE and SD-LMALS-NE discriminated most accurately the hypogranulation group against the other groups. Conclusion NE-SFL and NE-FSC and the combination of SD-V-NE and SD-LMALS-NE are useful in detecting cases with decreased granules in neutrophils. [ABSTRACT FROM AUTHOR]

Background Single-platform flow cytometry technology together with CD45-gating is becoming the method of choice for absolute CD4 T cell enumeration. Immunological assessment of HIV patients by monitoring CD4 can provide valuable information on antiviral treatment response and disease progression. Methods A total of 97 HIV-positive individuals were recruited from 2 hospitals in Tripoli, Libya, and 14 healthy blood donors. The HIV-infected individuals were classified by CD4+ count into HIV-positive (>200 cells/µL) or AIDS (≤200 cells/µL) groups. CD4+ and CD8+ cell counts were determined and compared among the groups and with similar published data. Results The mean ± SD CD4+ cell counts were 1106 ± 442.8 cells/µL in healthy individuals, 460 ± 219.7 cells/µL in the HIV-positive group, and 78 ± 64.3 cells/µL in the AIDS group. The mean ± SD CD4+/CD8+ ratio was 1.6 ± 0.58, 0.4 ± 0.22, and 0.1 ± 0.1, respectively. CD4+ counts in Libyan healthy adults might be higher than those reported in several studies in other regions, whereas CD4+ counts in Libyan AIDS patients seem lower. Conclusion Reference values for T lymphocyte counts in Libyan healthy individuals should be investigated more extensively, and the reasons why Libyan AIDS patients seem to have such lower CD4+ counts should be examined. [ABSTRACT FROM AUTHOR]

The two invasive Reynoutria species, Reynoutria japonica var. japonica and Reynoutria sachalinensis, and their hybrid Reynoutria x bohemica are often misidentified by managers and nonspecialists. The taxonomic confusions are all the more exacerbated by the infraspecific variability of introduced populations in terms of morphology, genetic diversity, and ploidy level. We resolved the identity of North‐Eastern French invasive populations using 4582 single‐nucleotide polymorphisms (SNPs) from a RADseq analysis, DNA contents estimated by flow cytometry, and 12 vegetative morphometric variables. The SNPs supported only one single genotype for R. japonica over 11 localities, while the nine localities of Reynoutria x bohemica were represented by one genotype each. Estimation of genome size using DAPI staining and flow cytometry revealed only octoploid cytotypes for R. japonica and hexaploid cytotypes for R. x bohemica, whereas R. sachalinensis was represented by tetraploid and hexaploid cytotypes. Among morphometric variables, no single one allows for a clear differentiation of the three taxa. We propose a combination of characters to easily and quickly identify these three invasive taxa based on six vegetative criteria including leaf and apex length, as well as leaf shape, leaf base, and apex shape, and the extrafloral nectaries on the node. [ABSTRACT FROM AUTHOR]

Objectives: The mitochondrial function in anti-MDA5 and TIF1-γ-positive dermatomyositis (DM) is relatively unknown. This study attempted to explore mitochondrial mass within the peripheral lymphocyte subsets of anti-MDA5 and TIF1-γ-positive DM. Methods: This cross-sectional study enrolled 109 DM patients and 32 healthy controls (HCs). The mitochondrial mass of peripheral lymphocyte subsets was analyzed via flow cytometry using median fluorescence intensity assessment. Results: Compared with HCs, there was an abnormal change in peripheral lymphocyte subsets in anti-MDA5 and anti-TIF1-γ-positive DM patients. Anti-MDA5 and anti-TIF1-γ-positive DM patients also exhibited a significantly elevated mitochondrial mass in peripheral lymphocyte subsets. Furthermore, anti-MDA5 antibody levels were positively associated with the mitochondrial mass of most lymphocyte subsets in anti-MDA5-positive DM patients. Univariate logistic regression analysis indicated that the increased mitochondrial mass in some peripheral lymphocyte subsets was related to the occurrence of anti-MDA5-positive DM and presence of anti-MDA5 antibodies. Similar results were obtained in anti-TIF1-γ-positive DM patients. Conclusions: Abnormal lymphocyte subset counts and percentages as well as altered mitochondrial mass in anti-MDA5 and TIF1-γ-positive DM patients were associated with anti-MDA5 and TIF1-γ antibodies. We believe that these results may provide novel mitochondria-based insights into DM pathogenesis. [ABSTRACT FROM AUTHOR]

