Whole Blood Of Healthy Donors Research Articles

A sensitive assay for measuring whole-blood responses to type I IFNs

Human inborn errors of the type I IFN response pathway and auto-Abs neutralizing IFN-α, -β, and/or -ω can underlie severe viral illnesses. We report a simple assay for the detection of both types of condition. We stimulate whole blood from healthy individuals and patients with either inborn errors of type I IFN immunity or auto-Abs against type I IFNs with glycosylated human IFN-α2, -β, or -ω. As controls, we add a monoclonal antibody (mAb) blocking the type I IFN receptors and stimulated blood with IFN-γ (type II IFN). Of the molecules we test, IP-10 (encoded by the interferon-stimulated gene (ISG) CXCL10) is the molecule most strongly induced by type I and type II IFNs in the whole blood of healthy donors in an ELISA-like assay. In patients with inherited IFNAR1, IFNAR2, TYK2, or IRF9 deficiency, IP-10 is induced only by IFN-γ, whereas, in those with auto-Abs neutralizing specific type I IFNs, IP-10 is also induced by the type I IFNs not neutralized by the auto-Abs. The measurement of type I and type II IFN-dependent IP-10 induction therefore constitutes a simple procedure for detecting rare inborn errors of the type I IFN response pathway and more common auto-Abs neutralizing type I IFNs.

Kinetics and toxicity of nanoplastics in ex vivo exposed human whole blood as a model to understand their impact on human health

The ubiquitous presence of nanoplastics (NPLs) in the environment is considered of great health concern. Due to their size, NPLs can cross both the intestinal and pulmonary barriers and, consequently, their presence in the blood compartment is expected. Understanding the interactions between NPLs and human blood components is required. In this study, to simulate more adequate real exposure conditions, the whole blood of healthy donors was exposed to five different NPLs: three polystyrene NPLs of approximately 50 nm (aminated PS-NH2, carboxylated PS-COOH, and pristine PS- forms), together with two true-to-life NPLs from polyethylene terephthalate (PET) and polylactic acid (PLA) of about 150 nm. Internalization was determined in white blood cells (WBCs) by confocal microscopy, once the different main cell subtypes (monocytes, polymorphonucleated cells, and lymphocytes) were sorted by flow cytometry. Intracellular reactive oxygen species (iROS) induction was determined in WBCs and cytokine release in plasma. In addition, hemolysis, coagulation, and platelet activation were also determined. Results showed a differential uptake between WBC subtypes, with monocytes showing a higher internalization. Regarding iROS, lymphocytes were those with higher levels, which was observed for different NPLs. Changes in cytokine release were also detected, with higher effects observed after PLA- and PS-NH2-NPL exposure. Hemolysis induction was observed after PS- and PS-COOH-NPL exposure, but no effects on platelet functionality were observed after any of the treatments. To our knowledge, this is the first study comprehensively evaluating the bloodstream kinetics and toxicity of NPL from different polymeric types on human whole blood, considering the role played by the cell subtype and the NPLs physicochemical characteristics in the effects observed after the exposures.

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active β tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and βΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Complement activating ABO anti-A IgM/IgG act synergistically to cause erythrophagocytosis: implications among minor ABO incompatible transfusions

BackgroundTypically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO ‘low titer’ products are used for minor ABO incompatible transfusions. We sought to understand the role of IgM/IgG and complement activation by anti-A on extravascular hemolysis.MethodsSamples evaluated included (i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, (ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N = 6) or not (N = 6), and (iii) Group O sera from 10 patients with anti-A hemolysin activity. A flow cytometric monocyte erythrophagocytosis assay was developed using monocytes isolated by immunomagnetic CD14-positive selection from ACD whole blood of healthy donors. Monocytes were frozen at − 80 °C in 10% dimethyl sulfoxide/FBS and then thawed/reconstituted on the day of use. Monocytes were co-incubated with anti-A-sensitized fluorescently-labeled Group A1 + RBCs with and without fresh Group A serum as a source of complement C3, and erythrophagocytosis was analyzed by flow cytometry. The dependency of IgM/IgG anti-A and complement C3 activation for RBC erythrophagocytosis was studied. Anti-A IgG subclass specificities were examined for specific samples.ResultsThe plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (> 10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (≥ 128) and IgG titers (> 1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2.ConclusionHigh anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fcγ- and CR1-receptors.

