Isolation and Assessment of a Highly-Active Anti-Inflammatory Exopolysaccharide from Mycelial Fermentation of a Medicinal Fungus Cs-HK1.

The purpose of this work was to fractionate the complex exopolysaccharide (EPS) from a medicinal fungus Ophiocordyceps sinensis Cs-HK1 based on the molecular weight (MW) range and to assess the in vitro anti-inflammatory activity of different EPS fractions in THP-1 cell culture. The lower MW fraction (EPS-LM-1) showed a much higher anti-inflammatory activity. EPS-LM-1 was identified as a heteropolysaccharide consisting of mannose, glucose, and galactose residues with an average MW of 360 kDa. EPS-LM-1 significantly inhibited the lipopolysaccharide-induced inflammatory responses with the effective concentrations for 50% inhibition below 5 µg/mL on a few major proinflammatory markers. With such a notable in vitro anti-inflammatory activity, EPS-LM-1 is a promising candidate for the development of a new anti-inflammation therapy.

Differences in Baseline Characteristics and White Blood Cell Ratios Between Racial Groups in Patients with Pancreatic Adenocarcinoma.

PURPOSE: Pancreatic adenocarcinoma remains a malignancy with poor prognosis. Black patients experience poorer overall survival compared with other races. Recent studies have elucidated certain prognostic factors at the time of diagnosis of pancreatic cancer which have largely not been studied for differences between racial groups. We present a study examining differences in blood levels between Black and non-Black patients and their effects on overall survival. METHODS: This is a retrospective cohort study. One hundred sixty-three patients were confirmed to carry a tissue diagnosis of pancreatic adenocarcinoma and included in analysis; 27 of the patients were self-identified as "Black"; 136 were analyzed together as "Non-Black" with the majority identifying as "White". Various blood markers were drawn at the time of diagnosis. Kaplan-Meier and multivariable Cox regression models were used to examine differences in these factors between Black and non-Black patients, as well as their effect on overall survival. RESULTS: Black patients were younger at diagnosis (p = 0.001) and were more likely to experience significant weight loss leading up to diagnosis (p = 0.009); Black patients also had a lower neutrophil-to-lymphocyte ratio (NLR) (p = 0.001) and higher lymphocyte-to-monocyte ratio (LMR) (p = 0.001) at diagnosis. In multivariable analysis, an NLR > 3.5 had a significantly negative impact on overall survival (p = 0.002), as did the presence of metastatic disease (p < 0.001). CONCLUSION: Black patients demonstrated a "favorable" white blood cell profile (higher LMR, lower NLR) compared with non-Black patients. This may suggest that the immune response in pancreatic adenocarcinoma is not what is driving disparately poor outcomes in Black patients. Further study is warranted to ascertain the role of immune response in pancreatic adenocarcinoma, the prognostic use of these measurements at diagnosis, and possible other factors, such as genetics, which may better explain poorer outcomes in Black patients.

Peripheral Absolute Lymphocyte Count: An Economical and Clinical Available Immune-Related Prognostic Marker for Newly Diagnosed Multiple Myeloma.

BACKGROUND To find economical and clinically available immune-related prognostic markers that could predict the overall survival (OS) of newly diagnosed multiple myeloma (NDMM) in the new drug era. MATERIAL AND METHODS Absolute lymphocyte count (ALC) and absolute monocyte count (AMC) were measured in routine blood samples from 102 patients with NDMM, and the lymphocyte-monocyte ratio (LMR) was derived. All the patients were receiving bortezomib-based chemotherapy as induction treatment. Log-rank testing was used for comparing the differences between groups. Univariate and multivariate tests were used to identify prognostic markers. RESULTS The median ALC and LMR values at diagnosis were 1.43×109/L and 3.7, respectively, and served as the cutoff point. As prognostic factors, ALC, LMR, and a new staging system combining ALC and the ISS staging system (L-ISS) were expected to have a significant impact on predicting OS. Furthermore, multivariate analysis showed that ALC ≥1.43×109/L (hazard ratio [HR]: 0.223; 95% confidence interval [CI]: 0.071-0.705; P=0.011), LMR ≥3.7 (HR: 0.363; 95% CI: 0.139-0.947; P=0.038), and L-ISS late stage (HR: 1.619; 95% CI: 1.065-2.743; P=0.027) were independent predictors for OS. CONCLUSIONS ALC and LMR can serve as surrogate markers for patients' antitumor immunity at the initial diagnosis of multiple myeloma. A new immune-related staging system, L-ISS, which combines ALC and the ISS staging system, can predict clinical outcomes in patients who are receiving bortezomib-based chemotherapy.

