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.

Allosensitisation in NHP results in cross-reactive anti-SLA antibodies not detected by a lymphocyte-based flow cytometry crossmatch.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

Inhibition of Cathepsin S in Autoimmune CD25KO Mouse Improves Sjögren Disease-Like Lacrimal Gland Pathology.

Purpose: CD25KO mice are a model of Sjögren disease (SjD) driven by autoreactive T cells. Cathepsin S (CTSS) is a protease crucial for major histocompatibility complex class II presentation that primes T cells. We investigated if a diet containing CTSS inhibitor would improve autoimmune signs in CD25KO mice. Methods: Four-week female CD25KO mice were randomly chosen to receive chow containing a CTSS inhibitor (R05461111, 262.5 mg/kg chow) or standard chow for 4 weeks. Cornea sensitivity was measured. Inflammatory score was assessed in lacrimal gland (LG) histologic sections. Flow cytometry of LG and ocular draining lymph nodes (dLNs) investigated expression of Th1 and Th17 cells. Expression of inflammatory, T- and B-cell, and apoptotic markers in the LG were assessed with quantitative PCR. The life span of mice receiving CTSS inhibitor or standard chow was compared. CD4+ T cells from both groups were isolated from spleens and adoptively transferred into RAG1KO female recipients. Results: Mice receiving CTSS inhibitor had better cornea sensitivity and improved LG inflammatory scores. There was a significant decrease in the frequency of CD4+ immune cells and a significant increase in the frequency of CD8+ immune cells in the dLNs of CTSS inhibitor mice. There was a significant decrease in Th1 and Th17 cells in CTSS inhibitor mice in both LGs and dLNs. Ifng, Ciita, and Casp8 mRNA in CTSS inhibitor mice decreased. Mice that received the CTSS inhibitor lived 30% longer. Adoptive transfer recipients with CTSS inhibitor-treated CD4+ T cells had improved cornea sensitivity and lower inflammation scores. Conclusions: Inhibiting CTSS could be a potential venue for the treatment of SjD in the eye and LG.

MYELOID-DERIVED SUPPRESSOR CELLS TWO YEARS AFTER HEPATITIS C VIRUS ERADICATION USING DIRECTLY ACTING ANTIVIRALS.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) have immature morphology, relatively weak phagocytic activity, as well as some immunosuppressive functions. The capacity of MDSCs to inhibit T-cell-mediated immunological responses is their most notable functional characteristic. Down-regulating antitumor immune surveillance is one way that the expansion and activation of MDSCs contribute significantly to the occurrence and progression of tumors. Increased levels of MDSCs in patients with chronic hepatitis C virus (HCV) infection could suppress T-cell responses, promoting viral escape and hepatitis progression. This may make HCV-infected individuals more vulnerable to severe infections, hepatic and extra-hepatic tumors, and a diminished capacity to react to immunization. It is still unknown if effective HCV eradication with directly acting antivirals (DAAs) can restore immune functions and immune surveillance capacity. OBJECTIVE: The purpose of this study was to observe the frequency of M-MDSCs (CD33+, CD11b+, and HLA-DR) in patients with a previous history of HCV, 2-3 years after virus eradication using DAA therapy. METHODS: This study was conducted on 110 subjects: fifty-five subjects without liver cirrhosis who were treated with HCV using DAAs and attained SVR for a period of 2-3 years and 55 age- and gender-matched healthy controls. The study was conducted during the period from January to July 2022. Patients were recruited from the National Viral Hepatitis Treatment Unit, Alexandria University Hepatology outpatient clinic, and the Alexandria University Tropical Medicine outpatient clinic. The frequencies of MDSCs (CD33+CD11b + HLA-DR-) by flow cytometry were assessed. RESULTS: Even after the virus had been eradicated for longer than two years, MDSC levels in HCV-treated individuals were found to be considerably higher. In the HCV-treated group, the median number of MDSCs was 5, with an interquartile range (IQR) of 3.79-7.69. In contrast, the median for the control group was 3.1, with an IQR of 1.4-3.2 (P˂0.001). CONCLUSION: Successful DAA therapy leads to slow and partial immunological reconstitution, as demonstrated by the failure to attain normal levels of MDSC's 2 years after successful HCV eradication despite the normalization of laboratory parameters as well as the absence of liver fibrosis. The clinical implications of these findings should be thoroughly studied.