Abstract A92: Assessing the T-cell exhaustion status of TCR discovery outputs

Abstract Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion. Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.

Влияние общего воздействия слабого низкочастотного вихревого магнитного поля на систему естественной противоопухолевой резистентности организма

Purpose: Experimental study of the antitumor mechanism of increasing the functional activity of phagocytes with a general effect on the body of a weak low-frequency vortex magnetic field (VMP).
 Material and methods: The functional activity of phagocytes was assessed upon activation by the intensity of chemiluminescence (CHL) on a Biolumat instrument (model LB 9500, by Berthold, Germany). Samples for in vitro and in vivo experiments were prepared according to generally accepted protocols. The impact of the VMP (Vmax = 3.0 mTl, f = 100 Hz) was performed using a Magnitoturbotron magnetotherapeutic installation (developed by NPF Az).
 Results: It was found in vitro experiments that the exposure of a suspension of VMP cells stimulated an increase in CHL by 58 % relative to the control, while the main contribution to the intensity of the signal of the CHL was made by neutrophils. In the study of the blood CHL of mice with subcutaneously inoculated melanoma B-16, it was established that the value of the specific CHL in the experiment with VFM by the end of the observation period (day 17) increased sharply (3 times) relative to the beginning of the observation and to the control at the same observation period was 3.3 times more. Experiments with whole blood of healthy donors and patients with breast cancer showed that the CHL curves over time were biphasic in nature and had two maxima, but the phase ratio was different. At donors, the main luminescence developed by the 100th minute, and a maximum of 30–40 min was mild. In patients with breast cancer, the first maximum at 30–40 min was the main, the second maximum was weak and came later than that of donors. Experiments with the effects of VMP on the organism of healthy and patients with breast cancer of people showed an increase in the functional activity of neutrophils as a result of exposure to VMP, but in patients with breast cancer, the level of CHL was significantly (3 times) higher than of healthy donors. Based on the research, it was concluded that exposure to the VMP is able to form a priming neutrophil.
 Сonclusion: It is shown that the overall effect of weak low-frequency VFM increases the level of nonspecific resistance of the organism to the tumor process, which expands the possibilities of rehabilitation of patients, allows expanding the compensatory capabilities of the body and reduce the risk of disease.

Abstract 2219: Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma of the bladder

