Fluorescent-labeled Antibodies Research Articles

Detection of human coronavirus-OC43 in nasopharyngeal swab specimens via immunofluorescence staining using human serum and an anti-human antibody.

Immunofluorescence methods to detect seasonal human coronavirus (HCoV)-OC43 in nasopharyngeal swab specimens using cell lines have not yet been established. A human rectal adenocarcinoma cell line (HRT-18) was exposed to the specimens obtained from patients with upper respiratory tract infections. Immunofluorescence staining was conducted with the combination of human serum containing the HCoV-OC43 anti-spike protein antibody and a fluorescence-labeled anti-human antibody. Positive staining in HRT-18 cells was detected after exposure to specimens obtained from nine of the eleven patients in which HCoV-OC43 RNA was detected using the FilmArray method. Increased supernatant viral RNA levels were also detected in HRT-18 cells exposed to specimens obtained from four of five patients. In contrast, positive staining was not detected in HRT-18 cells exposed to six patient specimens that tested negative for RNA from seventeen types and subtypes of respiratory viruses, including HCoV-OC43. The cells inoculated with the established strain HCoV-OC43 (ATCC VR-759) also showed positive staining. These findings suggest that the replication-competent HCoV-OC43 in the specimens could be detected via immunofluorescence staining of HRT-18 cells with human serum. It may be possible to obtain positive staining for viruses other than HCoV-OC43 using this method.

The potential of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit.

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR-dCas9-based genome labeling tool, known as CRISPR-FISH, allows efficient labeling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on further development of the CRISPR-FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an Arabidopsis centromere-specific guide RNA, we demonstrate target-specific labeling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labeled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9-ALFA-tag system was also instrumental in live cell imaging of telomeres in Nicotiana benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive tyramide signal amplification method with CRISPR-FISH, demonstrating effective labeling of Arabidopsis centromeres.

Human stem cell-derived neurons and astrocytes to detect novel auto-reactive IgG response in immune-mediated neurological diseases.

Up to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known central nervous system (CNS) antigens. We developed a cell-based assay (CBA) to screen for novel neural antibodies in serum and cerebrospinal fluid (CSF) using neurons and astrocytes derived from human-induced pluripotent stem cells (hiPSCs). Human iPSC-derived astrocytes or neurons were incubated with serum/CSF from 99 patients [42 with inflammatory neurological diseases (IND) and 57 with non-IND (NIND)]. The IND group included 11 patients with previously established neural antibodies, six with seronegative neuromyelitis optica spectrum disorder (NMOSD), 12 with suspected autoimmune encephalitis/paraneoplastic syndrome (AIE/PNS), and 13 with other IND (OIND). IgG binding to fixed CNS cells was detected using fluorescently-labeled antibodies and analyzed through automated fluorescence measures. IgG neuronal/astrocyte reactivity was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were used as CNS-irrelevant control target cells. Reactivity profile was defined as positive using a Robust regression and Outlier removal test with a false discovery rate at 10% following each individual readout. Using our CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19/99 subjects. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases, and to both cell types in two cases, as confirmed by microscopy single-cell analyses. Highlighting the significance of our comprehensive 96-well CBA assay, neural-specific antibody binding was more frequent in IND (15 of 42) than in NIND patients (4 of 57) (Fisher's exact test, p = 0.0005). Two of four AQP4+ NMO and four of seven definite AIE/PNS with intracellular-reactive antibodies [1 GFAP astrocytopathy, 2 Hu+, 1 Ri+ AIE/PNS)], as identified in diagnostic laboratories, were also positive with our CBA. Most interestingly, we showed antibody-reactivity in two of six seronegative NMOSD, six of 12 probable AIE/PNS, and one of 13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC-detected antibody binding in 13 versus zero patients, respectively, establishing the specificity of the detected antibodies for neural tissue. Our unique hiPSC-based CBA allows for the testing of novel neuron-/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing a diagnosis of such complex diseases.