Increased pathogenicity and pro-inflammatory capabilities of mucosal-associated invariant T cells involved in Oral Lichen Planus.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells assume pivotal roles in numerous autoimmune inflammatory maladies. However, scant knowledge exists regarding their involvement in the pathological progression of oral lichen planus (OLP). The focus of our study was to explore whether MAIT cells were altered across distinct clinical types of OLP. METHODS: The frequency, phenotype, and partial functions of MAIT cells were performed by flow cytometry, using peripheral blood from 18 adults with non-erosive OLP and 22 adults with erosive OLP compared with 15 healthy adults. We also studied the changes in MAIT cells in 15 OLP patients receiving and 10 not receiving corticosteroids. Surface proteins including CD4, CD8, CD69, CD103, CD38, HLA-DR, Tim-3, Programmed Death Molecule-1 (PD-1), and related factors released by MAIT cells such as Granzyme B (GzB), interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-17A, and IL-22 were detected. RESULTS: Within non-erosive OLP patients, MAIT cells manifested an activated phenotype, evident in an elevated frequency of CD69+ CD38+ MAIT cells (p < 0.01). Conversely, erosive OLP patients displayed an activation and depletion phenotype in MAIT cells, typified by elevated CD69 (p < 0.01), CD103 (p < 0.05), and PD-1 expression (p < 0.01). Additionally, MAIT cells exhibited heightened cytokine production, encompassing GzB, IFN-γ, and IL-17A in erosive OLP patients. Notably, the proportion of CD103+ MAIT cells (p < 0.05) and GzB secretion (p < 0.01) by MAIT cells diminished, while the proportion of CD8+ MAIT cells (p < 0.05) rose in OLP patients with corticosteroid therapy. CONCLUSIONS: MAIT cells exhibit increased pathogenicity and pro-inflammatory capabilities in OLP. Corticosteroid therapy influences the expression of certain phenotypes and functions of MAIT cells in the peripheral blood of OLP patients.

Monoclonal antibodies for equine IL-1ß enable the quantification of mature IL-1ß in horses.

Interleukin-1ß (IL-1ß) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1ß mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1ß are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1ß. The specificity of the new IL-1ß mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1ß in a fluorescent bead-based assay and for staining of IL-1ß-producing immune cells by flow cytometry. The bead-based assay for equine IL-1ß had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1ß after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1ß bead-based assay. A comparison of two commercial equine IL-1ß ELISA kits with the new IL-1ß fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1ß in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1ß recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1ß in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1ß secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1ß within the cells even when proteolytic cleavage for IL-1ß activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1ß in horses.

Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

Surra-affected dromedary camels show reduced numbers of blood B-cells and in vitro evidence of Trypanosoma-induced B cell death.

Trypanosomosis due to Trypanosoma evansi (surra) is one of the most important diseases with a significant impact on camel health and production. Trypanosoma-induced immunosuppression mechanisms, which are key factors of disease pathogenesis, have been characterized in several animal species. The present study investigated, therefore, the impact of trypanosomosis on the immunophenotype of blood leukocytes in camels. For this, the relative and absolute values of blood leukocyte populations, their expression pattern of cell surface molecules, and the numbers of the main lymphocyte subsets were compared between healthy camels and camels with clinical symptoms of chronic surra and serological evidence of exposure to Trypanosoma infection. Leukocytes were separated from the blood of healthy and diseased camels, labeled with fluorochrome-conjugated antibodies, and analyzed by flow cytometry. Compared to healthy camels, the leukogram of diseased camels was characterized by a slightly increased leukocyte count with moderate neutrophilia and monocytosis indicating a chronic inflammatory pattern that may reflect tissue injury due to the long-lasting inflammation. In addition, the analysis of lymphocyte subsets revealed a lower number and percentage of B cells in diseased than healthy camels. In vitro incubation of camel mononuclear cells with fluorochrome-labeled T. evansi revealed a higher capacity of camel B cells than T cells to bind the parasite in vitro. Furthermore, cell viability analysis of camel PBMC incubated in vitro with T. evansi whole parasites but not the purified antigens resulted in Trypanosoma-induced apoptosis and necrosis of camel B cells. Here we demonstrate an association between trypanosomosis in camels and reduced numbers of blood B cells. In vitro analysis supports a high potential of T. evansi to bind to camel B cells and induce their elimination by apoptosis and necrosis.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Mutagenesis and fluorescence-activated cell sorting of oleaginous Saccharomyces cerevisiae and the multi-omics analysis of its high lipid accumulation mechanisms.