Abstract Introduction: Immune checkpoint therapies with PD-1/PD-L1 inhibitors show durable responses in a subset of patients with metastatic urothelial carcinoma of the bladder (UCB). However, PD-L1 expression in tumor biopsies does not correlate with response to PD-1/PD-L1 inhibitors in all patients. In addition, determination of PD-L1 expression in tumor tissue has not been harmonized to date and biopsies from metastatic sites are often hardly accessible. Thus, a reliable predictive biomarker is still urgently needed. In this study, we perform detection of PD-L1 on circulating tumor cells (CTCs) in blood samples of patients with advanced UCB. Experimental procedures: Blood samples of 93 non-metastatic (M0) and 33 metastatic (M1) UCB patients were analyzed using the CellSearch® system. Following EpCAM-based enrichment, CK+/DAPI+/CD45- cells were identified as CTCs using the CTC-Kit. For PD-L1 detection, the CellSearch® CXC-Kit (optional fluorescence channel: PE) proved more suitable than the CellSearch® CTC-Kit (optional fluorescence channel: FITC). After enumeration of CTCs by the CTC-Kit, for nine CTC-positive patients (M1) a second blood sample was further analyzed for PD-L1 protein expression using the CXC-Kit. Specificity of the PE-labelled anti-PD-L1-antibody (clone E1L3N) was validated by Western blot analysis, IF and FACS. Results: Enumeration of CTCs by the CellSearch® CTC-Kit revealed significantly higher CTC-positivity in M1 than in M0 patients (51.5% vs. 22.6%, p=0.004 for ≥1 CTCs/7.5 mL blood; 30.3% vs. 3.2%, p<0.001 for ≥5 CTCs/7.5 mL blood). Also mean absolute CTC numbers were significantly higher in M1 than M0 patients (p<0.001). PD-L1 detection in the CellSearch® system was established using five UCB cell lines with different PD-L1 expression, according to Western blot analysis and FACS, which were spiked into whole blood of healthy donors. Using the CXC-Kit, ≥1 PD-L1+ CTCs were detected in 77.8% (7/9) of CTC-positive M1 patients. Thus, PD-L1 expression levels presented with intra- and inter-patient heterogeneity. The follow-up analysis of one patient who received anti-PD-L1 therapy showed substantial increases in CTC numbers as well as in the ratio and intensity of PD-L1+ CTCs in conjunction with disease progression under immunotherapy. The patient died within one month after the follow-up blood draw. Conclusion: This is the first study demonstrating the feasibility to detect and monitor PD-L1 expression on CTCs of advanced UCB patients in the CellSearch® system. The established assay is readily available for clinical application and should be implemented in future clinical trials to evaluate its relevance for predicting response to immune checkpoint inhibitors. Citation Format: Sonja Bergmann, Anja Coym, Gunhild von Amsberg, Melanie Janning, Armin Soave, Michael Rink, Klaus Pantel, Sabine Riethdorf. Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2219.

Calnexin Impairs the Antitumor Immunity of CD4+ and CD8+ T Cells

Elucidation of the mechanisms of T-cell-mediated antitumor responses will provide information for the rational design and development of cancer immunotherapies. Here, we found that calnexin, an endoplasmic reticulum (ER) chaperone protein, is significantly upregulated in oral squamous cell carcinoma (OSCC). Upregulation of its membranous expression on OSCC cells is associated with inhibited T-cell infiltration in tumor tissues and correlates with poor survival of patients with OSCC. We found that calnexin inhibits the proliferation of CD4+ and CD8+ T cells isolated from the whole blood of healthy donors and patients with OSCC and inhibits the secretion of IFNγ, TNFα, and IL2 from these cells. Furthermore, in a melanoma model, knockdown of calnexin enhanced the infiltration and effector functions of T cells in the tumor microenvironment and conferred better control of tumor growth, whereas treatment with a recombinant calnexin protein impaired the infiltration and effector functions of T cells and promoted tumor growth. We also found that calnexin enhanced the expression of PD-1 on CD4+ and CD8+ T cells by restraining the DNA methylation status of a CpG island in the PD-1 promoter. Thus, this work uncovers a mechanism by which T-cell antitumor responses are regulated by calnexin in tumor cells and suggests that calnexin might serve as a potential target for the improvement of antitumor immunotherapy.

Olive Oil Phenolics Prevent Oxysterol-Induced Proinflammatory Cytokine Secretion and Reactive Oxygen Species Production in Human Peripheral Blood Mononuclear Cells, Through Modulation of p38 and JNK Pathways.

ScopeThe aim of the present study was to investigate the ability of extra virgin olive oil (EVOO) polyphenols to counteract the proinflammatory effects induced by dietary and endogenous oxysterols in ex vivo immune cells.Methods and resultsPeripheral blood mononuclear cells (PBMCs), separated from the whole blood of healthy donors, were utilized and were stimulated with an oxysterols mixture, in the presence of physiologically relevant concentrations of the EVOO polyphenols, hydroxytyrosol, tyrosol, and homovanillic alcohol. Oxysterols significantly increased the production of proinflammatory cytokines, interleukin‐1β, regulated on activation, normal T‐cell expressed and secreted and macrophage migration inhibitory factor in ex vivo cultured PBMCs. Increased levels of reactive oxygen species (ROS) were also detected along with increased phosphorylation of the p38 and JNK. All phenolic compounds significantly reduced cytokine secretion induced by the oxysterols and inhibited ROS production and mitogen activated protein kinase phosphorylation.ConclusionsThese results suggest that extra virgin olive oil polyphenols modulate the immune response induced by dietary and endogenous cholesterol oxidation products in human immune cells and may hold benefit in controlling chronic immune and/or inflammatory processes.

