Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Impact of production methods and storage conditions on extracellular vesicles in packed red blood cells and platelet concentrates.

The use of blood and blood products can be life-saving, but there are also certain risks associated with their administration and use. Packed red blood cells (pRBCs) and platelet concentrates are the most commonly used blood products in transfusion medicine to treat anemia or acute and chronic bleeding disorders, respectively. During the production and storage of blood products, red blood cells and platelets release extracellular vesicles (EVs) as a result of the storage lesion, which may affect product quality. EVs are subcellular structures enclosed by a lipid bilayer and originate from the endosomal system or from the plasma membrane. They play a pivotal role in intercellular communication and are emerging as important regulators of inflammation and coagulation. Their cargo and their functional characteristics depend on the cell type from which they originate, as well as on their microenvironment, influencing their capacity to promote coagulation and inflammatory responses. Hence, the potential involvement of EVs in transfusion-related adverse events is increasingly recognized and studied. Here, we review the knowledge regarding the effect of production and storage conditions of pRBCs and platelet concentrates on the release of EVs. In this context, the mode of processing and anticoagulation, the influence of additive solutions and leukoreduction, as well as the storage duration will be addressed, and we discuss potential implications of EVs for the clinical outcome of transfusion.

Functionally impaired antibody response to BNT162b2 booster vaccination in CVID IgG responders.

BACKGROUND: Although previous studies described the production of IgG antibodies in a subgroup of patients with common variable immunodeficiency (CVID) following messenger RNA vaccinations with BNT162b2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (kdis) and the antibody response to booster immunization has not been studied. OBJECTIVE: We sought to analyze in CVID responders and healthy individuals, the avidity of anti-SARS-CoV-2 serum antibodies and their neutralization capacity as measured by surrogate virus-neutralizing antibodies in addition to IgG-, IgM-, and IgA-antibody levels and the response of circulating (peripheral blood) follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV-2 messenger RNA vaccine. METHODS: Binding IgG, IgA, and IgM serum levels were analyzed by ELISA in patients with CVID responding to the primary vaccination (CVID responders, n = 10) and healthy controls (n = 41). The binding avidity of anti-spike antibodies was investigated using biolayer interferometry in combination with biotin-labeled receptor-binding-domain of SARS-CoV-2 spike protein and streptavidin-labeled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. RESULTS: After the third vaccination with BNT162b2, IgG-, IgM-, and IgA-antibody levels, surrogate virus-neutralizing antibody levels, and antibody avidity were lower in CVID responders than in healthy controls. In contrast, anti-SARS-CoV-2 spike protein avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID responders was significantly reduced when compared with that in healthy individuals. CONCLUSIONS: Impaired affinity maturation during booster response provides new insight into CVID pathophysiology.

Interleukin-4 receptor alpha signaling regulates monocyte homeostasis.

Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα-/- ) and mice with a myeloid-specific deletion of IL-4Rα (IL-4Rαf/f LysMcre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4Rα-/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4Rα-/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4Rαf/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo.

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Mus musculus papillomavirus 1 is a key driver of skin cancer development upon immunosuppression.

Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.

Differential expression of Plg-RKT and its effects on migration of proinflammatory monocyte and macrophage subsets.

Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-RKT is expressed by immune cells, including monocytes and macrophages. Among monocytes and macrophages, distinct subsets can be distinguished based on cell surface markers and pathophysiological function. We investigated expression of Plg-RKT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14++CD16+ human monocytes and Ly6Chigh mouse monocytes expressed the highest levels of Plg-RKT and bound significantly more plasminogen compared with the other respective subsets. Proinflammatory human macrophages, generated by polarization with lipopolysaccharide and interferon-γ, showed significantly higher expression of Plg-RKT compared with alternatively activated macrophages, polarized with interleukin-4 and interleukin-13. Directional migration of proinflammatory monocytes was plasmin dependent and was abolished by anti-Plg-RKT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6Chigh monocyte recruitment was observed in Plg-RKT -/- compared with Plg-RKT +/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-RKT in vivo. Our data demonstrate higher expression of Plg-RKT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Neutrophil Extracellular Trap Degradation by Differently Polarized Macrophage Subsets.

OBJECTIVE: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden. CONCLUSIONS: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.

Deficiency of Cathelicidin-related Antimicrobial Peptide Promotes Skin Papillomatosis in Mus musculus Papillomavirus 1-infected Mice.

Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wild-type mice. These included a stronger reduction in CD4+ and CD8+ T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.

Storage of human whole blood, but not isolated monocytes, preserves the distribution of monocyte subsets.

Human monocytes include CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical) subsets with divergent roles in immune regulation and inflammation. Since the functional characterization of monocyte subsets is most commonly performed using isolated monocytes, we investigated the influence of different monocyte isolation protocols on the relative abundance of monocyte subsets. Using flow cytometric subset characterization directly in whole blood as a reference, we found that monocyte isolation by enrichment of peripheral blood mononuclear cells and subsequent depletion of non-monocytes by magnetic labeling did not alter the distribution of monocyte subsets. Particularly, we failed to detect a loss of CD16+ subsets upon monocyte isolation, although one of the negative depletion protocols used contained an anti-CD16 antibody to label granulocytes. Overnight storage of isolated monocytes induced a significant repartition of monocyte subsets towards CD14++CD16+ intermediate monocytes, which was barely seen in stored whole blood. We identified intermediate monocytes as main binding partners of platelet-derived extracellular vesicles (EVs) and propose that residual platelets contained in isolated monocyte preparations release EVs that induce the expression of the IgG receptor FcγRIII (CD16) on monocytes.

PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent.

OBJECTIVE: Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. APPROACH AND RESULTS: We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1-/- bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. CONCLUSIONS: We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1.

Analysis of platelet-derived extracellular vesicles in plateletpheresis concentrates: a multicenter study.

BACKGROUND: Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers. STUDY DESIGN AMD METHODS: Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry. RESULTS: During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p < 0.001) and PL-EVs increased in 74 PCs (A, C-E; p < 0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage. CONCLUSION: PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques.

The impact of citrate concentration on adhesion of platelets and leukocytes to adsorbents in whole blood lipoprotein apheresis.

Lipoprotein apheresis is applied to deplete low density lipoprotein and other apolipoprotein B containing lipoproteins in patients with severe familial hypercholesterolemia, hypertriglyceridemia associated pancreatitis, or lipoprotein (a)-hyperlipoproteinemia. Anticoagulation of the extracorporeal circuit may influence cellular activation, as evidenced by a reduction of inflammatory parameters during regional citrate anticoagulation with acid citrate dextrose A (ACD-A) commonly used in whole blood lipid apheresis. While the citrate concentration in the extracorporeal circuit has to ensure efficient anticoagulation, citrate infusion into the patient should be limited to avoid citrate overload. We assessed the influence of citrate concentration on cellular activation during in vitro circulation of whole blood containing 2.8 mM citrate (ACD-A 1:40), 5.6 mM citrate (ACD-A 1:20), or 13 mM citrate over polyacrylate-based adsorbents for lipoprotein apheresis. We found increased platelet adhesion for anticoagulation with 2.8 mM citrate as compared to 5.6 or 13 mM citrate, as shown by cell counting and confirmed by scanning electron microscopy of adsorbent beads as well as by elevated levels of platelet activation markers and of platelet-derived microvesicles. Leukocytes showed an equivalent adhesion pattern, while red blood cells remained unaffected at all citrate concentrations. Passage of blood over two consecutive columns resulted in enhanced platelet adhesion to the second column, presumably due to upstream preactivation. In conclusion, citrate influences activation and adhesion of platelets and leukocytes in a concentration-dependent manner, and ACD-A 1:20, equivalent to a citrate concentration of 5.6 mM in whole blood, ensures minimal cellular activation during passage of whole blood over polyacrylate-based adsorbents.

Release kinetics and mitogenic capacity of collagen barrier membranes supplemented with secretome of activated platelets - the in vitro response of fibroblasts of the periodontal ligament and the gingiva.

BACKGROUND: Platelet preparations can stimulate the healing process and have mitogenic properties. We hypothesized that collagen barrier membranes (CBM), clinically used in guided bone regeneration and guided tissue regeneration, can serve as carriers for platelet secretome. METHODS: Secretome was generated from washed platelets and unwashed platelets (washed/unwashed PSEC) and lyophilized onto CBM. Overall appearance of CBM was evaluated by scanning electron microscopy. The impact of PSEC on cell attachment was measured based on fluorescence microscopy with DiI-labeled cells. To assess the release kinetics, supernatants of CBM were collected and medium was replaced at hour 1-48. The mitogenic effect was evaluated with periodontal fibroblasts. Furthermore, the release of total protein, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF) ß1 was measured. RESULTS: CBM overall appearance and cell attachment was not modulated by PSEC. Supernatants taken after one hour induced a mitogenic response in fibroblasts and showed the highest levels of total protein, TGFß1 and PDGF-BB. These effects decreased rapidly in subsequent supernatants. While supernatants of CBM loaded with unwashed PSEC induced a stronger mitogenic response than supernatants of CBM loaded with washed PSEC this difference between the PSEC preparations was not observed when cells were seeded on 48-hours-washed CBM. CONCLUSIONS: CBM release platelet-derived factors in continuously declining release kinetics.

Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Association of HER2 codon 655 polymorphism with ovarian cancer.

The role of the human epidermal growth factor receptor 2 (HER2) codon 655 (Ile655Val) polymorphism in ovarian cancer is not fully understood. Two studies indicated a possible association between the Val allele and elevated risk or reduced prognosis of ovarian cancer. We investigated the HER2 codon 655 (rs1136201) polymorphism in 242 Austrian women-142 ovarian cancer patients and 100 healthy controls-by polymerase chain reaction and pyrosequencing. Associations between Ile655Val polymorphism and clinicopathological variables (e.g., age, FIGO stage, grading, serous vs. non-serous histology) were evaluated. The genotype distributions in ovarian cancer patients and controls were: AA; 66.2 %, AG; 25.35 %, GG; 8.45 %, and AA; 63 %, AG; 34 %, GG; 3.7 %, respectively (OR 1.15, CI 95 % 0.67-1.96). We observed a non-significant trend toward elevated cancer risk in Val/Val genotype (OR 2.98, CI 95 % 0.82-10.87, p = 0.10). Of note, 11 out of 12 Val/Val homozygotes were postmenopausal. The link between the Val/Val homozygosity and age over 50 years at diagnosis (OR 0.15, CI 95 % 0.02-1.2) was barely significant (p = 0.056). Summarizing, our data indicated a non-significant trend toward increased ovarian cancer risk in the Val/Val homozygosity, especially in women aged above 50 years. Further large-cohort studies focusing on the role of the HER2 codon 655 Val allele are needed.

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies.

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.