An Assessment of the Glyconutrient Ambrotose™ on Immunity, Gut Health, and Safety in Men and Women: A Placebo-Controlled, Double-Blind, Randomized Clinical Trial.

BACKGROUND: Certain dietary fibers have been reported to improve gut health and cellular immunity. Ambrotose is a glyconutrient supplement that contains mannose-rich polysaccharides (acemannan), reported to improve immune function. A more nutrient-dense version of this dietary supplement has been developed recently, with added aloe leaf gel powder (acemannan). The purpose of this study was to evaluate the impact of the traditional and newly developed Ambrotose products on immunity, gut health, and psychological well-being in healthy men and women. METHODS: Seventy-five men and women were randomly assigned in double-blind manner to one of five treatments, as follows: Ambrotose Advanced (AA) at 2 or 4 g daily, Ambrotose LIFE (AL) at 2 or 4 g daily, or placebo. Subjects ingested their assigned treatment daily for eight weeks. Resting heart rate, blood pressure, and measures of psychological well-being were analyzed before and after four and eight weeks of supplementation. Blood samples were collected at the same times and analyzed for zonulin, hematology measures, and cytokines-IL-6, IL-10, IL-1ß, and TNF-α (analyzed both with and without stimulation via lipopolysaccharide [LPS]). RESULTS: All Ambrotose treatments were well-tolerated. There were no differences among treatments in heart rate or blood pressure across time. Self-reported well-being scores were generally higher for the Ambrotose treatments but there were no changes of statistical significance across time (p > 0.05). Differences of statistical significance were noted for select biochemical variables, the most notable being a dramatic decrease in monocytes in the Ambrotose groups. No change was noted in the cytokine response to LPS stimulation in all groups, indicating a maintenance of a healthy immune response. Conclusion: Regular supplementation with Ambrotose is safe and can improve subclinical cellular adversity (as evidenced by a decrease in monocytes), without unnecessary activation of an immune response.

Assessment of a poly-epitope candidate vaccine against Hepatitis B, C, and poliovirus in interaction with monocyte-derived dendritic cells: An ex-vivo study.

Design and application of epitope-based polyvalent vaccines have recently garnered attention as an efficient alternative for conventional vaccines. We previously have reported the in silico design of HHP antigen which encompasses the immune-dominant epitopes of Hepatitis B surface antigen (HBsAg), Hepatitis C core protein (HCVcp) and Poliovirus viral proteins (VPs). It has been shown that the HHP has desirable conformation to expose the epitopes, high antigenicity and other desired physicochemical and immunological properties. To confirm the accuracy of these predictions, the ex-vivo immunogenicity of the HHP was assessed. The HHP gene was chemically synthesized in pET28a and expressed in E. coli (BL21). The expressed protein was purified and its immunological potency was evaluated on dendritic cells (DCs) as antigen presenting cells (APCs). Functional analysis was assessed in co-cultivation of autologous T-cells with matured DCs (mDCs). T-cell activation, proliferation and cytokines secretion were evaluated using flowcytometry and ELISA methods. Our results indicated that the HHP could induce the DC maturation. The mDCs were able to trigger T-cell activation and proliferation. In silico design and ex-vivo confirmation of immunological potential could pave the way to introduce efficient immunogens for further analysis. The ability of HHP in DC maturation and T-cell activation makes it an amenable vaccine candidate for further in-vivo studies.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms.

Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.

Assessment efficacy of neutrophil-lymphocyte ratio and monocyte-lymphocyte ratio in preeclampsia.

OBJECTIVE: Abnormal changes in immune-mediated inflammation contribute to the pathogenesis of preeclampsia (PE). We aim to investigate the value of systemic immune inflammation indices-neutrophil-lymphocyte ratio (NLR) and monocyte-lymphocyte ratio (MLR)-to identify and evaluate the prognosis of patients with PE. METHODS: This study reviewed clinical records of 367 PE patients (162 with mild PE and 205 with severe PE), in addition to a control group of 172 normal pregnancies. Blood cell counts were performed at the first diagnosis of PE, and NLR and MLR were calculated by absolute cell count. RESULTS: Absolute neutrophil, lymphocyte, and monocyte counts and NLR and MLR values in PE were significantly different from controls, although monocyte counts did not significantly differ between mild and severe PE. Receiver operating characteristics curve (ROC) analysis showed NLR and MLR had better diagnostic accuracy in distinguishing PE from controls [NLR area under the curve (AUC) = 0.70; MLR AUC = 0.78]. Further, NLR was the best predictor of disease severity (AUC = 0.71). Cutoff values of NLR > 4.198 or MLR > 0.325 for control and PE groups or a cutoff value of NLR > 4.182 for PE groups indicated that patients were more likely to encounter preterm delivery, have shorter admission-to-delivery interval, and develop maternal and neonatal complications. CONCLUSION: Secondary analyses of white blood cell differential count parameters effectively evaluate the systemic inflammatory/immune state. Compared with absolute cell counts, NLR and MLR offer more effective indicators of clinical assessment, disease severity evaluation, and prognosis evaluation of PE.

Monocyte HLA-DR Assessment by a Novel Point-of-Care Device Is Feasible for Early Identification of ICU Patients With Complicated Courses-A Proof-of-Principle Study.

Background: Critically ill patients, especially following trauma or extensive surgery, experience a systemic immune response, consisting of a pro-inflammatory as well as a counterbalancing anti-inflammatory response. Pro-inflammation is necessary for the initiation of homeostatic control and wound healing of the organism. However, when the counterbalancing mechanisms dominate, a condition of secondary immunodeficiency occurs, which renders the patient susceptible for opportunistic or secondary infections. However, the incidence of this condition is yet illusive. Methods: For a period of 3 months (May to July 2017), 110 consecutive patients admitted to the surgical ICU of the Heidelberg University Hospital, a tertiary university hospital, were enrolled in the study. Monocyte HLA-DR (mHLA-DR), a long-known surrogate of monocyte function, was assessed quantitatively once on admission utilizing a novel point-of-care flow cytometer with single-use cartridges (Accelix system). Patients were followed up for further 28 days and data on ICU stay, antibiotic therapy, microbiological findings, and mechanical ventilation were recorded. Statistical analysis was performed to evaluate the incidence of immunosuppression-defined by different thresholds-as well as its consequence in terms of outcome and clinical course. Results: Depending on the HLA-DR threshold applied for stratification (≤8,000/≤5,000/≤2,000 molecules/cell), a large group of patients (85.5/68.2/40.0%) already presented with a robust decrease of HLA-DR on admission, independent of the cause for critical illness. Analyzed for survival, neither threshold was able to stratify patients with a higher mortality. However, both thresholds of 2,000 and 5,000 were able to discriminate patients with longer ICU stay, ventilation time and duration of antibiotic therapy, as well as higher count of microbiological findings. Moreover, a mHLA-DR value ≤2,000 molecules/cell was associated with higher incidence of overall antibiotic therapy. Conclusion: Single assessment of mHLA-DR using a novel point-of-care flow cytometer is able to stratify patients according to their risk of a complicated course. Therefore, this device overcomes the technical boundaries for measuring cellular biomarkers and paves the way for future studies involving personalized immunotherapy to patients with a high immunological risk profile independent of their background. Trial Registration: German Clinical Trials Register; ID: DRKS00012348.