Abstract A014: Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients

Abstract Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. Mechanistic studies have demonstrated that Imprime forms an immune complex with endogenous IgG and IgM anti-β-glucan antibodies (ABA). The immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, to kill antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. In this study, we sought to investigate how the formation of this Imprime-ABA immune complex might influence patient selection strategies. We have shown that there is a threshold level of ABA required for binding of Imprime-ABA immune complex to the innate immune effectors. In human healthy volunteers (N=143) IgG ABA concentrations vary from 8 to 1448 μg/ml. Imprime treatment of whole blood from individuals with ABA concentrations above a threshold yields immune complex formation that allows both classical and alternative complement pathways leading to complement opsonization of the complex. This complex then binds innate immune cells, triggering the modulation of cell surface receptors and production of selective chemokines such as IL-8. We now show that the predominant subclass of endogenous IgG ABA specific to Imprime is IgG2. Additionally, we show that binding of Imprime to immune cells critically involves FcgRIIA, the only Fc receptor capable of binding IgG2 immune complexes. Blocking FcgRIIA significantly inhibits binding to both neutrophils and monocytes in whole blood. This observation was further confirmed by binding of Imprime to HEK cells overexpressing FcgRIIA. In order to evaluate the effect that ABA IgG subclass has on Imprime, chimeric ABAs with identical affinity but differing in the human IgG subclass (IgG1 vs. IgG2) were generated using the heavy and light chain variable domains from a mouse ABA hybridoma. IgG1 or IgG2 ABA added to whole blood of healthy donors with low ABA levels was able to increase Imprime binding, consistent with previous data showing that Imprime binding is dependent on ABA levels. However, the relative increase in binding due to the addition of IgG1 or IgG2 ABA varied among donors. While addition of either IgG1 or IgG2 ABA was equivalent in increasing binding in some donors, IgG1 was more efficacious than IgG2 in other donors. These results led us to look at allelic variants of the FCGR2A gene. In humans, there is a SNP in FCGR2A, R131H, that defines two alleles whose protein products differ in affinity for IgG2, with H variant designating the higher affinity allele and the R variant indicating lower affinity. The variable binding of Imprime in donors with addition of chimeric ABA was found to be related to the FCGR2A genotype. Individuals homozygous for the R genotype showed lower binding with IgG2 chimeric ABA than with IgG1 chimeric ABA. The findings were similar with exogenously added naturally occurring ABA purified from serum. The effect of the R131H SNP on Imprime binding in relation to endogenous ABA levels was further confirmed in a survey of 140 healthy volunteers. The prevalence and functional relevance of this SNP on Imprime binding to immune cells in cancer patients is being evaluated. Together, these data implicate both the concentration of ABA in serum and FCGR2A genotype as critical determinants of the ability of an individual to bind to Imprime and suggest that either or both may enable the selection of patients most likely to derive therapeutic benefit from Imprime treatment. Citation Format: Nandita Bose, Anissa Chan, Adria Jonas, Nadine Ottoson, Xiaohong Qiu, Lindsay Wurst, Steven Leonardo, Peter Maimonis, Katie Ertelt, Ben Harrison, Keith Gorden. Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A014.