We developed a flow cytometry (FC) assay enabling the rapid and accurate identification of bacterial and viral infections using whole blood samples. The streamlined flow cytometry assay is designed to be user-friendly, making it accessible even for operators with limited experience in FC techniques. The key components of the assay focus on the expression levels of specific surface markers—CD64 on polymorphonuclear neutrophils (PMN) as a marker for bacterial infection, and CD169 on monocytes (MO) for viral infection. The strong performance indicated by an area under the receiver operating characteristic (ROC) curve of 0.94 for both PMN CD64 positive predictive value (PPV) 97.96% and negative predictive value (NPV) 76.67%, and MO CD169 PPV 82.6% and NPV 86.9%, highlight the assay's robustness in differentiating between bacterial and viral infections accurately. The FC assay includes the assessment of immune system status through HLA-DR and IL-1R2 modulation in MO, providing a useful insight into the patients' immune response. The significant increase in the frequency of MO exhibiting reduced HLA-DR expression and elevated IL-1R2 levels in infected patients (compared to healthy controls) underscores the potential of these markers as indicators of infection severity. Although the overall correlation between HLA-DR and IL-1R2 expression levels was not significant across all patients, there was a trend in patients with more severe disease suggesting that these markers may have the potential to assist in stratifying patient risk. The present FC assay has the potential to become routine in the clinical microbiology laboratory community and to be helpful in guiding clinical decision making. [ABSTRACT FROM AUTHOR]

Background: Eosinophils are key therapeutic targets in severe asthma that are suppressed by IL5 (mepolizumab) and IL5 receptor (benralizumab) blockade. The effect of IL5 pathway biologics on recently described homeostatic (hEOs) and inflammatory (iEOs) eosinophil subsets is unknown. We aimed to determine the relative impact of mepolizumab and benralizumab treatment on eosinophil subset and phenotype, and explore clinical associations of eosinophil subsets with severe asthma characteristics and treatment response. Methods: We performed a cross‐sectional observational study of severe asthma (eosinophilic n = 32, non‐eosinophilic n = 23, mepolizumab‐treated n = 25), with longitudinal follow‐up of 30 eosinophilic participants at two timepoints (4–24 weeks, >24 weeks) post‐commencement of mepolizumab (n = 20) or benralizumab (n = 10). Blood hEOs and iEOs were measured by flow cytometry assessment of surface CD62L protein. Results: iEO proportion was significantly lower in mepolizumab‐treated participants in both the cross‐sectional and longitudinal study. Mepolizumab and benralizumab depleted iEOs to a similar extent, however a significantly greater number of hEOs remained in mepolizumab participants at follow‐up. Greater iEO proportion correlated with poorer asthma control in eosinophilic but not non‐eosinophilic asthma. Higher residual iEO proportion correlated with poorer asthma control in mepolizumab‐treated individuals. Reduced blood eosinophil viability was observed in around half of mepolizumab‐treated participants, which was associated with significantly better asthma control and spirometry. Conclusions: Mepolizumab depletes iEOs and reduces circulating eosinophil viability in severe asthma but preserves a residual population of circulatory hEOs. In contrast benralizumab depleted both iEOs and hEOs. Higher iEO abundance and eosinophil viability are associated with poorer clinical outcomes following mepolizumab‐treatment. Monitoring circulating eosinophil phenotype and viability may be useful to predict biologic treatment response in severe asthma. [ABSTRACT FROM AUTHOR]

