Multifunctional superparamagnetic MnO@SiO2 core/shell nanoparticles and their application for optical and magnetic resonance imaging

Highly biocompatible multifunctional nanocomposites consisting of monodisperse manganese oxide nanoparticles with luminescent silica shells were synthesized by a combination of w/o-microemulsion techniques and common sol–gel procedures. The nanoparticles were characterized by TEM analysis, powder XRD, SQUID magnetometry, FT-IR, UV/vis and fluorescence spectroscopy and dynamic light scattering. Due to the presence of hydrophilic poly(ethylene glycol) (PEG) chains on the SiO2 surface, the nanocomposites are highly soluble and stable in various aqueous solutions, including physiological saline, buffer solutions and human blood serum. The average number of surface amino groups available for lig…

UV Exposure Boosts Transcutaneous Immunization and Improves Tumor Immunity: Cytotoxic T-Cell Priming through the Skin

Immunologic approaches to combat cancer aim at the induction of tumor-reactive immune responses to achieve long-term protection. In this context, we recently developed a transcutaneous immunization (TCI) method using the Toll-like receptor (TLR) 7 agonist imiquimod and a peptide epitope. Application onto intact skin induces potent cytotoxic T lymphocyte (CTL) responses and protection against transplanted tumors. The purpose of this study was to explore the effects of UV irradiation on imiquimod-based TCI. Here we show that skin exposure to low-dose UV light before TCI with imiquimod strongly boosts specific CTL responses leading to memory formation and enhanced tumor protection. Toward the …

Distinct Signaling Cascades of TREM-1, TLR and NLR in Neutrophils and Monocytic Cells

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an important mediator of innate inflammatory responses in microbial infections and sepsis. TREM-1 ligation on neutrophils (PMN) or monocytes results in the production of proinflammatory cytokines. Engagement of TREM-1 induces the activation of MAP kinases as well as rapid Ca<sup>2+</sup> mobilization. However, a detailed understanding of TREM-1 signaling pathways is currently lacking. We evaluated the TREM-1 signaling hierarchy in monocytic cells and found that the acute myeloid leukemia cell line MUTZ-3 expresses TREM-1 in a natural and functional manner. We compared essential signaling molecules of the TREM-1, TLR an…

Donor and host B cell-derived IL-10 contributes to suppression of graft-versus-host disease

Graft-versus-host disease (GvHD) is a frequent life-threatening complication following allogeneic HSC transplantation (HSCT). IL-10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL-10-deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC-mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL-10 resulted in increased allogeneic T-cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL-10 was prominently p…

ERK3/MAPK6 controls IL-8 production and chemotaxis

ERK3 is a ubiquitously expressed member of the atypical mitogen activated protein kinases (MAPKs) and the physiological significance of its short half-life remains unclear. By employing gastrointestinal 3D organoids, we detect that ERK3 protein levels steadily decrease during epithelial differentiation. ERK3 is not required for 3D growth of human gastric epithelium. However, ERK3 is stabilized and activated in tumorigenic cells, but deteriorates over time in primary cells in response to lipopolysaccharide (LPS). ERK3 is necessary for production of several cellular factors including interleukin-8 (IL-8), in both, normal and tumorigenic cells. Particularly, ERK3 is critical for AP-1 signaling…

Current Progress in Particle-Based Systems for Transdermal Vaccine Delivery

Transcutaneous immunization (TCI) via needle-free and non-invasive drug delivery systems is a promising approach for overcoming the current limitations of conventional parenteral vaccination methods. The targeted access to professional antigen-presenting cell (APC) populations within the skin, such as Langerhans cells (LCs), various dermal dendritic cells (dDCs), macrophages, and others makes the skin an ideal vaccination site to specifically shape immune responses as required. The stratum corneum (SC) of the skin is the main penetration barrier that needs to be overcome by the vaccine components in a coordinated way to achieve optimal access to dermal APC populations that induce priming of…

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

Polymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Although it has long been known that marrow cellularity is inversely correlated with G-CSF levels, the mechanism linking peripheral clearance to production remains unknown. Herein, the feedback response to antibody induced neutropenia is characterized to consist of G-CSF–dependent shifts of marrow hematopoietic progenitor populations including expansion of the lin-/Sca-1/c-kit (LSK) and granulocyte macrophage progenitor (GMP) compartments at the expense of thrombopoietic and red cell precursors. Evidence is …

Herpes virus entry mediator synergizes with Toll-like receptor mediated neutrophil inflammatory responses

In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the f…

Updated Results from the German Mpnsg-0212 Combination Trial: Ruxolitinib Plus Pomalidomide in Myelofibrosis with Anemia