Experience in the production and clinical application of the cell-based medicinal product Easytense® for the repair of cartilage defects of the human knee

INTRODUCTION. The current cell-based cartilage repair methods, such as autologous chondrocyte transplantation, are not sufficiently effective, and the surgery is painful and traumatic. Therefore, there is a need for a more effective cell therapy product with a minimally invasive surgical procedure for its implantation into the patient.AIM. This study aimed to develop a manufacturing technology for the production of an autologous cell-based medicinal product (CBMP) comprising three-dimensional structures (3D-spheroids) based on chondrocytes isolated from the patient’s cartilage tissue, as well as to evaluate its clinical efficacy.MATERIALS AND METHODS. Autologous chondrocytes isolated from the patient’s cartilage biopsy were propagated in monolayer culture to obtain the required number of cells. Subsequently, the chondrocytes were cultivated on plates with a non-adhesive coating to form 3D spheroids. All CBMP production steps were performed under aseptic conditions in cell culture isolators. The authors used phase-contrast microscopy and immunohistochemical staining with specific fluorescence-labelled antibodies to characterise chondrocyte phenotypes at different stages of cultivation. Genetic stability was controlled by karyotyping. The efficacy of Easytense® was evaluated in a clinical trial using specialised functional tests and the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. The primary efficacy endpoint was a change in the overall score on the Knee Injury and Osteoarthritis Outcome Score (KOOS).RESULTS. A manufacturing technology without using animal sera, growth factors, cytokines, or other additives was developed for the production of the autologous CBMP Easytense®. Karyological data confirmed that the chondrocytes retained genetic stability for 3 passages in monolayer culture. When cultured as 3D spheroids, the chondrocytes produced cartilage extracellular matrix proteins (type II collagen, aggrecan), thus acquiring the ability to repair damaged cartilage. The clinical trial demonstrated a statistically significant improvement in knee cartilage 12 months after the transplantation of 3D spheroids derived from autologous chondrocytes. The mean change in the overall KOOS score was 23.8±15.9.CONCLUSIONS. The clinical trial results indicate that Easytense® is highly effective for cartilage repair. Based on these results, the CBMP has been granted marketing authorisation and introduced into clinical practice in the Russian Federation. Easytense® has the potential to replace endoprosthetics and expensive surgeries abroad.

Enhancing the sensitivity of rapid antigen detection test (RADT) of different SARS-COV-2 variants and lineages using fluorescence-labeled antibodies and a fluorescent meter

Enhancing the sensitivity of rapid antigen detection test (RADT) of different SARS-COV-2 variants and lineages using fluorescence-labeled antibodies and a fluorescent meter

CD20 + T lymphocytes in isolated Hashimoto’s thyroiditis and type 3 autoimmune polyendocrine syndrome: a pilot study

BackgroundCD20+ T cells represent up to 5% of circulating T lymphocytes. These cells have been shown to produce higher levels of IL-17A and IFN-γ than those of CD20− T lymphocytes. Some reports described the role of CD20+ T cells in autoimmune disorders such as multiple sclerosis and rheumatoid arthritis possibly due to their ability to produce these inflammatory cytokines. This study is aimed at describing the behavior of CD20+ T lymphocytes in the most frequent autoimmune disorder, i.e., Hashimoto’s thyroiditis (HT), presenting isolated or associated to further autoaggressive disorders in a frame of poly-autoimmunity.MethodsThe study group encompasses 65 HT patients: 23 presenting in isolated form (IT) and 42 with an associated non-endocrine autoimmune disorder [16 with chronic atrophic gastritis (CAG), 15 with nonsegmental vitiligo (VIT), and 11 with celiac disease (CD)]. Twenty healthy donors act as control group (HD). Chronic use of interfering drugs, severe or chronic disorders, and pregnancy and lactation were used as exclusion criteria. Whole blood samples (100 µl) were stained with fluorescent-labeled antibodies (anti-CD45, anti-CD3, anti-CD19, anti-CD16, anti-CD56, anti-CD4, anti-CD8, anti-CD20). Red blood cells were then lysed by adding 1 ml of hypotonic buffer, and samples were acquired on a Flow Cytometer.ResultsCD3+CD8+CD20+ T lymphocytes’ percentages, were significantly higher in the whole group of autoimmune patients compared to healthy donors (p = 0.0145). Dividing HT patients based on the type of presentation of autoimmune thyroiditis, CAG group showed the highest percentage of these cells as compared to HD and CD (p = 0.0058). IT patients showed higher percentages of CD3+ CD8+CD20+ cells than those of HD patients although not reaching statistical significance. However, dividing IT group based on thyroid function, hypothyroid patients showed higher CD8+CD20+ cell percentages than those of HD and euthyroid patients (p = 0.0111). Moreover, in IT patients, these cells were negatively correlated with FT4 levels (p = 0.0171; r = −0.4921).ConclusionsThese preliminary findings indicate that CD8+CD20+ T cells are activated in patients with autoimmune thyroiditis and may behave differently according to the presence of poly-autoimmunity and hypothyroidism.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Evaluation of Circulating Endothelial Cells as Direct Marker of Endothelial Damage in Allo-Transplant Recipients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome

Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a rare but potentially fatal complication following allogenic hematopoietic cell transplantation (allo-HCT). Timely identification of SOS/VOD to allow for prompt treatment is critical, but identifying a VOD-predictive biomarker remains challenging. Given the pivotal role of endothelial dysfunction in SOS/VOD pathophysiology, the CECinVOD study prospectively evaluated levels of circulating endothelial cells (CECs) in patients undergoing allo-HCT with a myeloablative conditioning (MAC) regimen to investigate the potential of CEC level in predicting and diagnosing SOS/VOD. A total of 150 patients from 11 Italian bone marrow transplantation units were enrolled. All participants were age >18 years and received a MAC regimen, putting them at elevated risk of developing SOS/VOD. Overall, 6 cases of SOS/VOD (4%) were recorded. CECs were detected using the Food and Drug Administration-approved CellSearch system, an immunomagnetic selection-based platform incorporating ferrofluid nanoparticles and fluorescent-labeled antibodies, and were defined as CD146+, CD105+, DAPI+, or CD45-. Blood samples were collected at the following time points: before (T0) and at the end of conditioning treatment (T1), at neutrophil engraftment (T2), and at 7 to 10 days postengraftment (T3). For patients who developed VOD, additional samples were collected at any suspected or proven VOD onset (T4) and weekly during defibrotide treatment (T5 to T8). A baseline CEC count >17/mL was associated with an elevated risk of SOS/VOD (P = .04), along with bilirubin level >1.5 mg/mL and a haploidentical donor hematopoietic stem cell source. Postconditioning regimen (T1) CEC levels were elevated (P = .02), and levels were further increased at engraftment (P < .0001). Additionally, patients developing SOS/VOD after engraftment, especially those with late-onset SOS/VOD, showed a markedly higher relative increase (>150%) in CEC count. Multivariate analysis supported these findings, along with a high Endothelial Activation and Stress Index (EASIX) score at engraftment (T2). Finally, CEC kinetics corresponded with defibrotide treatment. After the start of therapy (T4), CEC levels showed an initial increase in the first week (T5), followed by a progressive decrease during VOD treatment (T6 and T7) and a return to pre-SOS/VOD onset levels at resolution of the complication. This prospective multicenter study reveals a low incidence of SOS/VOD in high-risk patients compared to historical data, in line with recent reports. The results from the CECinVOD study collectively confirm the endothelial injury in allo-HCT and its role in in the development of SOS/VOD, suggesting that CEC level can be a valuable biomarker for diagnosing SOS/VOD and identifying patients at greater risk of this complication, especially late-onset SOS/VOD. Furthermore, CEC kinetics may support treatment strategies by providing insight into the optimal timing for discontinuing defibrotide treatment.

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

A Universal and Programmable Platform based on Fluorescent Peptide-Conjugated Probes for Detection of Proteins in Organelles of Living Cells.

Realizing protein analysis in organelles of living cells is of great significance for developing diagnostic and therapeutic methods of diseases. Fluorescent-labeled antibodies with well imaging performance and high affinity are classical biochemical tools for protein analysis, while due to the inability to effectively enter into cells, not to mention organelles and the uncontrollable reaction sites that might cause antibodies inactivation when chemically modification, they are hard to apply to living cells. Inspired by the structure of fluorescent-labeled antibodies, we designed as a universal detection platform that was based on the peptide-conjugated probes (PCPs) and consisted of three parts: a) a rotor type fluorescent molecular scaffold for conjugation and signal output; b) the cell penetration protein recognition unit; c) the subcellular organelle targeting unit. In living cells, PCPs could firstly localize at organelles and then proceed protein specific recognition, thus jointly leading to the restriction of twisted intramolecular charge transfer and activation of fluorescence signal. As a proof-of-concept, six different proteins in three typical intracellular organelles could be detected by our platform through simply replacing the recognition sequence of proteins and matching organelle targeting units. The position and intensity of fluorescence signals demonstrated specificity of PCPs and universality of the platform.

Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100–1000 nm) and exosomes (30–150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25–2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Rapid and Sensitive On-Site Detection of Fipronil in Foods Using Evanescent Wave Fluorescent Immunosensor

Fipronil (FIP), a broad-spectrum phenylpyrazole insecticide, is highly toxic and threatens human health and ecological balance. Developing convenient, rapid, portable analytical technology for on-site and high-frequency testing of FIP is essential to reduce its damage. Herein, a monoclonal antibody (Clone F-3F6) against FIP, with high affinity and specificity, was produced using a novel immunogen, FIP-BSA, which was simply and directly synthesized by conjugating FIP with bovine serum albumin (BSA). Among the previously reported antibodies, F-3F6 acts more specifically against FIP. The FIP metabolites fipronil desulfinyl, fipronil sulfide, and fipronil sulfone showed lower cross-reactivity, and other pesticides were not recognized. To achieve high-frequency and on-site measurements of FIP, an evanescent wave fluorescence biosensor was built by integrating evanescent wave fluorescence technology, a functionalized fiber bioprobe, and a fluorescence-labeled F-3F6 antibody. The detection limit of FIP was 0.032 μg/L. The detection results of real milk and water samples showed that all the coefficients of variation were less than 10%, and the recovery ranged from 90 to 120%. The high reusability and stability of functionalized fiber bioprobe enables the accurate, cost-effective, high-frequency, and facile quantitative detection of FIP. This highly specific and reliable evanescent wave fluorescence biosensor will be well suited to the sensitive and high-frequency on-site analysis of only FIP in food.

Megakaryocytes Have Immune Characteristics in Murine Immune Bone Marrow Failure

Introduction Megakaryocytes (MKs) have familiar roles in the bone marrow (BM) microenvironment, including platelet production, maintenance of hematopoietic stem and progenitor cells (HSPCs) function, and more recent evidence suggests MK engagement in immune responses. Immune aplastic anemia (AA) is characterized by oligoclonal expansion of T-cells and death of HSPCs leading to bone marrow failure (BMF). As MKs demonstrate niche maintenance and immune effector function, we explored the potential roles of MKs in the development of AA in a murine model of immune BMF. Methods BMF was induced in CByB6F1 mice by intravenous infusion of lymph node (LN) cells from major histocompatibility complex mismatched C57BL/B6 (B6) donor mice at a dose of 30-40x10 6 cells/recipient. No irradiation was given to recipients to avoid extra damage to MKs and CByB6F1 recipient mice were sacrificed at D7, D10, D14, and D17 following LN cell infusion (Chen J, et al. Blood. 2004). Peripheral blood (PB) samples, tibias, femurs, and sterna were harvested at each timepoint. Immunophenotyping of MKs and T cells was performed by flow cytometry on flushed BM cells and two-photon excitation microscopy (2P) imaging was performed on fixed sternal samples, using fluorescence-labelled CD41 antibody to visualize the physical location of MKs in BM. To enrich for MKs, BM cells were filtered through a 10 µm pluriStrainer™ through which most lymphocytes, red blood cells, and other small cells were eluted while MKs and other large size cells remained on the strainer. Large cells and residual small cells were subsequently assessed by single cell RNA sequencing (sc-RNAseq) on the Parse Bio Evercode™ platform based on combinatorial barcoding technology, which was able to accommodate the large size of MKs, as no partitioning step is needed in the analysis workflow. Results BMF was successfully induced as measured by decreasing PB counts from point of lymphocyte infusion accompanied by increasing expansion of CD4 and CD8 T-cells and marked reduction of total BM cells. MKs persisted throughout the course of BMF as measured by both flow cytometry and 2P (Figure 1A) despite PB thrombocytopenia and progressive marrow hypocellularity. Concomitantly, MKs identified by large size on forward scatter versus side scatter and CD41+ expression had progressive upregulation of immune effector markers IA-IE and CD53 over the course of BMF. Following sc-RNAseq and bioinformatics analysis of enriched BM cells at D14, cell clustering identified a distinct cell cluster representing MKs (Figure 1B). MK identity was supported by co-expression of transcripts encoding platelet factor 4, TUBB1, Glycoprotein 5, and Itga2b (CD41). This MK cell population was distinct from macrophages which highly expressed Fcgr4, Cd5l, Ccr5, Cd74 and Gbp5 genes. Gene expression profiling demonstrated that MKs in BMF mice showed upregulation of immune pathways such as interferon gamma and interferon alpha response and downregulation of pathways related to platelet activation and platelet homeostasis, as compared with normal mice. Conclusions In a murine model of BMF, using multiple modalities, we demonstrate that MKs cease platelet production, manifested by reduction in peripheral blood platelet count and downregulation of gene expression pathways related to platelet production, while concurrently acquiring characteristics of immune effector cells. This suggests that MKs are active participants in immune BMF.