Acquiring lipid-producing strains of Saccharomyces cerevisiae is necessary for producing high-value palmitoleic acid. This study sought to generate oleaginous S. cerevisiae mutants through a combination of zeocin mutagenesis and fluorescence-activated cell sorting, and then to identify key mutations responsible for enhanced lipid accumulation by multi-omics sequencing. Following three consecutive rounds of mutagenesis and sorting, a mutant, MU310, with the lipid content of 44%, was successfully obtained. Transcriptome and targeted metabolome analyses revealed that a coordinated response involving fatty acid precursor biosynthesis, nitrogen metabolism, pentose phosphate pathway, ethanol conversion, amino acid metabolism and fatty acid ß-oxidation was crucial for promoting lipid accumulation. The carbon fluxes of acetyl-CoA and NADPH in lipid biosynthesis were boosted in these pathways. Certain transcriptional regulators may also play significant roles in modulating lipid biosynthesis. Results of this study provide high-quality resource for palmitoleic acid production and deepen the understanding of lipid synthesis in yeast.

Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Bisphenol-A induced cyto-genotoxicity on retinal pigment epithelial cells is differentially modulated by a multi-supplement containing guarana, selenium, and L-carnitine.

Bisphenol A (BPA) may adversely affect human health by inducing oxidative stress and irreversible damage to cells. Bioactive compounds found in some functional foods, individually or in combination, can attenuate the negative effects of BPA exposure; an example is the multi-supplement containing guarana (Gua), selenium (Se), and L-carnitine (LC) -GSC- which has already demonstrated antioxidant, genoprotective, and immunomodulatory activities. This study aimed to determine the effect of GSC and its constituents on oxidative and genotoxic alterations triggered by BPA exposure in the retinal epithelial cell line. The cells exposed to BPA (0.001, 0.01, 0.1, 1, 3, and 10 µM) to determine the lowest concentration required to induce cyto-genotoxicity. ARPE-19 cells were then concomitantly exposed to the selected BPA concentration, GSC, and its components (Gua, 1.07 mg/mL; Se, 0.178 µg/mL; and LC, 1.43 mg/mL). Flow cytometry, biochemical assays, qRT-PCR, genotoxicity, apoptosis, and cellular proliferation. Based on our results, 10 µM of BPA could induce cyto-genotoxic and oxidative alterations. BPA did not alter the Bcl-2/BAX expression ratio but induced Casp3 and Casp8 overexpression, suggesting that apoptosis was induced mainly via the extrinsic pathway. GSC partially reversed the alterations triggered by BPA in ARPE-19 cells. However, Se had unexpected negative effects on ARPE-19 cells. The multi-supplement GSC may attenuate changes in oxidative and genotoxic markers related to exposure of ARPE-19 cells to BPA. our results revealed that the antioxidant, anti-apoptotic, and genoprotective properties of GSC were not universally shared by its individual, once Se did not exhibit any positive impact.

[Characterizing immune microenvironment cell composition of chronic rhinosinusitis with nasal polyps based on mass cytometry technology].

