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Abstract Background Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE). Objectives To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE. Animals Sixteen healthy dogs and 26 dogs were diagnosed with CIE. Methods Prospective case‐control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts. Results Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18‐273] CIE, 44/μL [16‐162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2‐257] CIE, 34/μL [1‐114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75‐18.67] CIE, 8.77% [3.79‐25.03] HC, P = .002), activated Th cells (0.16% [0.02‐0.83] CIE, 0.33% [0.05‐0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02‐0.39] CIE, 0.21 [0.07‐0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12‐77.12] CIE, 12.32% [1.21‐39.22] HC, P = .04) and interferon‐gamma (IFN‐γ) producing T lymphocytes (7.36% [0.63‐55.83] CIE, 1.44% [0.00‐10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00‐21.48] CIE, 3.52% [0.18‐10.52] HC, P = .02). Conclusions and Clinical Importance Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN‐γ producing T lymphocytes and the systemic immune‐inflamation index (SII) could potentially serve as biomarkers for the disease.

Abstract Myeloperoxidase (MPO) is the most specific marker of the myeloid lineage, essential for diagnosing acute myeloid leukemia and mixed phenotype acute leukemia with myeloid components. In this regard, we present a unique case of B‐acute lymphoblastic leukemia (B‐ALL) with isolated MPO expression in bone marrow blasts detected by flow cytometry and immunohistochemistry, while peripheral blood blasts were negative for MPO expression. In this report, our discussion encompasses diagnostic pitfalls from a laboratory testing perspective in similar cases and includes a literature review. Furthermore, we emphasize the necessity of conducting a comprehensive analysis for the accurate diagnosis of MPO‐positive B‐ALL cases.

Abstract Circulating cell‐free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell‐free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double‐stranded nucleic acids. Fluorescent staining of EVs isolated from cell‐conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs’ membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma‐derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.

Abstract Flow cytometry‐based immunophenotyping is a mainstay of diagnostics in acute myeloid leukaemia (AML). Aberrant CD56 and T‐cell antigen expression is observed in a fraction subset of AML cases, but the clinical relevance remains incompletely understood. Here, we retrospectively investigated the association of CD56 and T‐cell marker expression with disease‐specific characteristics and outcome of 324 AML patients who received intensive induction therapy at our centre between 2011 and 2019. We found that CD2 expression was associated with abnormal non‐complex karyotype, NPM1 wild‐type status and TP53 mutation. CD2 also correlated with a lower complete remission (CR) rate (47.8% vs. 71.6%, p = 0.03). CyTdT and CD2 were associated with inferior 3‐year event‐free‐survival (EFS) (5.3% vs. 33.5%, p = 0.003 and 17.4% vs. 33.1%, p = 0.02, respectively). CyTdT expression was also correlated with inferior relapse‐free survival (27.3% vs. 48.8%, p = 0.04). In multivariable analyses CD2 positivity was an independent adverse factor for EFS (HR 1.72, p = 0.03). These results indicate a biological relevance of aberrant T‐cell marker expression in AML and provide a rationale to further characterise the molecular origin in T‐lineage‐associated AML.

Abstract Background The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) immune response is crucial for disease management, although diminishing immunity raises the possibility of reinfection. Methods We examined the immunological response to SARS‐CoV‐2 in a cohort of convalescent COVID‐19 patients in matched samples collected at 1 and 6−8 months after infection. The peripheral blood mononuclear cells were isolated from enrolled study participants and flow cytometry analysis was done to assess the lymphocyte subsets of naive, effector, central memory, and effector memory CD4+ or CD8+ T cells in COVID‐19 patients at 1 and 6−8 months after infection. Immunophenotypic characterization of immune cell subsets was performed on individuals who were followed longitudinally for 1 month (n = 44) and 6−8 months (n = 25) after recovery from COVID infection. Results We observed that CD4 +T cells in hospitalized SARS‐CoV‐2 patients tended to decrease, whereas CD8+ T cells steadily recovered after 1 month, while there was a sustained increase in the population of effector T cells and effector memory T cells. Furthermore, COVID‐19 patients showed persistently low B cells and a small increase in the NK cell population. Conclusion Our findings show that T cell responses were maintained at 6−8 months after infection. This opens new pathways for further research into the long‐term effects in COVID‐19 immunopathogenesis.

Abstract Background Major histocompatibility complex (MHC) class II molecules expressed on B cells, monocytes and dendritic cells present processed peptides to CD4+ T cells as one of the mechanisms to combat infection and inflammation. Aim To study MHC II expression in a variety of nonhuman primate species, including New World (NWM) squirrel monkeys (Saimiri boliviensis boliviensis), owl monkeys (Aotus nancymae), common marmosets (Callithrix spp.), and Old World (OWM) rhesus (Macaca mulatta), baboons (Papio anubis). Methods Two clones of cross‐reactive mouse anti‐human HLADR monoclonal antibodies (mAb) binding were analyzed by flow cytometry to evaluate MHC II expression on NHP immune cells, including T lymphocytes in whole blood (WB) and peripheral blood mononuclear cells (PBMC). Results MHC class II antibody reactivity is seen with CD20+ B cells, CD14+ monocytes and CD3+ T lymphocytes. Specific reactivity with both clones was demonstrated in T lymphocytes: this reactivity was not inhibited by purified CD16 antibody but was completely inhibited when pre‐blocked with purified unconjugated MHC II antibody. Freshly prepared PBMC also showed reactivity with T lymphocytes without any stimulation. Interestingly, peripheral blood from rhesus macaques and olive baboons (OWM) showed no such T lymphocyte associated MHCII antibody reactivity. Discussion & Conclusion Our results from antibody (MHC II) reactivity clearly show the potential existence of constitutively expressed (with no stimulation) MHC II molecules on T lymphocytes in new world monkeys. These results suggest that additional study is warranted to evaluate the functional and evolutionary significance of these finding and to better understand MHC II expression on T lymphocytes in new world monkeys.