Background: The emergence of chemoresistance markedly compromised the treatment efficiency of human cancer, including non‐small cell lung cancer (NSCLC). In the present study, we aimed to explore the effects of ubiquitin‐specific peptidase 22 (USP22) and murine double minute 2 (MDM2) in gefitinib resistance in NSCLC. Methods: Immunohistochemistry (IHC) assay, quantitative real‐time polymerase chain reaction (qRT‐PCR) assay and western blot assay were carried out to determine the expression of USP22 and MDM2. Transwell assay and flow cytometry analysis were performed to evaluate cell migration and apoptosis. Cell Counting Kit‐8 (CCK‐8) assay was employed to assess gefitinib resistance. The phenomenon of ferroptosis was estimated by related commercial kits. The oxidized C11‐BODIPY fluorescence intensity by C11‐BODIPY staining. The relation between USP22 and MDM2 was analyzed by ubiquitination assay and co‐immunoprecipitation (Co‐IP) assay. Results: USP22 was abnormally upregulated in NSCLC tissues and cells, and USP22 silencing markedly repressed NSCLC cell migration and facilitated apoptosis and ferroptosis. Moreover, our results indicated that ferroptosis could enhance the suppressive effect of gefitinib on NSCLC cells. Besides, USP22 overexpression enhanced gefitinib resistance and ferroptosis protection in NSCLC cells. Mechanically, USP22 stabilized MDM2 and regulated MDM2 expression through deubiquitination of MDM2. MDM2 deficiency partially restored the effects of USP22 on gefitinib resistance and ferroptosis in NSCLC cells. Of note, we validated the promotional effect of USP22 on gefitinib resistance in NSCLC in vivo through establishing the murine xenograft model. Conclusion: USP22/MDM2 promoted gefitinib resistance and inhibited ferroptosis in NSCLC, which might offer a novel strategy for overcoming gefitinib resistance in NSCLC. [ABSTRACT FROM AUTHOR]

Background: Rotator cuff repair augmentation using biological materials has become popular in clinical practice to reduce the high retear rates associated with traditional repair techniques. Tissue engineering approaches, such as engineered tendon-fibrocartilage-bone composite (TFBC), have shown promise in enhancing the biological healing of rotator cuff tears in animals. However, previous studies have provided limited long-term data on TFBC repair outcomes. The effect of mechanical stimulation on TFBC has not been explored extensively. Purpose: To evaluate functional outcomes after rotator cuff repair with engineered TFBC subjected to mechanical stimulation in a 6-month follow-up using a canine in vivo model. Study Design: Controlled laboratory study. Methods: A total of 40 canines with an acute infraspinatus (ISP) tendon transection model were randomly allocated to 4 groups (n =10): (1) unilateral ISP tendon undergoing suture repair only (control surgery); (2) augmentation with engineered TFBC alone (TFBC); (3) augmentation with engineered TFBC and bone marrow-derived stem cells (BMSCs) (TFBC+C); and (4) augmentation with engineered TFBC and BMSCs, as well as mechanical stimulation (TFBC+C+M). Outcome measures—including biomechanical evaluations such as failure strength, stiffness, failure mode, gross appearance, ISP tendon and muscle morphological assessment, and histological analysis—were performed 6 months after surgery. Results: As shown in the mechanical test, the TFBC+C+M group exhibited higher failure strength compared with other repair techniques. The most common failure mode was avulsion fracture in the TFBC+C+M group, but tendon-bone junction rupture was observed predominantly in different groups. Engineered TFBC with mechanical stimulation showed over 70% relative failure strength compared with normal ISP, and the other groups showed about 50% relative failure strength. Histological analysis revealed less fat infiltration and closer-to-normal muscle fiber structure in the mechanical stimulation group. Conclusion: This study provides evidence that mechanical stimulation of engineered TFBC promotes rotator cuff regeneration, thus supporting its potential for rotator cuff repair augmentation. Clinical Relevance: This study provides valuable evidence supporting the use of a novel tissue-engineered material (TFBC) in rotator cuff repair and paves the way for advancements in the field of rotator cuff regeneration. [ABSTRACT FROM AUTHOR]