Background: Anemia remains one cardinal symptom associated with reduced quality of life (QoL) in patients (pts) with myelofibrosis (MF) which is normally not being addressed by ruxolitinib (RUX). In our previous MPNSG-0109 trial, single-agent pomalidomide (POM) improved cytopenia in 14% (POM 0.5 mg QD) and 29% (POM 2.0 mg QD) of MF pts, respectively. In the MPNSG-0212 study, we sought to investigate the potential synergism of RUX plus POM to improve anemia and QoL in MF pts. Study Design: MPNSG-0212 is an ongoing multicenter, open-label, single-arm phase-Ib/II trial with a target population of 90 pts following a two-stage design (NCT01644110). Pts 1-40 in cohort 1 (co1) were treated with RU…

Regulatory T Cells and IL-10 Independently Counterregulate Cytotoxic T Lymphocyte Responses Induced by Transcutaneous Immunization

Background: The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (Treg) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses. Methodology/Principal Findings: TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after…

Infectious complications in patients with myelodysplastic syndromes: A review of the literature with emphasis on patients treated with 5-azacitidine.

Myelodysplastic Syndromes are oligo-clonal stem cell disorders that are associated with cytopenias in the peripheral blood. Major causes for morbidity and mortality in myelodysplastic syndromes (MDS) patients are infections mostly due to bacteria or fungi. Beside leucopenia per se in affected patients, function of white blood cells particularly that of neutrophils seems to be impaired. Here we summarize the available data on infections in MDS patients in general and particularly those treated with 5-azacitidine.

Antifungal drugs influence neutrophil effector functions

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) re…

A Role for NFAT in Innate Immunity: Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation Under Cyclosporine Α Treatment

Abstract Background and Aims: Patients after allogeneic hematopoietic stem cell transplantation (HSCT) suffer from immunodeficiency, in part due to long-term immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA). Additionally, these patients have an increased risk for opportunistic fungal infections like invasive aspergillosis (IA). The nuclear factor of activated T cells (NFAT) is known as an important transcription factor in signaling-pathways downstream of calcineurin in the adaptive immune systems, e.g. in T cells, but also plays an important role in innate immune response as indicated by recent data in rodent models. These studies showed a relevant impac…

Dermal CD207-Negative Migratory Dendritic Cells Are Fully Competent to Prime Protective, Skin Homing Cytotoxic T-Lymphocyte Responses

Dendritic cells (DCs) are important inducers and regulators of T-cell responses. They are able to activate and modulate the differentiation of CD4+ and CD8+ T cells. In the skin, there are at least five phenotypically distinct DC subpopulations that can be distinguished by differential expression of the cell surface markers CD207, CD103, and CD11b. Previous studies have suggested that dermal CD11b−CD207+ conventional type 1 DCs are indispensable for the priming of a skin homing cytotoxic T-lymphocyte response. However, conventional type 1 DCs are also the only skin DC subset capable of cross-presenting exogenous antigens on major histocompatibility complex class I. Thus, it remained unclear…

Luspatercept Increases Hemoglobin and Reduces Transfusion Burden in Patients with Low-Intermediate Risk Myelodysplastic Syndromes (MDS): Long-Term Results from Phase 2 PACE-MDS Study

Abstract Background: Management of anemia is a common therapeutic challenge in patients with MDS. Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014). Aims: This is an ongoing, phase 2, multicenter, open-label, long-term extension study to evaluate the effects of luspatercept in patients (pts) with low-intermediate risk MDS. Endpoints include long-term safety and tolerability, erythroid respo…

Luspatercept Response in ESA-NaïVe/RS+ Patients and RS- Patients with Low-Intermediate Risk Myelodysplastic Syndromes (MDS)

Abstract Background: Management of anemia is a common therapeutic challenge in patients with myelodysplastic syndromes (MDS). Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014). Aims: This is an ongoing, phase 2, multicenter, open-label study to evaluate the effects of luspatercept in patient (pts) with low-intermediate risk MDS. Endpoints included erythroid response (IWG HI-E), RBC transfus…

Phenotypic and functional characterization of neutrophils and monocytes from patients with myelodysplastic syndrome by flow cytometry.

Myelodysplastic syndrome (MDS) is a clonal stem cell disorder frequently associated with inefficient granulopoiesis showing dysplastic polymorphonuclear neutrophils (PMNs). To assess PMN functionality in MDS in a clinical routine setting, 30 MDS patients and ten healthy volunteers were analyzed for PMN and monocyte phenotype and function (degranulation, CD62L shedding, oxidative burst and phagocytosis) upon stimulation with lipopolysaccharide by multi-color flow cytometry (MCFC). Our data show a heterogeneous pattern for CD66, CD16 and CD64 expression on PMNs of MDS patients. CD62L shedding rate and CD66 degranulation were reduced. Interestingly, we detected correlations between the WHO ada…