Abstract Studying acute changes in vascular endothelial cells in humans is challenging. We studied ten African American women and used the J‐wire technique to isolate vein endothelial cells before and after a four‐hour lipid and heparin infusion. Dynamic changes in lipid‐induced oxidative stress and inflammatory markers were measured with fluorescence‐activated cell sorting. We used the surface markers CD31 and CD144 to identify human endothelial cells. Peripheral blood mononuclear cells isolated from blood were used as a negative control. The participants received galantamine (16 mg/day) for 3 months. We previously demonstrated that galantamine treatment effectively suppresses lipid‐induced oxidative stress and inflammation. In this study, we infused lipids to evaluate its potential to increase the activation of endothelial cells, as assessed by the levels of CD54+ endothelial cells and expression of Growth arrest‐specific 6 compared to the baseline sample. Further, we aimed to investigate whether lipid infusion led to increased expression of the oxidative stress markers IsoLGs and nitrotyrosine in endothelial cells. This approach will expedite the in vivo identification of novel pathways linked with endothelial cell dysfunction induced by oxidative stress and inflammatory cytokines. This study describes an innovative method to harvest and study human endothelial cells and demonstrates the dynamic changes in oxidative stress and inflammatory markers release induced by lipid infusion.

Abstract Background Quantitative measurement of minimal residual disease (MRD) is the “gold standard” for estimating the response to therapy in childhood B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP‐ALL, in terms of genetic subgroups with relation to clinically defined risk groups. Methods A total of 485 children with non‐high‐risk BCP‐ALL with available cytogenetic data and MRD studied at the end‐of‐induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard‐risk (SR) of intermediate‐risk (ImR) regimens of “ALL‐MB 2008” reduced‐intensity protocol. Results and Discussion Among all study group patients, 203 were found to have low‐risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC‐MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC‐MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low‐(0.03%) and intermediate (0.01%) cytogenetic risk groups. Conclusion Our data show that combining clinical risk factors with MFC‐MRD measurement is the most useful tool for risk group stratification of children with BCP‐ALL in the reduced‐intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

The mammary gland is a vital exocrine organ that has evolved in mammals to secrete milk and provide nutrition to ensure the growth and survival of the neonate The mouse mammary gland displays extraordinary plasticity each time the female undergoes pregnancy and lactation, including a sophisticated process of tertiary branching and alveologenesis to form a branched epithelial tree and subsequently milk‐producing alveoli. Upon the cessation of lactation, the gland remodels back to a simple ductal architecture via highly regulated involution processes. At the cellular level, the plasticity is characterised by proliferation of mammary cell populations, differentiation and apoptosis, accompanied by major changes in cell function and morphology. The mammary epithelium requires a specific stromal environment to grow, known as the mammary fat pad. Mammary adipocytes are one of the most prominent cell types in the fat pad, but despite their vast proportion in the tissue and their crucial interaction with epithelial cells, their physiology remains largely unknown. Over the past decade, the need to understand the properties and contribution of mammary adipocytes has become more recognised. However, the development of adequate methods and protocols to study this cellular niche is still lagging, partially due to their fragile nature, the difficulty of isolating them, the lack of reliable cell surface markers and the heterogenous environment in this tissue, which differs from other adipocyte depots. Here, we describe a new rapid and simple flow cytometry protocol specifically designed for the analysis and isolation of mouse mammary adipocytes across mammary gland developmental stages.

Abstract The water chestnut Trapa bispinosa Roxb. has been domesticated in China and has been reported as the only domesticated species of this genus. To understand the origin of T. bispinosa and its evolution pathway, we compared the genetic similarity and seed morphology of domesticated water chestnut T. bispinosa with three wild species T. natans, T. incisa, and T. japonica along with archeological seed samples from the Tianluoshan site (approximately 7000–6300 cal BP) in China. The largest seed size was observed only in the domesticated species, whereas other wild species showed smaller size including T. natans L. genetically close to the domesticated type, and T. incisa was the smallest in size. The volumes of the seed capsule and endosperm were measured using X ray CT scans, showing the ratios of total volumes between T. bispinosa and wild species ranged from 4.2 to 4.5. The ratios of endosperm volume ranged from 3.3 to 3.7. Both measurements showed domesticated species have larger seed volume. Genome size was indirectly estimated by flow cytometry. Domesticated species with larger seed size was estimated as diploid, as were the wild species except for tetraploid species T. japonica. Domesticated species clearly showed the largest edible organs, but it was not a result of ploidy level changes. Maternal lineages traced using complete whole chloroplast sequences, suggested that T. natans is the closest to T. bispinosa, both of which are close to T. japonica. The result was confirmed by PCR genotyping with chloroplast insertion/deletion (cpINDEL) markers developed in the study. T. incisa showed distinct plastid types within the species, and T. japonica showed a unique plastid genotype. Our study concludes the largest volumes for the edible endosperm have been accomplished through nearly 6000 years of artificial selection, but the domestication did not involve ploidy level changes.