Objective: To analyze the cellular composition characteristics of the nasal tissue immune microenvironment in patients with control, chronic rhinosinusitis without nasal polyps (CRSsNP), non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP), and eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) using mass cytometry flow technology. Methods: Thirteen CRS patients who underwent endoscopic nasal surgery at the Department of Otorhinolaryngology Head and Neck Surgery of Peking Union Medical College Hospital from March to December 2022 were recruited, including 8 males and 5 females, aged 22.3 to 58.3 years. Three control mucosae were obtained from normal ethmoid or sphenoid sinuses of patients with benign tumors of the temporal fossa or non-functional pituitary adenomas who underwent endoscopic surgery, excluding allergic rhinitis and sinusitis. Sixteen clinical tissue samples (3 of control, 3 of CRSsNP, 4 of neCRSwNP, and 6 of eCRSwNP) were prepared into single-cell suspensions. Mass cytometry flow detection was performed using a combination of 42 molecular markers to analyze the differences in cell subpopulations among the groups. Data were analyzed using GraphPad Prism 9. Results: Based on the mass cytometry flow results, cells from control, CRSsNP, neCRSwNP, and eCRSwNP were divided into seven main cell subgroups, with detailed subgrouping of T/NK cells and myeloid cells. In T/NK cells, compared with the control group, the number of NK CD56bright cells increased in the CRSsNP group, while NK CD56dim cells decreased; compared with the CRSsNP group, the eCRSwNP group showed a decrease in NKT cells and CD4+Tem cells; compared with the CRSsNP group, the eCRSwNP group showed a significant increase in CD25 expression within Treg cells; compared with the CRSsNP group, the eCRSwNP group showed a significant decrease in Tbet expression in CD8+Teff cells and CD8+TRM cells; in eCRSwNP, the expression of CD103 in CD8+TRM cells was significantly lower than in CRSsNP. In myeloid cells, compared with the other three groups, the eCRSwNP group showed a significant increase in macrophages and a significant decrease in cDC1 and monocytes; compared with the control group and CRSsNP, the eCRSwNP group also showed a significant decrease in resting state macrophages; compared with the CRSsNP group, the eCRSwNP group showed a significant decrease in the level of CX3CR1 within cDC2 and monocytes; the expression levels of NLRP3 in cDC2 and macrophages in the eCRSwNP group were significantly higher than in the other three groups; compared with the control group, the expression levels of Gata3 in cDC2 and macrophages in the eCRSwNP group were also significantly increased; additionally, the expression of CCR2 within monocytes in the eCRSwNP group was lower than in the CRSsNP group. In ILC, compared with the control group, the expression of CCR6 decreased in the eCRSwNP group. Conclusions: Compared with the control group, CRSsNP, and neCRSwNP, eCRSwNP shows an increase in macrophage number, a decrease in cDC1 and resting state macrophages, and depletion of protective cells CD103+CD8+TRM. Additionally, the expression levels of CCR2 and CX3CR1 in monocytes of eCRSwNP are decreased.

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

Identifying antibiotic-resistant strains via cell sorting and elastic-light-scatter phenotyping.

The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: • Individual bacteria cells are placed on gradient agar plates by a cell sorter • Laser-light scatter patterns are used to recognize antibiotic-resistant organisms • Scatter patterns formed by colonies correspond to AMR-associated phenotypes.

Severe CSF immune cell alterations in cryptococcal meningitis gradually resolve during antifungal therapy.

Cryptococcal meningitis (CM) is a severe fungal disease in immunocompromised patients affecting the central nervous system (CNS). Host response and immunological alterations in the cerebrospinal fluid (CSF) after invasion of Cryptococcus neoformans to the central nervous system have been investigated before but rigorous and comprehensive studies examining cellular changes in the CSF of patients with cryptococccal meningitis are still rare. We retrospectively collected CSF analysis and flow cytometry data of CSF and blood in patients with CM (n = 7) and compared them to HIV positive patients without meningitis (n = 13) and HIV negative healthy controls (n = 7). Within the group of patients with CM we compared those with HIV infection (n = 3) or other immunocompromised conditions (n = 4). Flow cytometry analysis revealed an elevation of natural killer cells and natural killer T cells in the CSF and blood of HIV negative patients with CM, pointing to innate immune activation in early stages after fungal invasion. HIV positive patients with CM exhibited stronger blood-CSF-barrier disruption. Follow-up CSF analysis over up to 150 days showed heterogeneous cellular courses in CM patients with slow normalization of CSF after induction of antifungal therapy.

Performance of immunological assays for universal and differential diagnosis of HTLV-1/2 infection in candidates for blood donations from the Brazilian Amazon.