Rapid and accurate quantification of viable Bacillus cereus in foods with a Propidium monoazide (PMA) - Fluorescence in situ hybridization (FISH) - Flow cytometry (FCM) method

Fluorescence-labeled antibodies and viability indicators are routinely employed in conjunction with flow cytometry (FCM) to rapidly quantify viable target bacteria, ensuring food safety. However, the specific detection of Bacillus cereus, a prominent bacterium causing food poisoning, proves challenging because of its close phylogenetic relationship with other Bacillus species. Moreover, the accuracy of viability indicators in identifying viable B. cereus has been underexplored. This study established a rapid and specific method for quantifying viable B. cereus. The membrane-permeable dye propidium monoazide was identified as an effective means to differentiate between viable and non-viable B. cereus cells. Subsequently, fluorescence in situ hybridization was introduced to specifically identify viable B. cereus cells. Furthermore, FCM was utilized for the rapid and accurate quantification of labeled viable B. cereus cells. Following optimization, the method demonstrated notable specificity and robust anti-interference ability, with B. cereus recovery rates in milk powder and chicken breast after pretreatment ranging from 96.3% to 97.7%. Viable B. cereus cells were accurately quantified within 1.5 h, with a linear range of 102 to 107 cells/g. In conclusion, this developed method enables the rapid and specific quantification of viable B. cereus, offering a novel approach to the detection of specific foodborne pathogens.

Localized Increased Permeability of Blood-Brain Barrier for Antibody Conjugates in the Cuprizone Model of Demyelination.

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Immunofluorescence Microscopy.

Visualizing fluorescence-tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence-labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super-resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such as Tetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly-l-lysine-coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super-resolution images with SRRF microscopy.

Structural Changes in the Skeletal Muscle of Pigs after Long-Term Administration of Testosterone, Nandrolone and a Combination of the Two.

Anabolic steroid hormones (AASs) are used in most countries of the world to accelerate the growth of animals, increase the volume of their muscles and thereby increase meat production. However, there is a strict ban on the use of AASs in the fattening of all animals in all countries of the European Union, and there must therefore be effective methods of detection and control of these substances. Methods based on chromatography and mass spectrometry may no longer be completely effective when faced with new synthetic steroids of unknown chemical structures and low concentrations. Therefore, there is an effort to develop new methods of AAS detection, based primarily on the monitoring of biological changes at the level of gene expression or changes in metabolism or structure at the cellular level. More detailed knowledge of the mechanisms of action of AASs on tissues is essential for these methods, and histological changes are one of them. In this study, we report histological changes in muscle structure after AAS application, specifically in the size of muscle fibers, the amount of endomysium and the number of nuclei and satellite cells in muscle fibers. A pig model was also intentionally used for the study, as no such study has been carried out on this species, and at the same time, pork is one of the most consumed meats across Europe. The results of histology and fluorescent antibody labeling showed that AASs increased the diameter and surface area of muscle fibers and also significantly increased the number of satellite cells on the fiber surface. The evident correlations between the number of satellite cells, all nuclei and the diameters of muscle fibers between some experimental groups provide evidence that the selected histological parameters could be additional detection mechanisms for screening a large number of samples and indicate the possibility of the presence of AASs in pork meat in the future.

Exosome GLUT1 derived from hepatocyte identifies the risk of non-alcoholic steatohepatitis and fibrosis.

