Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Mechanism of action of Coptidis Rhizome in treating periodontitis based on network pharmacology and in vitro validation.

OBJECTIVE: Explore the therapeutic mechanism of Coptidis Rhizome (CR) in periodontitis using network pharmacology, and validate it through molecular docking and in vitro experiments. METHODS: Screened potential active components and target genes of CR from TCMSP and Swiss databases. Identified periodontitis-related target genes using GeneCards. Found common target genes using Venny. Conducted GO and KEGG pathway analysis. Performed molecular docking and in vitro experiments using Berberine, the main active component of CR, on lymphocytes from healthy and periodontitis patients. Assessed effects on inflammatory factors using CCK-8, flow cytometry, and ELISA. RESULTS: Fourteen active components and 291 targets of CR were identified. 30 intersecting target genes with periodontitis were found. GO and KEGG analysis revealed oxidative stress response and IL-17 signaling pathway as key mechanisms. Molecular docking showed strong binding of Berberine with ALOX5, AKT1, NOS2, and TNF. In vitro experiments have demonstrated the ability of berberine to inhibit the expression of Th17 + and other immune related cells in LPS stimulated lymphocytes, and reduce the secretion of IL-6, IL-8, and IL-17. CONCLUSION: CR treats periodontitis through a multi-component, multi-target, and multi-pathway approach. Berberine, its key component, acts through the IL-17 signaling pathway to exert anti-inflammatory effects.

Unravelling B cell heterogeneity: insights into flow cytometry-gated B cells from single-cell multi-omics data.

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches. Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases. Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data (AlliGateR). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations. Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets.

Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Diagnosis of rare entity CD138 negative plasma cell multiple myeloma: the synergy between laboratory and clinician makes the difference.

Plasma cell multiple myeloma (MM) is a multiform clinical entity characterized by different laboratory hallmarks. This case shows a rare entity of plasma cell myeloma: the entire plasma cell population lack the CD138 expression. In this case, a careful analysis of laboratory finding, particular flow cytometry gating strategies and the use of other ancillary laboratory tests, guide the clinicians to correct diagnosis. The correct evaluation of pre-analytical phase and the correct gating strategy are the necessary conditions to produce robust and solid flow cytometric results. The diagnostic implications of CD138-negative plasma cell are strictly linked to stem cell-like clonogenic features, such as possible more aggressive clinical behaviour and increasing probability of chemotherapy resistance. At this time, clinical laboratory remains the main reference point to MM diagnosis.

Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19- in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency.

Background: Primary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined. Methods: In this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH -/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy). Results: Results from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively. Conclusions: Multivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

Changes in subset distribution and impaired function of circulating natural killer cells in patients with colorectal cancer.

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing.

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Microfluidics identify moso bamboo and henon bamboo by leaf protoplast subpopulations with single-cell analysis.

Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09 (± 0.39) × 108 V/m2 and 6.54 (± 0.26) × 108 V/m2, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(± 0.51) × 108 V/m2 suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus.

Objectives: Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Methods: Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Results: Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Conclusion: Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE.

Foxp3+CD39+CD73+ regulatory T-cells are decreased in the peripheral blood of women with deep infiltrating endometriosis.

Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms.

Comparative study of two laboratory techniques for the detection of HLA-B27 in patients with axial spondyloarthritis: a cross-sectional analysis.

BACKGROUND: The diagnostic and prognostic relevance of Human Leukocyte Antigen B-27 (HLA-B27) in Axial Spondyloarthritis (AxSpA) is undeniable, with 70% of Ankylosing Spondylitis (AS) patients carrying the B27 gene, contrasted with a mere 4.35% in the general population. Flow cytometry (FC) and Polymerase Chain Reaction (PCR) have emerged as the predominant techniques for routine HLA-B27 typing. While various studies have compared these methods, none have catered to the unique characteristics of the Brazilian demographic. Therefore, this research aims to compare FC and PCR in a Brazilian cohort diagnosed with AxSpA. METHODS: An analytical cross-sectional study was undertaken involving 62 AxSpA outpatients from a Brazilian University Hospital. Both FC and PCR-SSP assays were utilized to ascertain HLA-B27 typing. The outcomes (either confirming or refuting the allele's presence) underwent rigorous scrutiny. Agreement between the methodologies was assessed using the kappa statistic. A p-value of < 0.05 was deemed statistically significant. RESULTS: Of the participants, 90.3% (n = 56) were HLA-B27 positive according to FC, while 79% (n = 49) were identified as positive using the PCR method. FC exhibited a sensitivity rate of 98% paired with a specificity of 38.5%. The Positive Predictive Value for FC stood at 85.7%, and the Negative Predictive Value was 83.5%. Consequently, the overall accuracy of the FC method was gauged at 85.5%. A kappa coefficient of κ = 0.454 was derived. CONCLUSIONS: FC demonstrated noteworthy sensitivity and satisfactory accuracy in HLA-B27 detection, albeit with a reduced specificity when contrasted with PCR-SSP. Nevertheless, given its cost-effectiveness and streamlined operation relative to PCR, FC remains a pragmatic option for preliminary screening in clinical practice, especially in low-income regions. To optimize resource allocation, we advocate for a refined algorithm that initiates by assessing the relevance of HLA-B27 typing based on Choosing Wisely recommendations. It then leans on FC, and, if results are negative yet clinical suspicion persists, advances to PCR. This approach aims to balance diagnostic accuracy and financial prudence, particularly in regions contending with escalating medical costs.