The present study compares the ability of distinct immunological assays (chemiluminescence immunoassay-CLIA, western blot-WB and flow cytometry-FC-Simplex and Duplex) to detect anti-HTLV (human T-lymphotropic virus) antibodies in candidates for blood donations at the Amazonas State Blood Center (Brazil) between January 2018 and December 2022. Overall, 257,942 samples from candidates for blood donations were screened using CLIA, which led to 0.15% seropositivity for HTLV (409 samples). A total of 151 candidates for blood donations were enrolled for retesting with CLIA followed by additional testing using WB and FC-Simplex and Duplex analysis. Our results demonstrated that 62% (93/151), 20% (30/151) and 17% (26/151) of the samples presented positive results with retesting using CLIA, WB and FC-Simplex analysis, respectively. Additional analysis of the CLIA, WB and FC-Simplex results revealed an overall agreement of 56% for CLIA and WB (22 co-negative; 30 co-positive samples), 48% for CLIA and FC-Simplex (21 co-negative; 24 co-positive samples) and 80% for WB and FC-Simplex (51 co-negative; 23 co-positive samples). Considering the WB as the reference standard for the diagnosis of infection with HTLV-1/2, we observed that the CLIA results of ≤3.0 RLU and >10.0 RLU in the retest can be used define a negative or positive result, respectively, and could be used as new specific cut-off values. The overall agreement between WB and FC-Duplex for accomplishing the differential diagnosis was evaluated and demonstrated 100% correspondence for the diagnosis of HTLV-1 (15/15) and HTLV-2 (7/7). Our findings demonstrate that gaps in the diagnosis of infection with HTLV-1/2 could be overcome by the simultaneous use of distinct immunological assays during retesting of candidates for blood donations.

Diagnostic landscape of first-time cytometric screening for paroxysmal nocturnal hemoglobinuria in Poland in 2013-2022.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by PIG-A mutations, leading to glycophosphatidylinositol (GPI)-anchored proteins deficiency that triggers hemolysis - a hallmark of the disease. PNH diagnostics is based on high-sensitivity multicolor flow cytometry (MFC), enabling to detect even small populations of PNH cells. In this single-center, retrospective study, we aimed to characterize a cohort of PNH clone-positive patients first time screened from January 1st, 2013 until December 31st, 2022 with MFC according to International Clinical Cytometry Society PNH Consensus Guidelines. RESULTS: Out of 2790 first-time screened individuals, the presence of PNH clone in neutrophils was detected in 322 patients, including 49 children and 273 adults. Annual incidence was stable at a median of 31 patients (14 and 19 with clone sizes ≤ 1% and > 1%, respectively), with a decline in number of patients with clone sizes > 1% observed in 2020, potentially influenced by the COVID-19 pandemic. The most common screening indications were aplastic anemia and other cytopenias. CONCLUSIONS: A significant underrepresentation of hemolytic patients was observed as compared to the published cohorts suggesting that these patients are missed in diagnostic process and classic PNH remains underdiagnosed in Poland.

JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission.

OBJECTIVE: To investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission. METHODS: Peripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry. RESULTS: Compared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased. CONCLUSION: RA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

Circulating/cerebrospinal T lymphocytes as indicators of clinical prognosis in intracerebral hemorrhage: A prospective study.

Secondary injury of cerebral hemorrhage is induced by systemic inflammatory cascades, which are related to perihematomal brain edema, cellular apoptosis, and the disruption of the blood-brain barrier. This study was to specifically elaborate the relationship of circulating/cerebrospinal T lymphocytes and Glasgow Coma Scale (GCS) score at 6 months after intracerebral hemorrhage (ICH). The enrolled patients were divided into 2 groups based on GCS score: the favorable prognosis group (GCS > 12) and unfavorable prognosis group (GCS ≤ 12). T lymphocyte subpopulations were analyzed by flow cytometry. A total of 30 samples of peripheral blood and 17 samples of cerebrospinal fluid were collected and analyzed, including 19 cases and 12 cases in the favorable prognosis group (GCS > 12) respectively. Both CD3+ and CD3+CD4+ T lymphocyte counts on Day 1 after ICH were lower in the peripheral blood of patients with unfavorable prognosis (GCS ≤ 12) (P = .025 and .022, respectively). There were correlation trends between the GCS scores and CD3+ T lymphocyte count (P = .0144), and CD3+CD4+ T lymphocyte count (P = .0135). In cerebrospinal fluid, there was a close correlation between the GCS scores and CD3+CD4+ percentage, CD4+/CD8+ ratio, CD3+ and CD3+CD4+ T lymphocyte counts. The area under the curve of CD4+/CD8+ T lymphocyte ratio was the largest among them (P = .000 and area under the curve = 0.917), with a significantly high specificity and sensitivity (0.917 and 1.000). Based on cerebrospinal fluid samples, the CD4+/CD8+ T lymphocyte ratio on Day 1 after ICH may be a more significant indicator to predict the short-term prognosis at 6 months after ICH.