The role of the reduction of extracellular calcium ion concentrations in activation of human peripheral blood T cells

Calcium plays a fundamental role in many essential functions of a living organism. It is well known that many infections can reduce extracellular calcium concentrations ([Ca2+]o) (hypocalcaemia) in human blood. The importance of Ca2+ for the T lymphocyte activation has been known for a long time, but the optimal range of [Ca2+]o levels has not been found yet. The main goal of this work was the study of functional activities of CD4+ cells in human peripheral blood (HPB) in vitro in the conditions of hypocalcemia. Polyclonal activation was induced in whole blood of healthy donors by immobilized monoclonal antibodies (mAbs) toward CD3 molecules (epsilon chain of the T cell receptor) and dissolved mAbs toward CD28 (the major molecule of T cell costimulation). Dependencies of T-cell activation parameters on varied [Ca2+]o in HPB in vitro were studied. Production of interleukins-2 (IL-2), IL-4, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) as well as CD4+CD69+, CD4+CD25+, and CD4+(CYTOKINE)+ T cells were used as activation parameters of the T cell polyclonal activation in HPB. EGTA was used as a tool for the reduction of [Ca2+]o levels. The results demonstrated that the EGTA-stimulated binding of extracellular calcium in HPB caused a nonlinear dependence of T cell activation on [Ca2+]o. No EGTA influence on resting T cells was found. The dependence of the number of CD4+CD69+ T cells on [Ca2+]o was represented as a nonlinear curve with two maxima and a local minimum in between. One of the maxima was in the area of the HPB lower [Ca2+]o normal level, whereas the other one was even lower, which is characteristic for pathological states. The T cell-induced production of IL-2, IL-4, IL-17A, IFN-γ, and TNF-α as well as the populations of CD4+CD25+ and CD4+(CYTOKINE)+ T cells were analyzed in HPB in vitro under the same activation conditions. All the dependencies obtained were similar in shape to that of the CD4+CD69+ T cell population versus EGTA concentration in HPB in vitro and was observed as curves with two maxima. The dependencies of this type were described first. It is likely that the presence of two maxima and a local minimum in between for the dependence of HPB T cell activation parameters on [Ca2+]o concentrations is of great physiological importance because hypocalcemia has been associated with many pathological states.

Effects of berberine and red yeast on proinflammatory cytokines IL-6 and TNF-α in peripheral blood mononuclear cells (PBMCs) of human subjects.

Background and Aims: Obesity is a condition associated with chronic or acute inflammatory response characterized by an increase of proinflammatory cytokine levels. Peripheral blood mononuclear cells (PBMCs) migrate in adipose tissue inducing synthesis and secretion of adipocytokines as IL-6 and TNF-α. The aim of this study was to investigate the effect of berberine (a natural alkaloid) and red yeast (a natural antioxidant) on IL-6 and TNF-α cytokines release and gene expression, in circulating lipopolisaccarides (LPS) stimulated PBMCs.Methods and Results: PBMCs isolated from whole blood of healthy donors were stimulated with LPS to induce cytokines production; simultaneously cells were treated with increasing doses of berberine and red yeast. The substances were administered alone or in association. IL-6 and TNF-α protein levels in the culture medium and their mRNA levels were assessed by ELISA and real time PCR, respectively. Berberine and red yeast treatment prevented the LPS induction of IL-6 release in the culture medium of PBMCs. In addition, berberine plus red yeast treatment showed a synergic inhibitory effect on IL-6 release at low concentration. Berberine and red yeast showed an inhibitory effect also on LPS induction of TNF-α release exerting a synergic effect mainly at high concentrations. On the contrary, berberine and red yeast did not significantly affect IL-6 and TNF-α mRNA levels induced by LPS. In this case, only concomitant treatment of PBMCs with high doses of berberine and red yeast inhibits LPS induced IL-6 or TNF-α mRNA levels.Conclusions: The results of our study show that both berberine and red yeast were able to carry out anti-inflammatory action through an inhibition of proinflammatory IL-6 and TNF-α protein release. Moreover, when given in combination these substances were able to inhibit IL-6 and TNF-α gene expression in PBMCs activated by LPS. Therefore, these substances could represent a useful pharmacological treatment to reduce the proinflammatory status accompanied with obesity.