It is particularly important to identify the progression of non-alcoholic fatty liver disease (NAFLD) for prognosis evaluation and treatment guidance. The aim of this study was to explore the clinic use of exosomal protein-based detection as a valuable non-invasive diagnostic method for NAFLD. Exosomes were extracted from plasma of patients with NAFLD using Optima XPN-100 ultrafast centrifuge. The patients were recruited from outpatients and inpatients of Beijing Youan Hospital Affiliated to Capital Medical University. The exosomes were stained with fluorescent-labeled antibody and determined by ImageStream® X MKII imaging flow cytometry. Generalized linear logistic regression model was used to evaluate the diagnostic value of hepatogenic exosomes in NAFLD and liver fibrosis. The percentage of hepatogenic exosomes glucose transporter 1 (GLUT1) in patients with non-alcoholic steatohepatitis (NASH) was significantly higher than that in patients with non-alcoholic fatty liver (NAFL). According to liver biopsy, we found that the percentage of hepatogenic exosomes GLUT1 in patients with advanced NASH (F2-4) was significantly higher than that in patients with early NASH (F0-1), and the same trend was observed in exosomes with CD63 and ALB. Compared with other clinical fibrosis scoring criteria (FIB-4, NFS, etc.), the diagnostic performance of hepatogenic exosomes GLUT1 was the highest and the area under the receiver-operating curves (AUROC) was 0.85 (95% CI 0.77-0.93). Furthermore, the AUROC of hepatogenic exosomes GLUT1 combined with fibrosis scoring was as high as 0.86-0.91. Hepatogenic exosome GLUT1 can be a molecular biomarker for early warning of NAFLD to distinguish the NAFL and NASH, and it also can be used as a novel non-invasive diagnostic biomarker for the staging liver fibrosis in NAFLD.

Enhancing the sensitivity of rapid antigen detection test (RADT) of different SARS-CoV-2 variants and lineages using fluorescence-labeled antibodies and a fluorescent meter

RT-qPCR is considered the gold standard for diagnosis of COVID-19; however, it is laborious, time-consuming, and expensive. RADTs have evolved recently as relatively inexpensive methods to address these shortcomings, but their performance for detecting different SARS-COV-2 variants remains limited. RADT test performance could be enhanced using different antibody labeling and signal detection techniques. Here, we aimed to evaluate the performance of two antigen RADTs for detecting different SARS-CoV-2 variants: (i) the conventional colorimetric RADT (Ab-conjugated with gold beads); and (ii) the new Finecare™ RADT (Ab-coated fluorescent beads). Finecare™ is a meter used for the detection of a fluorescent signal. 187 frozen nasopharyngeal swabs collected in Universal transport (UTM) that are RT-qPCR positive for different SARS-CoV-2 variants were selected, including Alpha (n = 60), Delta (n = 59), and Omicron variants (n = 108). Sixty flu and 60 RSV-positive samples were included as negative controls (total sample number = 347). The conventional RADT showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 62.4% (95%CI: 54–70), 100% (95%CI: 97–100), 100% (95%CI: 100-100), and 58% (95%CI: 49–67), respectively. These measurements were enhanced using the Finecare™ RADT: sensitivity, specificity, PPV, and NPV were 92.6% (95%CI: 89.08–92.3), 96% (95%CI: 96–99.61), 98% (95%CI: 89–92.3), and 85% (95%CI: 96–99.6) respectively. The sensitivity of both RADTs could be greatly underestimated because nasopharyngeal swab samples collected UTM and stored at −80 °C were used. Despite that, our results indicate that the Finecare™ RADT is appropriate for clinical laboratory and community-based surveillance due to its high sensitivity and specificity.

Investigation of the frequencies of various B cell populations in non-responder healthcare workers in comparison with responders to hepatitis B virus vaccination.

Hepatitis B is a major global health problem. More than 90% of hepatitis B-vaccinated immunocompetent adults become fully immune. The main purpose of vaccination is immunization. Whether non-responders have a lower percentage of total or antigen-specific memory B cells in comparison with responders is still controversial. We aimed to assess and compare the frequency of various B cell subpopulations in non-responders and responders. Fourteen responders and 14 non-responders of hospital healthcare workers were enrolled in this study. We used flow cytometry to evaluate various CD19+ B cell subpopulations using fluorescent-labeled antibodies against CD19, CD10, CD21, CD27 and IgM and ELISA to evaluate total anti-HBs antibodies. We found no significant differences in the frequency of various B cell subpopulations between the non-responder and responder groups. Furthermore, the frequency of the isotype-switched memory B cell population was significantly higher in the atypical memory B cell subset compared with the classical memory B cell subset in the responder and total groups (p=0.010 and 0.003, respectively). Responders and non-responders to HBsAg vaccine had comparable memory B cell populations. Whether anti-HBs Ab production has a correlation with the level of class switching in B lymphocytes in healthy vaccinated individuals needs further investigation.