Abstract Objectives Inflammatory markers such as erythrocyte sedimentation rate (ESR) and C‐reactive protein (CRP) are poorly informative about interferon (IFN)‐related disorders. In these conditions, the measure of the interferon score (IS), obtained by measuring the expression of IFN‐stimulated genes, has been proposed. Flow cytometry‐based assays measuring sialic‐acid‐binding Ig‐like lectin 1 (Siglec‐1) expression could be a more practical tool for evaluating IFN‐inflammation. The study compared Siglec‐1 measures with IS and other inflammatory indexes. We compared Siglec‐1 measures with IS and other inflammatory indexes in real‐world paediatric rheumatology experience. Methods We recruited patients with immuno‐rheumatological conditions, acute infectious illness and patients undergoing orthopaedic surgery as controls. Siglec‐1 expression was measured in all samples, and IS, ESR and CRP were also recorded if available. Results Overall, 98 subjects were enrolled in the study, with a total of 104 measures of Siglec‐1. Compared with IS, Siglec‐1 expression showed good accuracy (86.0%), specificity (72.7%) and sensitivity (85.7%). The measure of the percentage of Siglec‐1‐positive cells performed best at low levels of IFN‐inflammation, while the measure of mean fluorescence intensity performed best at higher levels. Ex vivo studies on IFN‐stimulated monocytes confirmed this behaviour. There was no link between Siglec‐1 expression and either ESR or CRP, and positive Siglec‐1 results were found even when ESR and CRP were normal. A high Siglec‐1 expression was also recorded in subjects with acute infections. Conclusion Siglec‐1 measurement by flow cytometry is an easy tool to detect IFN‐related inflammation, even in subjects with normal results of common inflammation indexes.

Abstract Objectives The aim of this study was to characterise the dynamic immune profile of paediatric burn patients for up to 18 months post‐burn. Methods Flow cytometry was used to measure 25 cell markers, chemokines and cytokines which reflected both pro‐inflammatory and anti‐inflammatory immune profiles. Peripheral blood mononuclear cells from 6 paediatric burn patients who had returned for repeated burn and scar treatments for > 4 timepoints within 12 months post‐burn were compared to four age‐matched healthy controls. Results While overall proportions of T cells, NK cells and macrophages remained relatively constant, over time percentages of these immune cells differentiated into effector and proinflammatory cell phenotypes including Th17 and activated γδ T cells. Circulating proportions of γδ T cells increased their expression of pro‐inflammatory mediators throughout the burn recovery, with a 3–6 fold increase of IL‐17 at 1–3 weeks, and NFκβ 9–18 months post‐burn. T‐regulatory cell plasticity was also observed, and Treg phenotype proportions changed from systemically reduced skin‐homing T‐regs (CCR4+) and increased inflammatory (CCR6+) at 1‐month post‐burn, to double‐positive cell types (CCR4+CCR6+) elevated in circulation for 18 months post‐burn. Furthermore, Tregs were observed to proportionally express less IL‐10 but increased TNF‐α over 18 months. Conclusion Overall, these results indicate the circulating percentages of immune cells do not increase or decrease over time post‐burn, instead they become highly specialised, inflammatory and skin‐homing. In this patient population, these changes persisted for at least 18 months post‐burn, this ‘immune distraction’ may limit the ability of immune cells to prioritise other threats post‐burn, such as respiratory infections.

Abstract Plants demonstrate exceptional variation in genome size across species, and their genome sizes can also vary dramatically across individuals and populations within species. This aspect of genetic variation can have consequences for traits and fitness, but few studies attributed genome size differentiation to ecological and evolutionary processes. Biological invasions present particularly useful natural laboratories to infer selective agents that might drive genome size shifts across environments and population histories. Here, we test hypotheses for the evolutionary causes of genome size variation across 14 invading populations of yellow starthistle, Centaurea solstitialis, in California, United States. We use a survey of genome sizes and trait variation to ask: (1) Is variation in genome size associated with developmental trait variation? (2) Are genome sizes smaller toward the leading edge of the expansion, consistent with selection for “colonizer” traits? Or alternatively, does genome size increase toward the leading edge of the expansion, consistent with predicted consequences of founder effects and drift? (3) Finally, are genome sizes smaller at higher elevations, consistent with selection for shorter development times? We found that 2C DNA content varied 1.21‐fold among all samples, and was associated with flowering time variation, such that plants with larger genomes reproduced later, with lower lifetime capitula production. Genome sizes increased toward the leading edge of the invasion, but tended to decrease at higher elevations, consistent with genetic drift during range expansion but potentially strong selection for smaller genomes and faster development time at higher elevations. These results demonstrate how genome size variation can contribute to traits directly tied to reproductive success, and how selection and drift can shape that variation. We highlight the influence of genome size on dynamics underlying a rapid range expansion in a highly problematic invasive plant.

A new label‐free imaging flow cytometry method for noninvasive and automated biological cell classification is presented. Each cell is rolled during flow, and its off‐axis holograms from multiple viewpoints are acquired. Using the reconstructed quantitative phase profiles of the cell projections, highly discriminating features, enabling cell detection, classification, and differentiation, are extracted via a modified ResNet‐18 deep convolutional neural network architecture. The model is first validated by classifying metastatic breast carcinoma cells (MCF‐7) and normal human mammary epithelial cells (MCF‐10A). An increase in classification accuracy by 1% is achieved when processing five interferometric projections versus processing a single interferometric projection. This model is further tested on four types of white blood cells and exhibits an accuracy increase of 5% when processing 12 interferometric projections versus processing a single interferometric projection. This approach is shown to be superior to that of using conventional 2D‐rotation augmentation, and can be used to decrease substantially the number of cell examples needed for training the classification model without impairing the results. This novel concept has great potential to be incorporated into label‐free imaging flow cytometry and improve cell classification, and be used to detect various types of medical conditions and diseases.