Expression of CD11b and CD18 on polymorphonuclear neutrophils stimulated with interleukin-2.

BackgroundInterleukin-2 (IL-2) is a lymphocyte-activating and growth-promoting factor, and has been widely studied on T-cells and NK-cells. However, the interaction of polymorphonuclear neutrophils (PMNs) with IL-2 is poorly studied and thus, this study aimed at defining IL-2 participation in the expression of CD11b and CD18 on PMNs.Material and methodsPMNs were isolated from heparinized whole blood of healthy donors. Purified cells were incubated with IL-2 (10 ng/ml) for 24 hours at 37°C in a humidified incubator with 5% CO2. After 24 hours’ incubation, surface molecules (CD11b and CD18) were measured by flow cytometry.ResultsInterestingly, the antibodies of IL-2Rβ chain (CD122-FITC) were found in all observed cells. The induction of CD11b mean fluorescence intensity (MFI) in highly purified PMNs stimulated with IL-2 was clearly increased recording 43% in comparison to the freshly isolated PMNs and the un-stimulated PMNs which were found to be 23% and 28% of CD11b, respectively. Furthermore, flow cytometry analysis demonstrated that the highly purified PMNs exposed to IL-2 showed an increase in CD18 MFI, recording 47% with respect to that of the freshly isolated PMNs and PMNs cultured with the medium alone which showed a small amount of 38% and 27%, respectively.ConclusionsResults demonstrated that CD11b and CD18 had been acquired on the surface of the IL-2-in vitro-activated PMNs. These findings indicated that IL-2 may play a crucial role in PMNs migration.

Abstract 2858: Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action.

Abstract Immunotherapy of neuroblastoma with anti-GD2 antibodies in combination with interleukin-2 (IL2) emerges as an important treatment option as shown in several clinical trials. In consequence, an antibody-IL2 fusion protein (hu14.18-IL2; immunocytokine) was designed that combines the targeting and effector functions of an anti-GD2 antibody and the immune modulation activity of IL2. In addition to mediation of typical effector functions such as CDC and ADCC this immunocytokine targets IL2 to GD2+ tumor cells and thereby links tumor cells to IL2-receptor bearing effector cells via an Fc-gamma independent mechanism. This concept has already shown encouraging results in Phase I/II trials in relapsed/refractory neuroblastoma patients. Here, we report the antigen binding characteristics and in vitro anti-neuroblastoma activity of a new GMP preparation of hu14.18-IL2 (APN301). Binding of APN301 to its nominal antigen GD2 was analyzed by solid phase ELISA and compared to other antibodies of the 14G2a family. Affinities of APN301 to GD2 and to anti-14.18 anti-idiotype antibodies were also determined by Biacore® analysis. GD2 specific ADCC, CDC and whole blood cytotoxicity were analyzed in cytotoxicity assays in vitro (calcein-release, 51Cr release) using GD2+ neuroblastoma target cell lines. We demonstrate similar binding characteristics of APN301 to GD2 compared to parent 14.18 antibodies (murine and chimeric). The anti-14.18 anti-id antibody ganglidiomab fully inhibits binding of APN301 to the nominal antigen GD2, thereby proving internal image properties. The dissociation constants of APN301 to GD2 and ganglidiomab as determined in Biacore analyses are in the 10-9M and 10-7M range, respectively, similar to those determined for the naked chimeric 14.18 antibody. Relevant for the prediction of clinical activity we demonstrate potent specific lysis of GD2+ human neuroblastoma cells mediated by APN301 in a whole blood cytotoxicity assay in vitro, utilizing whole blood of healthy donors as well as whole blood of high-risk neuroblastoma patients in advanced disease stage. Further clinical trials with APN301 in neuroblastoma patients are being planned, taking into account the found potent whole blood cytolytic activity and the pharmakokinetic properties of this immunocytokine. Citation Format: Hans Loibner, Manfred Schuster, Evelyne Janzek, Stefan Stranner, Bernhard Peball, Susanne Wiederkum, Oliver Mutschlechner, Nikolai Siebert, Holger Lode. Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2013-2858 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

