Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis.

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38-38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

Development and validation of a flow cytometry antibody test for Lawsonia intracellularis.

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (106 bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Cell-based flow cytometry assay for simultaneous detection of multiple autoantibodies in a single serum sample.

Accurate serologic evaluation of autoantibodies in patients with autoimmune diseases is critical. In the present study, we established a live cell-based assay for simultaneous detection of multiple autoantibodies in a single serum sample. Autoantibody seropositivity was determined by 3-color flow cytometry using live Chinese hamster ovary cells transiently expressing a target protein of interest fused to enhanced green fluorescent protein and labeled with Alexa Fluor 647 and Hoechst 33342. As a representative example, we applied the strategy for simultaneous detection of 2 recently established biomarkers for central nervous system autoimmune inflammatory demyelinating disorders, anti-aquaporin-4 autoantibody and anti-myelin oligodendrocyte glycoprotein autoantibody, in a single serum sample. This analysis revealed the coexistence of these 2 autoantibodies. We demonstrated that this assay can simultaneously detect 3 different autoantibodies. We propose a quadrant gating strategy of flow cytometry contour plots to clearly distinguish seropositive sera from seronegative sera regardless of the extent of the background signal level or the autoantibody titer. This novel and practical method using a combination of fluorescent proteins and fluorochromes to simultaneously detect multiple autoantibodies improves the efficiency of evaluating serum samples, and therefore provides significant benefits to both the patient and the healthcare professionals performing autoantibody testing.

Mapping Cell Phenomics with Multiparametric Flow Cytometry Assays.

Phenomics explores the complex interactions among genes, epigenetics, symbiotic microorganisms, diet, and environmental exposure based on the physical, chemical, and biological characteristics of individuals and groups. Increasingly efficient and comprehensive phenotyping techniques have been integrated into modern phenomics-related research. Multicolor flow cytometry technology provides more measurement parameters than conventional flow cytometry. Based on detailed descriptions of cell phenotypes, rare cell populations and cell subsets can be distinguished, new cell phenotypes can be discovered, and cell apoptosis characteristics can be detected, which will expand the potential of cell phenomics research. Based on the enhancements in multicolor flow cytometry hardware, software, reagents, and method design, the present review summarizes the recent advances and applications of multicolor flow cytometry in cell phenomics, illuminating the potential of applying phenomics in future studies.

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Brucellosis is a serious zoonosis occurring mainly in developing countries, and its diagnosis is largely dependent on serologic detection and bacterial culture. In this study, we developed the murine monoclonal antibodies (mAbs) against a conserved and major outer membrane protein 25 (Omp25) of Brucella species (B. spp.) for use in clinical diagnosis. The mAbs to Omp25 were produced by hybridoma technique, which were utilized for developing various immunoassays for detection of Brucellae, including Western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunochemical staining (ICS), immunofluorescence staining (IFS), and flow cytometry assay (FCM). A number of five mAbs (2B10, 4A12, 4F10, 6C12, and 8F3) specific to Omp25 were selected, including 2 IgG1, 2 IgG2a, and 1 IgG2b. Among them, mAbs 6C12, 8F3, and 4A12 reacted highly with B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2). No cross-reactivity with Yersinia enterocolitica O:9, Salmonella spp., and Escherichia coli was found. By mapping Omp25 epitopes, mAb 6C12 was found as reacting with a semi-conformational epitope, and mAbs 4A12 and 8F3 as recognizing a different linear epitope, respectively. The paired mAbs were tested for detecting Brucella species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Brucella Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of Brucellae in clinical samples with various immunoassays. mAb 6C12-based FCM could be potentially used for the monitoring of therapeutic efficacy for brucellosis in clinical practice.

Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro.

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses. The monitoring of NK cells function is important in various physiological and pathological conditions. Different approaches have been used to directly or indirectly evaluate NK cells activities. The purpose of this study was to investigate the correlation between the number of NK cells and cytotoxic activity of NK cells and to determine whether NKp46+NK cells reflect NK cytotoxicity status. In our study, we retrospectively analyzed laboratory data on NK cytotoxicity and NK lymphocyte levels of 4896 infertile women which underwent routine immunology investigation after IVF failures. In healthy women, NKp46 expression was assessed on NK cells (n = 214) and cytotoxicity activity was evaluated with regard to NKp46 expression. We found that despite a significant correlation coefficient (n = 4689, r = 0.447), the correlation with cytotoxicity is maintained only within the zones with a low or high NK cells frequency. NK cells frequency has no significant prognostic value for their cytotoxicity - within the medium NK frequency zone the samples may have any cytotoxicity, both reduced and elevated. However, our data demonstrate that NKp46+NK cells frequency correlates with cytotoxicity activity even more significantly than the NK cells frequency (n = 214, r = 0.67 and r = 0.62, respectively) and has significant prognostic value for the abnormal NK cytotoxicity status indications, both low and increased. Our results further support an important role of NKp46 in NK cells killing and afford grounds for using the measurement of the NKp46+NK cells frequency as an alternative method for abnormal NK cytotoxicity status indication, which is responsive, simple and reliable.

Insights into the candidacidal mechanism of Ctn[15-34] - a carboxyl-terminal, crotalicidin-derived peptide related to cathelicidins.

PURPOSE: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]. METHODOLOGY: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis. CONCLUSION: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration.

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

Robust Ki67 detection in human blood by flow cytometry for clinical studies.