Abstract Background B‐cell chronic lymphocytic leukemia (B‐CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double‐negative T (DNT) cells, double‐positive T (DPT) cells, and natural killer T (NKT)‐cells may be involved in tumor surveillance. Methods A detailed immunophenotypic analysis of the peripheral blood T‐cell compartment of 50 patients with B‐CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain‐lyse‐no wash technique and a comprehensive six‐color antibody panels. Results Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B‐CLL, as already reported. In particular, DNT, DPT, and NKT‐like percentages were significantly lower than in the controls, except for NKT‐like in the low‐risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low‐risk prognostic group of NKT‐like cells was found. A significant correlation of the absolute values of NKT‐like cells in the intermediate‐risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T‐cell subset plays a key role in the immune T response in B‐CLL. Conclusion These early results supported that DNT, DPT, and NKT‐like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Abstract We investigated the effect of a 31‐day ketogenic diet (KD) compared with a habitual, carbohydrate (CHO)‐based diet on total circulating natural killer (NK) CD3−CD56+, dim and bright subset count, and antigen‐stimulated CD3−CD56+ cell activation (CD69+) in response to exhaustive running. In a randomised, repeated‐measures, cross‐over study, eight trained, male endurance athletes ingested a 31‐day low‐CHO KD or their habitual diet (HD). On day 31, participants ran to exhaustion at 70% V̇O2max (∼3.5–4 h, ∼45–50 km). A low‐CHO ( 0.05). In conclusion, adaptation to a KD may alter the number of circulating NK cells but not their ability to activate to an antigenic challenge.

Abstract We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre‐freezing and post‐thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post‐thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre‐freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p

Abstract Background Several studies have attempted to characterise the origin of canine transmissible venereal tumour (CTVT). However, the participation of cancer stem cells (CSC) has not been reported Objectives Herein we describe the expression patterns of CSC markers CD44, CD34, CD90 and CD133 in CTVT Methods Thirty‐eight samples were selected and assessed through flow cytometry and immunohistochemistry Results and conclusions Twenty‐two tumours were classified as plasmacytoid and 16 as mixed. Almost all tumours showed high CD44 and low CD34 levels. CD133 and CD90 expression varied among tumours. Cytological groups did not differ in the proportion of CSC markers. Our results suggest that CSC subpopulations might participate in CTVT.

Saccharomyces cerevisiae is an important model organism and a typical fungal representative for studies of eukaryotes. The cell cycle of yeast can be analyzed by flow cytometry, and refining the cytometric resolution of the cell cycle with this technique is important. Such refinement is potentially influenced by multiple factors, including enzymatic treatment and variations in the culture media used, although this has been subject to only limited investigation. Here, we examined the effect of different enzymatic pre‐treatments and various media on cytometric resolution. We show that cytometric resolution is significantly altered by both enzymatic conditions and the media used. Culture media with different amino nitrogen concentrations potentially impact the protein content in the yeast cell wall, which may affect the permeability of the cell wall and alter cytometric resolution. The present study provides beneficial technical information about the influence of media and enzymes on the cytometric resolution of the yeast cell cycle and most likely other fungi, which should be considered in future research.

Abstract We present the case of a 70‐year‐old man diagnosed with acute myelomonocytic leukemia, albeit that his leukemic blasts at initial presentation had scant cytoplasm, inconspicuous cytoplasmic granules, and morphologically mimicked lymphoblasts. We would like to raise the recognition that acute myelomonocytic leukemia can actually present with atypical blast morphology.

Abstract Background The aim of the present study is to investigate the clinical value and characteristics of peripheral blood lymphocyte subsets in patients with pulmonary tuberculosis (PTB) using flow cytometry. Methods The absolute counts of T, CD4+T, CD8+T, natural killer (NK), NKT and B lymphocytes in 217 cases of PTB were detected, and the variations in lymphocyte subset counts between different ages and genders and between aetiological detection results and chest radiography results were analysed. Results In 75.3% of the patients with PTB, six subset counts were lower than the normal reference range, and 44% showed lower‐than‐normal CD4+T lymphocyte levels. The counts of T, CD4+T, CD8+T and B lymphocytes were significantly lower in patients aged >60 years, and the NKT cell counts were significantly lower in female patients than in male patients. Among the patients with positive aetiological results, 40.8% had reduced CD8+T counts; these were significantly lower than those in patients with negative aetiological results (P = 0.0295). The cell counts of T, CD4+T, CD8+T and B lymphocytes reduced as lesion lobe numbers increased. The counts of T, CD4+T and CD8+T lymphocytes were significantly higher in the group with lesions affecting one lobe than in the groups with two to three lobes or four to five lobes, and the counts of B lymphocytes were significantly higher in the group with one lobe and the group with two to three lobes than in the group with four to five lobes. The counts of CD4+T and CD8+T lymphocytes were highest in the no cavity group and showed a downward trend with the increase in cavities; the T lymphocyte count was significantly higher in the no cavity group than in the group with five or more cavities (P = 0.014), and the CD8+T lymphocyte count was significantly higher in the no cavity group than in the group with one to two cavities and the group with five or more cavities (P = 0.001 and 0.01, respectively). Conclusions In most patients with tuberculosis, immune function is impaired. The absolute counts of peripheral blood lymphocyte subsets are closely related to the aetiological results and lesion severity in patients with PTB; this could be used as evidence for immune intervention and monitoring curative effects.

Abstract Background P16INK4A is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co‐test for detecting high‐grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high‐throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention. Methods P16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A‐knockout) standards. Since 2018, 24 100‐women (HPV‐positive/‐negative, Pap‐normal/‐abnormal) have been enrolled nationwide for two‐tier validation work. In cross‐sectional studies, age‐ and viral genotype‐dependent expression of p16INK4A was investigated, and optimal diagnostic parameter cut‐offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2‐year prognostic values of p16INK4A were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV‐positive Pap‐normal, Pap‐abnormal biopsy‐negative and biopsy‐confirmed LSIL. Results P16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A‐positive ratio was 13.9 ± 1.8% among HPV‐negative NILM women and peaked at the ages of 40–49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions (HPV‐negative: 17.7 ± 5.0–21.4 ± 7.2%; HPV‐positive: 18.0 ± 5.2–20.0 ± 9.9%). Extremely low expression of p16INK4A was observed in women with HSILs. As the HPV‐combined double‐cut‐off‐ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co‐test. The p16INK4A‐abnormal situation was an independent HSIL+ risk factor for 2‐year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3–7.2). Conclusions FCM‐based p16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk‐stratification‐based interventions.