AQuantitative Evaluation OfRedox-Active Compounds In Human Blood Lipids

Endogenous low molecular weight redox active compounds (RACs) comprise antioxidants, pro-oxidants, transition metal cations and metal chelators. Traditional electrochemical methods of measuring RACs are limited to aqueous solutions, thus providing information of only hydrophilic RAC pools. In a large number of diseases associated with oxidative stress and/or with metal toxicity, redox states of hydrophilic as well as hydrophobic compartments are modified, and therefore development of methods for their detection is both necessary and important. The pools of lipid soluble RACs in reduced and oxidized forms in n-hexane extracts obtained from blood plasma, erythrocytes and whole blood of healthy donors were determined by spectrophotometric detection of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, which stoichiometrically interacts with hydrogen donors in non polar solutions. Measurements of RACs in extracts before and after treatment with NaBH4 provided information about the levels of both reduced and oxidized RACs. Vitamin E was also determined using a fluorescence method. The results have shown that vitamin E is the major RAC in blood plasma lipids but not in blood cell lipids, where other phenols and quinones appear to predominate.

Abstract 4901: Preliminary investigation of circulating NSCLC cells using dielectrophoresis-based instrumentation

Abstract Aims: The study of rare cell populations is an extremely important field in cancer research. Our main aim was to carry out a preliminary investigation into a new technological platform capable of identifying and studying rare tumor cells in whole blood samples from patients with advanced cancer. Methods: The study was performed using DEPArray™, a new dielectrophoresis-based instrumentation capable of detecting, examining and sorting circulating tumor cells (CTCs) in a very small quantity (microliters) of enriched whole blood. Analyses were conducted on 3 blood samples from patients with EGFR-mutated metastatic NSCLC and on 15 samples composed of A549 cells spiked into the whole blood of healthy donors. Whole blood starting volume was 7.5 ml. After Ficoll-Paque centrifugation, tumor cells were immunomagnetically enriched via EpCam positive selection or CD45 negative depletion and then fixed and stained with Hoechst and fluorescent conjugated antibodies against CD45 and pan-cytokeratin. Cell recovery was followed by EGFR gene mutation evaluation through sequence analysis. Results: Preliminary data showed that DEPArray™ detected, isolated and sorted enriched CTCs with 100% purity, with no lymphocyte contamination in subsequent analyses. Single or multiple cell sorting was possible, making it simple to perform different downstream molecular assays. In particular, the EGFR gene mutation status of NSCLC was studied to find out whether mutations were present or whether gene status remained that observed in the primary tumor tissue. Although it was decided to work on fixed cells during the investigation of this first set of samples, DEPArray™ is also capable of sorting live cells with high specificity. Conclusions: The study of infrequent tumor or tumor-related cells such as circulating tumor cells, cancer stem cells and/or circulating endothelial cells is an increasingly important area of research from both a preclinical and clinical point of view. A growing number of studies have evaluated different methods and variables involved in the detection of rare cells, in particular CTCs, but there is still no general consensus on the most accurate method to use. DEPArray™ is an innovative dielectrophoresis-based instrument capable of identifying, manipulating and sorting rare live and fixed cells with extremely high purity. Although the most accurate markers to discriminate between normal epithelial cells and tumor cells have yet to be defined, DEPArray™ has already proven to be extremely versatile in the field of rare cell studies, introducing new possibilities such as 100%-pure retrieval of live cells and furthering its potential as a promising new strategy for cancer cell detection and characterization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4901. doi:10.1158/1538-7445.AM2011-4901