AIM: Ki67 is a prognostic and/or predictive biomarker in patients with malignancies. Flow cytometry is a powerful technology for single-cell multiparameter analysis. RESULTS: We developed and validated a multicolor quantitative flow cytometry assay for detection of intracellular Ki67 expression in various immune cell subsets from human blood. The assay was optimized and showed excellent precisions. Assessment of the sample stability indicated that percentage changes from the fresh sample for the reportable results of interest were within 20%, up to 72 h after blood collection in the Cyto-Chex® BCT tube. CONCLUSION: The validated assay is sufficiently robust to analyze clinical samples. Easy access to peripheral blood enables continuous monitoring of Ki67 expression in blood as a biomarker, for example, for immunotherapy studies.

Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses.

8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells.

Alkannin inhibits growth and invasion of glioma cells C6 through IQGAP/mTOR signal pathway.

OBJECTIVE: This study aims to explore the effect of alkannin on the growth and invasion of glioma cells and its mechanism. METHODS: The effects of alkannin on the growth and invasion of glioma cells were detected with MTT assay, clone forming test and transwell assay. The effects of alkannin on the cell cycle were detected with flow cytometry assay. The changes of cyclin, MMPs and IQGAP/mTOR signal pathway related proteins were detected with western blotting methods. RESULTS: Alkannin (1 µM, 3 µM and 10 µM) can significantly inhibit the growth, proliferation, migration and invasion of glioma cells C6 with dose dependent. Alkannin can block cell cycle in G1 phase with the increased concentration, which was related with the down-regulation of cyclinA1, cyclinA2 and cyclinD1 expression. Alkannin can also down-regulate the expression of MMP 2, MMP 9 and IQGAP. Alkannin has no effect on mTOR but can inhibit the phosphorylation of mTOR. CONCLUSIONS: Alkannin can inhibit the growth and invasion of glioma cells C6 through IQGAP/mTOR signal pathway.

The frequency of heparin-induced thrombocytopenia in Taiwanese patients undergoing cardiopulmonary bypass surgery.

BACKGROUND/PURPOSE: There are few studies on heparin-induced thrombocytopenia (HIT) reported from Taiwan and Asian countries. We conducted a prospective study to investigate the frequency of HIT in patients undergoing cardiopulmonary bypass surgeries. METHODS: A cohort of 54 patients was enrolled from January 01, 2010 to October 31, 2011. Patients' clinical information was obtained for 4T score classification. Plasma (2-4 mL) was also collected before surgery and on Days 5 and 10 following heparin administration during the bypass procedure. This was tested for anti-heparin/PF4 antibodies and functional assay using flow cytometry (FC). RESULTS: The mean platelet count for this cohort followed the expected pattern in the postoperative setting. Seven of the 54 (13%) patients had positive antibodies assays before bypass surgery. This increased to 32% on Day 5 and was markedly elevated to 63% on Day 10 after surgery. Only one of the 54 patients (1.8%) was found to have both positive antibody assay and platelet activation, but no clinical HIT/thrombosis developed. CONCLUSION: Our study is the first report on the rates of HIT in the setting of cardiopulmonary bypass surgery in Taiwan and demonstrated no clinical HIT occurrence, despite the high frequency of HIT antibody in our cohort.

Proapoptotic activity of aflatoxin B1 and sterigmatocystin in HepG2 cells.

Aflatoxin B1 (AFB1) and sterigmatocystin (ST) are two hepatocarcinogenic mycotoxins that are commonly coexisted in cereal grains, and their co-proapoptotic activity in HepG2 cells was studied. The values of IC50, which is the dosage of mycotoxin resulting in a 50% cell growth inhibition measured by a sulforhodamine B (SRB) colorimetric assay, were 16.9 µM and 7.3 µM for AFB1 and ST, respectively. Additively and dose-dependently, cell apoptosis-related toxicity endpoints of double strand DNA and ATP content were decreased while the intracellular ROS and mitochondria membrane permeability (MMP) were increased. Consistently, when cell cycle is arrest at G0/G1 or S phase by AFB1 and/or ST, the experimental results from flow cytometry assay demonstrated that the rate of cell apoptosis and mitochondrial membrane potential were also additively increased and decreased, respectively, in a dose-dependent manner. Thus, the integrity of mitochondria (MMP and membrane potential) that is the central component of cell apoptosis is disrupted by AFB1 and ST in an additive manner. With the immunocytochemistry analysis showing increased expression of apoptosis-related proteins of Bax, Caspase-3 and p53 and decreased expression of Bcl-2 protein, an additive nature of the co-proapoptotic activity of AFB1 and ST was revealed.

Comparison of Two Quantitative Assays for Determination of Rh-anti-CD20 zumab and Their Application to Pharmacokinetic Study / 分析化学

An enzyme linked immunosorbent assay ( ELISA ) and a flow cytometry assay ( FCA ) based on Wil2-S cells were developed and systematically compared for quantification of recombinant anti-CD20 humanized monoclonal antibody ( rh-anti-CD20zumab) in biological matrix. The specificity, precision and accuracy of each method at correspondingly different linear range showed good results. For ELISA, the precisions of intra-day and inter-day were both <19 . 5%, the relative error was from-18 . 2% to 17 . 6%;For FCA, the precisions of intra-day and inter-day were both <19. 0%, the relative error was from -18. 9% to 18. 4%. The sensitivity of ELISA was significantly higher than that of FCA. The quantitative ranges of ELISA and FCA methods were 0. 04-5. 0 mg/L and 3. 1-200 mg/L, respectively. The concentrations in serum samples and pharmacokinetics analysis were determined by both of two methods after vein drip administration of rh-anti-CD20zumab in rhesus monkeys. Pharmacokinetics data showed that there was excellent consistency between results obtained by two methods at the given dose. We believe that the novel FCA with high speed and high sensitivity can be used to perform PK and PD study of cell surface antigen-targeted antibody derivatives.