Background Immune cells play a vital role in the pathology of ischemic stroke. Neutrophils and polymorphonuclear myeloid‐derived suppressor cells share a similar phenotype and have attracted increasing attention in immune regulation research, yet their dynamics in ischemic stroke remain elusive. Methods and Results Mice were randomly divided into 2 groups and intraperitoneally treated with anti‐Ly6G (lymphocyte antigen 6 complex locus G) monoclonal antibody or saline. Distal middle cerebral artery occlusion and transient middle cerebral artery occlusion were applied to induce experimental stroke, and mice mortality was recorded until 28 days after stroke. Green fluorescent nissl staining was used to measure infarct volume. Cylinder and foot fault tests were used to evaluate neurological deficits. Immunofluorescence staining was conducted to confirm Ly6G neutralization and detect activated neutrophils and CD11b+Ly6G+ cells. Fluorescence‐activated cell sorting was performed to evaluate polymorphonuclear myeloid‐derived suppressor cell accumulation in brains and spleens after stroke. Anti‐Ly6G antibody successfully depleted Ly6G expression in mice cortex but did not alter cortical physiological vasculature. Prophylactic anti‐Ly6G antibody treatment ameliorated ischemic stroke outcomes in the subacute phase. Moreover, using immunofluorescence staining, we found that anti‐Ly6G antibody suppressed activated neutrophil infiltration into parenchyma and decreased neutrophil extracellular trap formation in penumbra after stroke. Additionally, prophylactic anti‐Ly6G antibody treatment reduced polymorphonuclear myeloid‐derived suppressor cell accumulation in the ischemic hemisphere. Conclusions Our study suggested a protective effect of prophylactic anti‐Ly6G antibody administration against ischemic stroke by reducing activated neutrophil infiltration and neutrophil extracellular trap formation in parenchyma and suppressing polymorphonuclear myeloid‐derived suppressor cell accumulation in the brain. This study may provide a novel therapeutic approach for ischemic stroke.

Abstract Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV‐containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt‐EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.

Background: Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum., Methods: Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry., Results: Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients., Conclusions: IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis., (© 2024 The Author(s). European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Background: In acute myeloid leukemia (AML), further investigation is warranted to integrate measurable residual disease (MRD) with genetic characteristics for formulating a dynamic prognostic system for predicting response and selecting appropriate postremission therapeutic strategies., Methods: The authors incorporated MRD with genetic risk classification and assessed its impact on transplantation decision making within different risk cohorts, comprising 769 patients with newly diagnosed AML across three clinical trials. Only patients who achieved complete remission (CR) within two courses of chemotherapy were selected., Results: In the favorable-risk and intermediate-risk groups, patients who underwent transplantation according to the protocol experienced significant 3-year overall survival (OS) benefits compared with those who did not (favorable-risk group: hazard ratio [HR], 0.38; 95% confidence interval [CI], 0.20-0.73l p = .004; intermediate-risk group: HR, 0.53; 95% CI, 0.33-0.85; p = .008). In the intermediate-risk group, early detection of MRD positivity, even after the initial course of chemotherapy, was associated with a significantly elevated cumulative incidence of relapse (47.2% vs. 36.0%; p = .009) and a notable extension of OS with allogeneic hematopoietic stem cell transplantation (HR, 0.47; 95% CI, 0.28-0.79; p = .004). Conversely, patients who achieved MRD negativity at either of the two time points had comparable OS in the favorable-risk and intermediate-risk groups, regardless of whether they underwent transplant or not. In the adverse-risk group, allogeneic hematopoietic stem cell transplantation led to improvements in OS irrespective of MRD status (HR, 0.51; 95% CI, 0.38-0.69; p < .001)., Conclusions: Early clearance of MRD demonstrated significant prognostic value, particularly for patients in the favorable-risk and intermediate-risk groups. Positive MRD status after two courses of intensive chemotherapy were associated with a higher relapse rate and inferior OS, necessitating allogeneic hematopoietic stem cell transplantation., (© 2024 American Cancer Society.)

Background: Allergy immunotherapy (AIT), a treatment approach for allergic rhinitis (AR), is recognized for its potential to modify the disease course beyond mere symptom relief. Interleukin-36 γ (IL-36 γ ), a key player in immune responses, has been implicated in promoting eosinophilic inflammation in AR by activating eosinophils. We aimed to investigate the effect of IL-36 γ on group II lymphoid cell (ILC2) in AR patients who underwent sublingual immunotherapy (SLIT). Methods: Twenty-four AR patients were enrolled and administered with SLIT. Serum proteins of IL-36 γ , interleukin-5 (IL-5), and interleukin-13 (IL-13) during SLIT were quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). The proportion of ILC2 was determined by flow cytometry. Sorted ILC2s were stimulated by IL-36 γ and ILC2 cell differentiation, and type II cytokines expression were examined. Results: SLIT treatment decreased the serum protein levels of IL-36 γ , IL-5, IL-13, and the proportion of ILC2 significantly. IL-36 γ suppressed the proliferation of ILC2 by inhibiting the levels of ILC2 transcription factor. IL-36 γ also inhibited IL-5 and IL-13 expression from ILC2. Conclusion: The changes of IL-36 γ during SLIT were related to the inhibited function of ILC2, implying that IL-36 γ may be used as a new biomarker for monitoring the efficacy of SLIT in AR., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Xiaowei Qin et al.)

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., (© 2024 the Australian and New Zealand Society for Immunology, Inc.)