Hemopexin Is Protective Against Intravascular Hemolysis–Induced Vascular Damage

AbstractAbstract 3207 Background:Intravascular hemolysis renders hemoglobin (Hb), usually protected by the red blood cell environment, susceptible to oxidation to its ferric (metHb) form. Haptoglobin (Hp) provides first aid to protect the vasculature from acellular metHb havoc. However, once Hp is consumed, ferrous Hb readily undergoes oxidation to metHb, in which the globin to heme affinity is reduced. MetHb releases the loosely bound heme allowing it to execute its oxidative activity. While nucleated cells, like endothelial cells, are equipped with the heme oxygenase-1 defence system, the nucleus lacking red blood cells (RBC) and low density lipoproteins (LDL) particles have no defence from heme-induced oxidation. Therefore the sole defence from heme oxidative damage remains the plasma protein hemopexin (Hx), which binds hemin with high affinity comparable to that of globin. Following hemin binding, Hx undergoes conformational changes rendering the heme inaccessible thereby inactive. Aim:The current study is focused on the ability of plasma Hx to defend the vasculature from acellular Hb induced damage. Results:Hx defence from metHb damage was studied in the following projects: a) preventing in vitro LDL oxidation by metHb, b) decreasing in vitro lysis of red cells created by hypotonic and mechanical stress. (c) Exploring consumption of Hx in hemodialysis (HD) treated patients by measuring their Hx levels. (a) Methemoglobin iron is a central cause of LDL oxidation. This process initiates formation of blood vessel plaques and atherosclerosis development. This part of the study examined the effect of apoHx (lacking heme) on LDL oxidation by metHb. Incubation of isolated native LDL (nLDL) or vitamin E depleted (dLDL) with low concentration of metHb range (1-10μM) resulted in covalent apoB inter-particle crosslinking in both LDL types, dLDL being more sensitive than nLDL to the oxidative crosslinking. Addition of apoHx abolished metHb-induced oxidation even in the dLDL. This Hx activity (comparable to that of haptoglobin) was confirmed by both SDS-PAGE and fluorescent assay of bityrosines formation. The fact that Hx protects LDL, especially vitamin E depleted particles, shed light on the controversial antioxidant vitamin E properties by indicating its efficacy in defence against acellular Hb induced LDL oxidation. (b) Like LDL the red cell membrane is exposed to metHb-induced oxidative damage. Acellular Hb was formed by hypotonic shock or mechanical shear stress to fresh whole blood of healthy donors. The effect of Hx on acellular Hb levels was measured spectrophotometrically. Reduction of osmotic pressure in fresh blood to 40% of its normal value resulted in formation of ∼300 μM acellular Hb. Supplementation of exogenous apoHx (20μM) to blood before exposure to hypotonicity reduced acellular Hb formation by half. The specificity of Hx was proved by addition of up to 80μM albumin, which had no effect on acellular Hb concentration. Acellular Hb was formed also by applying shear stress on circulating fresh blood in narrow channels using a peristaltic pump. Within 4 hours acellular Hb reached ∼500μM. However, in presence of 20μM apoHx hemolysis was significantly reduced (up to 50%). This reduction may be attributed to Hx's ability to block the hemolysis amplifying effect of metHb on the RBC membrane. (c) In HD treated patients RBCs are exposed to the effects of recurrent mechanical shear stress. The last part of the research was dedicated to quantisation of apoHx levels (by titration with hemin) in sera of HD patients (n=60) prior to and immediately after a single dialysis procedure versus normal controls (n=20). Sera apoHx levels in HD patients prior to a dialysis session were lower compared to healthy controls (10.91±3.19 μM vs. 14.95±2.63 μM, p<0.05) and were even lower (7.49±3.8 μM) immediately after the treatment. These findings indicate that in the plasma of HD treated patients Hx consumption rate exceeds synthesis rate leading to a chronically low level of Hx. Moreover, reduction of Hx was even more prominent in patients on long term HD treatment of above five years. Conclusion:Hx is specifically effective in attenuation of acellular Hb damage to the vasculature. These results point to the option that treating HD patients with exogenous Hx may slow down the rate of atherosclerotic cardiovascular disease development, the major cause of morbidity and mortality in these patients. Disclosures:No relevant conflicts of interest to declare.

Human peripheral γδ T cells possess regulatory potential

SummaryDeficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.