Immunophenotype of myeloid granulocytes in Chinese patients with BCR::ABL1-negative myeloproliferative neoplasms.

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

Trichostatin A-modified vaccine provides superior protection against ovarian cancer formation and development.

More attention has been paid to immunotherapy for ovarian cancer and the development of tumor vaccines. We developed a trichostatin A (TSA)-modified tumor vaccine with potent immunomodulating activities that can inhibit the growth of ovarian cancer in rats and stimulate immune cell response in vivo. TSA-treated Nutu-19 cells inactivated by X-ray radiation were used as a tumor vaccine in rat ovarian cancer models. Prophylactic and therapeutic experiments were performed with TSA-modified tumor vaccine in rats. Flow cytometry and ELISpot assays were conducted to assess immune response. Immune cell expression in the spleen and thymus were detected by immunohistochemical staining. GM-CSF, IL-7, IL-17, LIF, LIX, KC, MCP-1, MIP-2, M-CSF, IP-10/CXCL10, MIG/CXCL9, RANTES, IL-4, IFN-γ, and VEGF expressions were detected with Milliplex Map Magnetic Bead Panel immunoassay. TSA vaccination in therapeutic and prophylactic models could effectively stimulate innate immunity and boost the adaptive humoral and cell-mediated immune responses to inhibit the growth and tumorigenesis of ovarian cancer. This vaccine stimulated the thymus into reactivating status and enhanced infiltrating lymphocytes in tumor-bearing rats. The expression of key immunoregulatory factors were upregulated in the vaccine group. The intensities of infiltrating CD4+ and CD8+ T cells and NK cells were significantly increased in the vaccine group compared to the control group (P<0.05). This protection was mainly dependent on the IFN-γ pathway and, to a much lesser extent, by the IL-4 pathway. The tumor cells only irradiated by X-ray as the control group still showed a slight immune effect, indicating that irradiated cells may also cause certain immune antigen exposure, but the efficacy was not as significant as that of the TSA-modified tumor vaccine. Our study revealed the potential application of the TSA-modified tumor vaccine as a novel tumor vaccine against tumor refractoriness and growth. These findings offer a better understanding of the immunomodulatory effects of the vaccine against latent tumorigenesis and progression. This tumor vaccine therapy may increase antigen exposure, synergistically activate the immune system, and ultimately improve remission rates. A vaccine strategy designed to induce effective tumor immune response is being considered for cancer immunotherapy.

Assessing the toxicity of one-step-synthesized PEG-coated gold nanoparticles: in vitro and in vivo studies.

OBJECTIVE: To evaluate the in vitro and in vivo toxicities of polyethylene glycol-coated gold nanoparticles synthesized using a one-step process. METHODS: Gold nanoparticles were prepared via a co-precipitation method using polyethylene glycol, and the synthesis product was characterized. For the in vitro evaluation, a flow cytometry analysis with Annexin V and iodide propidium staining was used to assess cytotoxicity in MG-63 cells labeled with 10, 50, and 100µg/mL of nanoparticle concentration. For the in vivo evaluation, nanoparticles were administered intraperitoneally at a dose of 10mg/kg dose in 10-week-old mice. Toxicity was assessed 24 hours and 7 days after administration via histopathological analysis of various tissues, as well as through renal, hepatic, and hematopoietic evaluations. RESULTS: Synthesized nanoparticles exhibited different hydrodynamic sizes depending on the medium: 51.27±1.62nm in water and 268.12±28.45nm (0 hour) in culture medium. They demonstrated a maximum absorbance at 520nm and a zeta potential of -8.419mV. Cellular viability exceeded 90%, with less than 3% early apoptosis, 6% late apoptosis, and 1% necrosis across all labeling conditions, indicating minimal cytotoxicity differences. Histopathological analysis highlighted the accumulation of nanoparticles in the mesentery; however, no lesions or visible agglomeration was observed in the remaining tissues. Renal, hepatic, and hematopoietic analyses showed no significant differences at any time point. CONCLUSION: Polyethylene glycol-coated gold nanoparticles exhibit extremely low toxicity and high biocompatibility, showing promise for future studies.