Abstract Background Although precursor‐targeted immune‐mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA. Hypothesis Dogs with PIMA have increased nRBC IgG and PS, and dogs with immune‐mediated hemolytic anemia (IMHA) have increased RBC PS compared to healthy dogs. Animals Blood from 20 healthy dogs and from dogs with IMHA (11) or other (non‐IMHA) conditions (9), and marrow aspirates with or without blood from 10 healthy dogs and from dogs with PIMA (17) or other (non‐IMHA, non‐PIMA) conditions (7). Methods Marrow nRBC stages were separated by density gradient. Flow cytometry was used to assess the percentage of RBCs or nRBCs with increased IgG or PS. Results Red blood cell (RBC) IgG positivity was increased in 9/11 IMHA dogs and 0/9 non‐IMHA dogs. Red blood cell PS positivity was increased in 10/11 IMHA dogs and 2/9 non‐IMHA dogs. Five of 17 PIMA dogs had increased nRBC IgG positivity in mid‐ or late‐stage fractions, whereas all 7 non‐PIMA dogs were negative. Mid‐ and late‐stage erythroid precursor PS was significantly higher in PIMA dogs compared to healthy dogs. Five of 14 PIMA dogs had increased RBC IgG positivity. Conclusions Immunoglobulin G and PS may promote destruction of nRBCs in PIMA dogs; PS may promote destruction of RBCs in IMHA dogs.

Abstract Extranodal marginal zone lymphoma of mucosa‐associated lymphoid tissue (MALT) is a low‐grade B‐cell lymphoma. MALT lymphomas involving the sublingual gland are extremely rare. Herein, we report a case of MALT lymphoma of the sublingual gland. Additionally, we discuss challenging diagnostic aspects as well as current treatment strategies.

Abstract Background: Hamburg is a city state of approximately 1.9 Mio inhabitants in Northern Germany. Currently, the COVID‐19 epidemic that had largely subsided during last summer is resurging in Hamburg and in other parts of the world, underlining the need for additional tools to monitor SARS‐CoV‐2 antibody responses. Aim: We aimed to develop and validate a simple, low‐cost assay for detecting antibodies against the native coronavirus 2 spike protein (CoV‐2 S) that does not require recombinant protein or virus. Method: We transiently co‐transfected HEK cells or CHO cells with expression vectors encoding CoV‐2 S and nuclear GFP. Spike protein‐specific antibodies in human serum samples bound to transfected cells were detected with fluorochrome conjugated secondary antibodies by flow cytometry orimmunofluorescence microscopy. We applied this assay to monitor antibody development in COVID‐19 patients, household contacts, and hospital personnel during the ongoing epidemic in the city state of Hamburg. Results: All recovered COVID‐19 patients showed high levels of CoV‐2 S‐specific antibodies. With one exception, all household members that did not develop symptoms also did not develop detectable antibodies. Similarly, lab personnel that worked during the epidemic and followed social distancing guidelines remained antibody‐negative. Conclusion: We conclude that high‐titer CoV‐2 S‐specific antibodies are found in most recovered COVID‐19 patients and in symptomatic contacts, but only rarely in asymptomatic contacts. The assay may help health care providers to monitor disease progression and antibody responses in vaccination trials, to identify health care personnel that likely are resistant to re‐infection, and recovered individuals with high antibody titers that may be suitable asplasma and/or antibody donors.

Abstract Moreau score has been used to differentiate chronic lymphocytic leukemia (CLL) from other mature B‐cell neoplasms. However, it showed limitations in Asian patients. Therefore, we conducted a new score system replacing CD5 and CD23 with CD43 and CD180 to evaluate its diagnostic value of CLL. 237 untreated samples diagnosed with mature B‐cell neoplasms were collected and were randomly divided into an exploratory and a validation cohort by a 2:1 ratio. The expression of CD5, CD19, CD20, CD23, CD43, CD79b, CD180, CD200, FMC7, and surface immunoglobulin (SmIg) were analyzed among all the samples. A proposed score was developed based on the logistic regression model. The sensitivity and specificity of the proposed score were calculated by ROC curves. CD43/CD180, CD200, FMC7, and CD79b were included in our new CLL score, which showed a sensitivity of 91.8% and a specificity of 83.1%. These results were confirmed in a validation cohort with a sensitivity of 90.5% (p = 0.808) and a specificity of 79.5% (p = 0.639). In CD5 negative or CD23 negative CLL group, the new CLL score displayed improved sensitivity of 79.4% compared to Moreau score and CLLflow score (41.2% and 47.1%, respectively). In atypical CLL group, the new CLL score showed improved sensitivity of 84.2% compared to Moreau score and CLLflow score (61.4% and 64.9%, respectively). This proposed atypical CLL score helped to offer an accurate differentiation of CLL from non‐CLL together with morphological and molecular methods, particularly in Chinese patients with atypical immunophenotype.

Abstract Background B‐cell chronic lymphocytic leukemia (BCLL) in dogs generally is considered an indolent disease, but previous studies indicate a wide range in survival times. Objectives We hypothesized that BCLL has a heterogeneous clinical course, similar to chronic lymphocytic leukemia in humans. We aimed to assess presentation and outcome in dogs with BCLL and evaluate the prognostic relevance of clinical and flow cytometric factors. Animals One hundred and twenty‐one dogs with BCLL diagnosed by flow cytometry. Three breed groups were represented: small breed dogs (n = 55) because of increased risk of BCLL; Boxers (n = 33) because of preferential use of unmutated immunoglobulin genes; and other breeds (n = 33). Methods Retrospective study reviewing signalment, clinicopathologic data, physical examination findings, treatment, and survival of dogs with BCLL. Cellular proliferation, determined by the percentage of Ki67‐expressing CD21+ B‐cells by flow cytometry, was measured in 39 of 121 cases. Clinical and laboratory variables were evaluated for association with survival. Results The median survival time (MST) for all cases was 300 days (range, 1‐1644 days). Boxers had significantly shorter survival (MST, 178 days) than non‐Boxers (MST, 423 days; P 40% Ki67‐expressing B‐cells) had significantly shorter survival (MST, 173 days) than did cases with 60 000 lymphocytes/μL) or clinical signs at presentation had significantly shorter survival. Conclusions and Clinical Importance B‐cell chronic lymphocytic leukemia had a variable clinical course and Boxer dogs and cases with high Ki67 had more aggressive disease.