Relationship between monocytes to lymphocytes ratio and axial spondyloarthritis.

BACKGROUND: Axial spondyloarthritis (axSpA) is a progressive, chronic, inflammatory skeletal disorder affecting the spine and sacroiliac joints. Many studies have shown that neutrophils, lymphocytes, monocytes, platelets, and red blood cells (RBCs) play important roles in the inflammatory process of axSpA. Neutrophils to lymphocytes ratio (NLR) and red blood cell distribution width (RDW) have been reported to be simple and inexpensive markers to indicate the disease activity of axSpA. However, the role of monocytes to lymphocytes ratio (MLR) and platelets to lymphocytes ratio (PLR) in axSpA was rarely mentioned. OBJECTIVE: The study's aim was to determine the role of MLR and PLR in axSpA patients and to investigate their relationships with disease severity. METHODS: AxSpA patients who fulfilled the Assessment in Ankylosing Spondylitis International Society classification criteria published in 2009 were enrolled in this study and divided into nonradiographic axial spondyloarthritis (nr-axSpA) group and ankylosing spondylitis (AS) group. Healthy age and gender-matched subjects were also enrolled as control group. MLR, PLR, NLR, RDW, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR) level were assessed. The correlation between the variables with finger-to-floor distance, Modified Schober test, and occiput-to-wall distance were tested with Pearson correlation. Furthermore, area under curve (AUC) value, sensitivity, specificity, and the optimal cutoff values were determined using receiver operating characteristic (ROC) curves. RESULTS: A total of 148 axSpA patients (67 nr-axSpA patients and 81 AS patients) and 58 healthy subjects were included in the study. The MLR, NLR, PLR, and RDW in axSpA group were higher than those in the control group (P < 0.05). Among them, MLR and RDW were highly increased in AS group compared with the nr-axSpA group (P < 0.05). MLR, NLR, PLR, and RDW were all positively correlated with ESR level and CRP level (P < 0.05). MLR and RDW were positively correlated with finger-to-floor distance and negatively correlated with Modified Schober test (P < 0.05). RDW was positively correlated with occiput-to-wall distance (P < 0.05). ROC curve results showed MLR yielded a higher AUC than NLR, PLR, and RDW (P < 0.05). In addition, the optimal cutoff value of MLR for axSpA was 0.22, with a specificity of 70.9% and sensitivity of 68.4%. CONCLUSIONS: MLR was elevated in AS patients compared to nr-axSpA patients and had a close relationship with CRP level, ESR level, and spine movements. MLR may be a reliable, cost-effective, and novel potential parameter to evaluate disease severity in axSpA.

In vitro assessment of silver nanoparticles immunotoxicity.

This study aimed to characterize unwanted immune effects of nanoparticles (NP) using THP-1 cells, human whole blood and enriched peripheral blood monocytes. Commercially available silver NP (AgNP < 100 nm, also confirmed by Single Particle Extinction and Scattering) were used as prototypical NP. Cells were treated with AgNP alone or in combination with classical immune stimuli (i.e. LPS, PHA, PWM) and cytokine assessed; in addition, CD54 and CD86 expression was evaluated in THP-1 cells. AgNP alone induced dose-related IL-8 production in all models, with higher response observed in THP-1 cells, possibly connected to different protein corona formation in bovine versus human serum. AgNP potentiated LPS-induced IL-8 and TNF-α, but not LPS-induced IL-10. AgNP alone induced slight increase in IL-4, and no change in IFN-γ production. While responses to PHA in term of IL-4 and IFN-γ production were not affected, increased PWM-induced IL-4 and IFN-γ production were observed, suggesting potentiation of humoral response. Reduction in PHA-induced IL-10 was observed. Overall, results indicate immunostimulatory effects. THP-1 cells work as well as primary cells, representing a useful and practical alternative, with the awareness that from a physiological point of view the whole blood assay is the one that comes closest to reality.

Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates.

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40-CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40-CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe-/-) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe-/- mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.

Bioinformatic Assessment of Macrophage Activation by the Innate Immune System.

Weighted gene co-expression network analysis (WGCNA) allows for the identification and characterization of cell type-specific gene modules in complex transcriptome datasets. Here, we use a microarray dataset of human macrophages comprising 29 conditions and 299 samples generated by differentiation of CD14+ monocytes into macrophages followed by in vitro stimulations to identify stimulation-specific gene modules. These gene modules can be used for experimental validation, as well as further bioinformatic analysis to determine key pathways or upstream transcription factors.

CCR2+ Inflammatory Monocytes Are Recruited to Yersinia pseudotuberculosis Pyogranulomas and Dictate Adaptive Responses at the Expense of Innate Immunity during Oral Infection.

Murine Ly6Chi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c+ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis Previously, we obtained evidence that IMs are important for a dominant CD8+ T cell response to the epitope YopE69-77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis Here we challenged CCR2+/+ or CCR2-/- mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2-/- mice did not have reduced survival but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster and with reduced lymphadenopathy compared to controls. Enhanced bacterial clearance in CCR2-/- mice correlated with reduced numbers of IMs in spleens and increased numbers of neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that green fluorescent protein-positive (GFP+) IMs accumulated at the periphery of neutrophil-rich Yersinia-containing pyogranulomas. GFP+ IMs colocalized with CD11c+ cells and YopE69-77-specific CD8+ T cells in MLNs, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2-/- mice had reduced numbers of splenic DCs, YopE69-77-specific CD8+ T cells, CD4+ T cells, and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.

Recovery and assessment of leukocytes from LR Express filters.

Leukocytes, or white blood cells, are used for a variety of investigational purposes and they offer advantages over laboratory-adapted cell lines. Leukocytes that are typically discarded by blood banks during the collection of red blood cells, platelets, and plasma can often be obtained for research use. However, the available leukocytes are frequently contained within a blood filtration device, such as the Terumo LR Express (TLRE) filter. In this study, procedures were evaluated for the ability to elute viable leukocytes from TLRE filters. The recovered leukocytes were assessed for composition, growth, and functionality. The large majority (>70%) of leukocytes were eluted with a single reverse-elution procedure and the recovered cells contained representative populations of the major leukocyte subsets. Purified T cells exhibited diverse T cell receptor repertoires, characteristic growth upon mitogen stimulation, and CD4+ T cells were able to support HIV-1 propagation. Purified monocytes were able to be differentiated into phenotypically characteristic populations of macrophages and dendritic cells. Overall, TLRE filters offer an attractive source of primary human cells for research and possibly clinical purposes.

Assessment of Vascular Dysfunction and Inflammation Induced by Angiotensin II in Mice.

Vascular inflammation in cardiovascular diseases is recognized to be linked with immune cell activation. Recruitment of immune cells into the vessel wall is an early step in angiotensin II-induced vascular dysfunction and arterial hypertension. Exploring the role of monocytes and macrophages in angiotensin II-induced hypertension and vascular inflammation in mouse models highlights the importance of these pathophysiological processes. Here we describe our routinely used protocols concerning angiotensin II-induced hypertension, assessment of blood pressure, vascular function, and immune cell infiltration.

Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells.

Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5ß1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.

Imaging flow cytometry in the assessment of leukocyte-platelet aggregates.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants.

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.

Assessment of Inflammasome Formation by Flow Cytometry.

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow-sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.