Background: Dysfunctional immune regulation plays a crucial role in the pathogenesis of airway allergies. Macrophages are one of the components of the immune regulation cells. The aim of this study is to elucidate the role of lysine demethylase 5 A (KDM5A) in maintaining macrophages' immune regulatory ability. Methods: DNA was extracted from Lactobacillus rhamnosus GG to be designated as LgDNA. LgDNA was administered to the mice through nasal instillations. M2 macrophages (M2 cells) were isolated from the airway tissues using flow cytometry. Results: We found that airway M2 cells of mice with airway Th2 polarization had reduced amounts of IL-10 and KDM5A. Mice with Kdm5a deficiency in M2 cells showed the airway Th2 polarization. The expression of Kdm5a in airway M2 cells was enhanced by nasal instillations containing LgDNA. KDM5A mediated the effects of LgDNA on inducing the Il10 expression in airway M2 cells. Administration of LgDNA mitigated experimental airway allergy. Conclusions: M2 macrophages in the airway tissues of mice with airway allergy show low levels of KDM5A. By upregulating KDM5A expression, LgDNA can increase Il10 expression and reconcile airway Th2 polarization. [ABSTRACT FROM AUTHOR]

Treatments targeting the immune system only benefit a subset of patients with bladder cancer (BC). Biomarkers predictive of BC progression and response to specific therapeutic interventions are required. We evaluated whether peripheral blood immune subsets and expression of clinically relevant immune checkpoint markers are associated with clinicopathologic features of BC. Peripheral blood mononuclear cells isolated from blood collected from 23 patients with BC and 9 age‐matched unaffected‐by‐cancer control donors were assessed using a 21‐parameter flow cytometry panel composed of markers of T, B, natural killer and myeloid populations and immune checkpoint markers. Patients with BC had significantly lower numbers of circulating CD19+ B cells and elevated circulating CD4+CD8+ T cells compared with the control cohort. Immune checkpoint markers programmed cell death protein 1 (PD‐1) and T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3) were elevated in the total peripheral immune cell population in patients with BC. Within the BC cohort, PD‐1 expression in T and myeloid cells was elevated in muscle‐invasive compared with non–muscle‐invasive disease. In addition, elevated T, B and myeloid PD‐1 cell surface expression was significantly associated with tumor stage, suggesting that measures of peripheral immune cell exhaustion may be a predictor of tumor progression in BC. Finally, positive correlations between expression levels of the various immune checkpoints both overall and within key peripheral blood immune subsets collected from patients with BC were observed, highlighting likely coregulation of peripheral immune checkpoint expression. The peripheral blood immunophenotype in patients with BC is altered compared with cancer‐free individuals. Understanding this dysregulated immune profile will contribute to the identification of diagnostic and prognostic indicators to guide effective immune‐targeted, personalized treatments. [ABSTRACT FROM AUTHOR]

Flow cytometry is a sophisticated technology used widely in both basic research and as a routine tool in clinical diagnosis. The technology has progressed from single parameter detection in the 1970s and 1980s to high end multicolor analysis, with currently 30 parameters detected simultaneously, allowing the identification and purification of rare subpopulations of cells of interest. Flow cytometry continues to evolve and expand to facilitate the investigation of new diagnostic and therapeutic avenues. The present review gives an overview of basic theory and instrumentation, presents and compares the advantages and disadvantages of conventional, spectral and imaging flow cytometry as well as mass cytometry. Current methodologies and applications in both research, pre- and clinical settings are discussed, as well as potential limitations and future evolution. This finding encourages the reader to promote such relationship between basic science, diagnosis and multidisciplinary approach since the standard methods have limitations (e.g., in differentiating the cells after staining). Moreover, such path inspires future cytometry specialists develop new/alternative frontiers between pre- and clinical diagnosis and be more flexible in designing the study for both human as well as veterinary medicine. [ABSTRACT FROM AUTHOR]

Background The presence of ACPA significantly increases the risk of developing RA. Dysregulation of lymphocyte subpopulations was previously described in RA. Our objective was to propose the predictive model for progression to clinical arthritis based on peripheral lymphocyte subsets and ACPA in individuals who are at risk of RA. Methods Our study included 207 at-risk individuals defined by the presence of arthralgias and either additional ACPA positivity or meeting the EULAR definition for clinically suspect arthralgia. For the construction of predictive models, 153 individuals with symptom duration ≥12 months who have not yet progressed to arthritis were included. The lymphocyte subsets were evaluated using flow cytometry and anti-CCP using ELISA. Results Out of all individuals with arthralgia, 41 progressed to arthritis. A logistic regression model with baseline peripheral blood lymphocyte subpopulations and ACPA as predictors was constructed. The resulting predictive model showed that high anti-CCP IgG, higher percentage of CD4+ T cells, and lower percentage of T and NK cells increased the probability of arthritis development. Moreover, the proposed classification decision tree showed that individuals having both high anti-CCP IgG and low NK cells have the highest risk of developing arthritis. Conclusions We propose a predictive model based on baseline levels of lymphocyte subpopulations and ACPA to identify individuals with arthralgia with the highest risk of progression to clinical arthritis. The final model includes T cells and NK cells, which are involved in the pathogenesis of RA. This preliminary model requires further validation in larger at-risk cohorts. [ABSTRACT FROM AUTHOR]