Abstract Context The detection of low‐level persistent or relapsed B‐cell neoplasms, particularly post‐therapy, can be challenging, often requiring multiple testing modalities. Objective Here we investigate the utility of CD19‐based selection of neoplastic B‐cells (CD19S) as an enrichment strategy to improve the detection rate of cytogenetic abnormalities in post‐therapy samples of B‐cell neoplasms, especially those with low‐level disease. Design In a cohort largely comprised of post‐therapy B‐ALL and CLL samples, we performed fluorescence in situ hybridization (FISH) analysis on CD19‐selected cells (CD19S FISH) in 128 specimens from 88 patients, and on non‐selected cells (NS FISH) in a subset of cases. The FISH findings were compared with the concurrent flow cytometry (FC) results in all samples and molecular analysis in a subset. Results CD19S FISH was able to detect cytogenetic aberrations in 86.0% of post‐therapy samples with evidence of disease as determined by routine or MRD FC, compared to 59.1% of samples by NS FISH. CD19S FISH detected significantly higher percentages of positive cells compared to NS FISH (p

Abstract This study describes a simple method for the large‐scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive.

Abstract The Quercus species serve as a powerful model for studying introgression in relation to species boundaries and adaptive processes. Coexistence of distant relatives, or lack of coexistence of closely relative oak species, introgression may play a role. In the current study, four closely related oak species were found in Zijinshan, China. We generated a comprehensive genome size (GS) database for 120 individuals of four species using flow cytometry‐based approaches. We examined GS variability within and among the species and hybridization events among the four species. The mean GSs of Q. acutissima, Q. variabilis, Q. fabri, and Q. serrata var. brevipetiolata were estimated to be 1.87, 1.92, 1.97, and 1.97 pg, respectively. The intraspecific and interspecific variations of GS observed among the four oak species indicated adaptation to the environment. Hybridization occurred both within and between the sections. A hybrid offspring was produced from Q. fabri and Q. variabilis, which belonged to different sections. The GS evolutionary pattern for hybrid species was expansion. Hybridization between the sections may be affected by habitat disturbance. This study increases our understanding of the evolution of GS in Quercus and will help establish guidelines for the ecological protection of oak trees.

Abstract Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

Abstract Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid‐based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB‐containing lipoproteins, identified by the use of fluorophore‐labelled polyclonal ApoB‐antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein‐bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid‐based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins.

Abstract This report highlights the importance of integrating clinical, radiological, genetic, and pathological laboratory findings to make a correct diagnosis especially with challenging and rare entities.

Abstract Opioid receptor activation was shown to enhance the efficacy of anti‐neoplastic drugs in several human cancer cell lines. In these cell lines, doxorubicin increased the number of opioid receptors and methadone concurrently enhanced cellular doxorubicin uptake. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti‐cancer therapy. Especially in veterinary medicine, where side effects of chemotherapy are tolerated to a lesser extent and hence smaller doses are given, agents potentiating chemotherapeutic agents would be an optimal approach to treatment. Canine transitional cell carcinoma cells (TCC, K9TCC), canine osteosarcoma cells (OSA, Abrams) and canine hemangiosarcoma cells (HSA, DAL‐4) were incubated with different combinations of methadone, buprenorphine and doxorubicin, in order to test inhibition of cell proliferation. Opioid receptor density was assessed with fluorescence‐activated cell sorting in drug native and doxorubicin pretreated cells. In TCC and OSA cell lines opioid receptor density increased after doxorubicin pretreatment. In combination treatment, however, we did not find significant potentiation of doxorubicin's inhibitory effect on proliferation in these cell lines. Neither was there a significant increase of the effect of doxorubicin when the opioids were added 24 hr before doxorubicin. Hence, we could not confirm the hypothesis that opioids increase the anti‐proliferative effect of the anti‐neoplastic drug doxorubicin in any of these canine tumour cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer.

Abstract Background Programed cell death protein 1 (PD‐1) is a key mediator for the development of T cell exhaustion that develops in response to persistent antigen stimulation. Aim In this study, we measured PD1 expression on CD3 positive bone marrow T‐lymphocytes in newly diagnosis AML patients and its relation to clinical/ prognostic outcomes in addition to response to induction therapy (day 28). Methods This study was conducted on 59 newly diagnosed AML patients and 20 healthy controls. Complete blood counts, flow cytometry using acute leukemia panel in addition to PD1 monoclonal antibodies were performed on bone marrow lymphocytes (CD3+), whereas cytogenetic/molecular studies were used to determine risk group. The patients’ remission status following induction therapy was determined. Results PD1 was brightly expressed in 91.5% of the cases than control sample with highly significant difference (P = .001). A cutoff of 3.5 for mean fluorescence intensity was used to divide patients into two groups (higher vs normal PD1 expression). A significant difference between the two groups regarding platelet count and aberrant CD7 expression (P = .007 and .023, respectively) was found. Those normally expressed PD1 respond better to induction therapy. Conclusion PD1 expression on BM T‐cells had a predictive value and providing an immunotherapeutic target for AML.

Abstract Background The CLN3 disease spectrum ranges from a childhood‐onset neurodegenerative disorder to a retina‐only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well‐known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. Methods Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal‐associated membrane protein 1 (LAMP‐1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. Results Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole‐shaped LAMP‐1 expression, suggesting the use of LAMP‐1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP‐1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in‐depth revealing that intracellular LAMP‐1 expression was most pronounced in T‐cells of classical‐protracted CLN3 disease while it was most pronounced in B‐cells of “retina‐only” CLN3 disease. Conclusion Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

Abstract The shallow Caroline Seamount is located in the tropical western Pacific Ocean. Its summit is 57 m below the surface and penetrates the euphotic zone. Therefore, it is ideal for the study of the influence of seamount on plankton distribution. Here, virioplankton abundance and distribution were investigated by flow cytometry (FCM) in the Caroline Seamount in August and September 2017. The total abundance of virus‐like particles (VLP) was in the range of 0.64 × 106–18.77 × 106 particles/ml and the average was 5.37 ± 3.75 × 106 particles/ml. Three to four distinct viral subclusters with similar side scatter but different green fluorescence intensities were identified. Above the deep chlorophyll maximum (DCM), two medium fluorescence virus (MFV) subclusters were discriminated. Between the DCM and the deeper layers, only one MFV subcluster was resolved. In general, low fluorescence viruses (LFV) comprised the most abundant subclusters. In the 75–150 m water column, however, the MFV abundance was higher than the LFV abundance. High fluorescence viruses (HFV) constituted the least abundant subcluster throughout the entire water column. Virioplankton abundance was significantly enhanced at the seamount stations. Environmental factors including water temperature and nitrate concentration were the most correlated with the variation in virioplankton abundance at the seamount stations. Interactions between shallow seamounts and local currents can support large virus standing stocks, causing a so‐called indirect “seamount effect” on the virioplankton.

Abstract Background Antiplatelet antibodies are detected in multiple diseases including primary immune thrombocytopenia (ITP). Dynamics of how these antibodies change over time in ITP is unknown in dogs. Hypothesis/Objectives Antiplatelet antibodies (APA) will be detected in thrombocytopenic dogs with multiple etiologies and dynamics of APA in dogs with ITP can be used to evaluate response to treatment and relapse. Determine APA at the time of diagnosis in thrombocytopenic dogs and serially in primary ITP dogs. Animals Seventy‐nine thrombocytopenic dogs and 28 primary ITP dogs. Methods Direct flow cytometry was performed in thrombocytopenic dogs at initial evaluation and serially in suspected primary ITP dogs. In primary ITP dogs, a 2‐tailed Fisher's exact test was performed comparing survival to discharge between dogs with and without melena and to relate response to treatment and relapse to changes in APA and platelet count (repeated measures analysis, Spearman correlation). Results Twenty percent (16/79) of thrombocytopenic non‐ITP dogs with infectious, neoplastic, or other diseases and all primary ITP dogs were positive for APA. Melena at initial evaluation was associated with decreased survival to discharge (odds ratio 0.06; P = .01). Persistence of APA was not associated with response to treatment, but recurrence of antibodies was associated with relapse (odds ratio 205.0; P

Abstract Introduction CD64 expression increases on neutrophils during bacterial infections. Recently an increase in CD169 expression has been discovered on monocytes during viral infections. Generally, interferons α (IFNsα) and IFNsγ are key drivers of the infectious host immune response. The purpose of this study was to explore if a link exists between these IFNs and both biomarkers. Methods Whole blood samples from healthy volunteers were stimulated with either IFNs, interleukins, or infectious extracts, to mimic an infectious state. Expressions of CD64 and CD169 were assessed in these samples by multiple flow cytometry methods, over precise kinetics. Results The expression of CD64 was statistically higher in samples stimulated with IFNγ, and CD169 in those stimulated with IFNα (and all other type I IFNs). Surface expressions are directly induced by their respective IFNs via Janus kinase/signal transducer and activator of transduction pathways within 6 to 8 hours of incubation. Mixing both types of IFNs seemed to indicate that they partially inhibit each other. Conclusions The induction of CD169 on monocytes and CD164 on neutrophils by type I and type II IFNs confirms the relevance of these markers for assessing between a viral‐ vs bacterial‐oriented immune response.

Abstract Background Lymphocytosis is relatively common in cats, but few studies describe lymphocyte populations or the clinical course associated with different immunophenotypic expansions. Hypothesis/Objectives We hypothesized that cats frequently develop non‐neoplastic lymphocytosis and that different neoplastic immunophenotypes have variable prognoses. We aimed to characterize the lymphocyte expansions in a large population of cats with lymphocytosis and to assess clinical presentation and outcome in a subset. Animals Three cohorts of cats older than 1 year with lymphocytosis (>6000/μL) were examined to define immunophenotypic categories (n = 146), evaluate outcome (n = 94), and determine prevalence of immunophenotypes (n = 350). Methods Retrospective study of cats with blood submitted for flow cytometry. Medical records (n = 94) were reviewed for clinical data, treatment, and survival information. Results Five major immunophenotypic categories were identified: B cell, heterogeneous (≥2 lineages expanded), CD4+ T cell, CD4−CD8− (double negative [DN]) T cell, and CD5‐low‐expressing T cell. B‐cell and heterogeneous phenotypes were more consistent with a non‐neoplastic process, having polyclonal antigen receptor gene rearrangements, younger age at presentation, lower lymphocyte counts, and prolonged survival. The neoplastic phenotypes, CD4+ T cell, DN T cell, and CD5 low T cell, had different median survival times (752 days [n = 37], 271 days [n = 7], 27.5 days [n = 12], respectively). Among CD4+ T‐cell cases, cats with abdominal lymphadenopathy, intestinal involvement, or both and females had shorter survival. Among 350 cats with lymphocytosis, CD4+ T‐cell lymphocytosis was most common, followed by heterogeneous and B‐cell phenotypes. Conclusions and Clinical Importance Neoplastic CD4+ T‐cell lymphocytosis is common in cats and has a prolonged clinical course compared to aberrant T‐cell phenotypes. Cats with heterogeneous and B‐cell lymphocyte expansions commonly have non‐neoplastic disease.