Objectives ANA-associated rheumatic and musculoskeletal (MSK) diseases (RMDs) [SLE, primary SS (pSS), scleroderma, inflammatory myositis, MCTD and UCTD] make up a disease spectrum with overlapping clinical and immunological features. MSK inflammation is common and impactful across ANA-associated RMDs. The objectives of this study were to evaluate MSK inflammation (ANA-associated arthritis) prevalence in a multidisease ANA-associated RMD study, assess its clinical impact across ANA-associated RMD diagnoses, propose new basket groupings of patients, and evaluate immunological profiles in legacy and new basket contexts. Methods An observational study enrolled patients with ANA-associated RMDs. Demographic variables, comorbidities, therapies, disease activity instruments [BILAG, SLEDAI, the EULAR SS disease activity index (ESSDAI), physician visual analogue scale (VAS)], patient-reported outcomes [SF36, FACIT-Fatigue, EQ5D, ICECAP-A, Work Productivity and Activity impairment (WPAI), patient VAS] and the biomarker profile (six-gene expression scores, flow cytometry, autoantibody profile) were analysed. Reclustering utilized Gaussian mixture modelling (GMM). The clinical and immune features of new and legacy clusters were compared. Results Inflammatory MSK symptoms were prevalent across ANA-associated RMDs, in 213/294 patients. In ANA-associated arthritis patients, most variables did not differ between diagnoses, with the exception of the EQ5D-5L index and mobility domains (lower in MCTD/pSS, both P  < 0.05). FM and OA prevalence were similar across diagnoses. Therapy use differed significantly, the use of biologics being greatest in SLE (P  < 0.05). GMM yielded two multidisease clusters: High MSK disease activity (n  = 89) and low MSK disease activity (n  = 124). The high MSK disease activity cluster included all patients with active joint swelling, and they had significantly higher prednisolone usage, physician global assessment (PGA), Sm/RNP/SmRNP/chromatin positivity, Tetherin mean fluorescence intensity (MFI), and IFN Score-A activity, along with numerically lower FM and OA prevalence. Conclusion We defined ANA-associated arthritis, a more clinically and immunologically homogeneous population than existing RMD populations for trials, and a more prevalent population for therapies in the clinic. [ABSTRACT FROM AUTHOR]

Several approaches can be used to genetically modify the human malaria parasite Plasmodium falciparum to express reporter proteins, such as fluorescent proteins and luciferases, and their expression can be controlled using stage-specific or constitutive promoters. The expression of fluorescent proteins in malaria parasites enables molecular analyses, such as live imaging, flow cytometry, and cell sorting. The number of parasites at each stage can be quantified using the luciferase luminescent signal to evaluate the antimalarial effects of the compounds of interest and the inhibitory effects of antibodies. Further parasite reporter lines are needed to unveil the biological aspects of human malaria parasites and boost drug discovery against this deadly pathogen. The human malaria parasite Plasmodium falciparum causes the most severe form of malaria in endemic regions and is transmitted via mosquito bites. To better understand the biology of this deadly pathogen, a variety of P. falciparum reporter lines have been generated using transgenic approaches to express reporter proteins, such as fluorescent proteins and luciferases. This review discusses the advances in recently generated P. falciparum transgenic reporter lines, which will aid in the investigation of parasite physiology and the discovery of novel antimalarial drugs. Future prospects for the generation of new and superior human malaria parasite reporter lines are also discussed, and unresolved questions in malaria biology are highlighted to help boost support for the development and implementation of malaria treatments. [ABSTRACT FROM AUTHOR]