Idelalisib Impairs TREM-1 and TLR Mediated Neutrophil Activation

Abstract Introduction: Polymorphonuclear neutrophils (PMN) play an essential role in innate inflammatory processes. Their functions are strictly regulated and many activating / inhibiting mechanisms along with their pathways are only incompletely understood. Besides toll-like receptors (TLR) and NOD-like receptors (NLR), triggering receptor expressed on myeloid cells (TREM)-1 is implicated in innate immune activation of these cells and plays a role in infectious as well as non-infectious conditions. Activation of TREM-1 results in release of pro-inflammatory chemokines and cytokines, increased surface expression of cell activation markers and degranulation. In TREM-1 downstream pathways and…

Biomarkers of Ineffective Erythropoiesis Predict Response to Luspatercept in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS): Final Results from the Phase 2 PACE-MDS Study

Abstract Background: Luspatercept is a fusion protein (modified activin receptor IIB-IgG Fc) being investigated for the treatment of anemias with ineffective erythropoiesis. MDS patients have increased Smad2/3 signaling in the bone marrow, leading to ineffective erythropoiesis. Luspatercept inhibits Smad2/3 signaling and promotes late-stage erythroid differentiation, thereby correcting ineffective erythropoiesis. Aims: This completed, 3-month, phase 2, multicenter, open-label study evaluated the effects of luspatercept on anemia in patients with low/int-1 risk MDS (IPSS classification). Study outcomes include erythroid response of increased hemoglobin (Hb) in low transfusion burden (LTB) pa…

Host-Derived CD8+ Dendritic Cells Protect Against Acute Graft-versus-Host Disease after Experimental Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a frequent life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT) and induced by donor-derived T cells that become activated by host antigen-presenting cells. To address the relevance of host dendritic cell (DC) populations in this disease, we used mouse strains deficient in CD11c(+) or CD8α(+) DC populations in a model of acute GVHD where bone marrow and T cells from BALB/c donors were transplanted into C57BL/6 hosts. Surprisingly, a strong increase in GVHD-related mortality was observed in the absence of CD11c(+) cells. Likewise, Batf3-deficient (Batf3(-/-)) mice that lack CD8α(+) DCs also displayed a strongly incr…

Neutrophil extracellular traps impair fungal clearance in a mouse model of invasive pulmonary aspergillosis

Abstract Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. T…

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue da…

Clinical Presentation of Patients with Adult Late-Onset Telomere Biology Disorders - Results from the Aachen Telomeropathy Registry

Abstract Introduction: Telomere biology disorders (TBD) are caused by mutations affecting proper telomere maintenance resulting in premature telomere shortening. Telomere length (TL) assessment is currently being used for screening and diagnosis of TBD of which Dyskeratosis congenita (DKC) is the most prominent TBD subtype typically found in children and adolescents. In adults, TBDs are characterized by a broad spectrum of more "cryptic" diverging mono- or oligosymptomatic clinical manifestations such as bone marrow failure (BMF), hepatopathy or interstitial lung disease (ILD). However, despite growing general clinical awareness and exertion of improved TL screening strategies, insufficient…

Quizartinib versus salvage chemotherapy in relapsed or refractory FLT3-ITD acute myeloid leukaemia (QuANTUM-R): a multicentre, randomised, controlled, open-label, phase 3 trial

Background Patients with relapsed or refractory FLT3 internal tandem duplication (FLT3-ITD)-positive acute myeloid leukaemia have a poor prognosis, including high frequency of relapse, poorer response to salvage therapy, and shorter overall survival than those with FLT3 wild-type disease. We aimed to assess whether single-agent quizartinib, an oral, highly potent and selective type II FLT3 inhibitor, improves overall survival versus salvage chemotherapy. Methods QuANTUM-R is a randomised, controlled, phase 3 trial done at 152 hospitals and cancer centres in 19 countries. Eligible patients aged 18 years or older with ECOG performance status 0-2 with relapsed or refractory (duration of first …

A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells.

Abstract The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I–restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results …

MDS-191: Long-Term Efficacy and Safety of Luspatercept in Lower-Risk Myelodysplastic Syndromes (MDS): Phase 2 PACE-MDS Study

Background: Luspatercept, a first-in-class erythroid maturation agent, has been investigated in patients with LR-MDS and ring sideroblasts (RS) (MEDALIST; Fenaux and Platzbecker NEJM 2020) and in an ongoing Phase 3 trial regardless of RS status (COMMANDS, NCT03682536 ). The previously reported Phase 2 trial of luspatercept (Platzbecker Lanc Onc 2017) includes subtypes of LR-MDS with and without RS, regardless of prior ESA exposure, and various EPO levels. Aims: Evaluate the long-term safety and efficacy of luspatercept in LR-MDS. Methods: Patients were IPSS low/int-1, age ≥ 18 years, Hgb NCT01749514 ; NCT02268383 ). Results: As of 13July2019, 115 patients were enrolled, of whom 108 were tre…

Genomic Landscape and Molecular Risk in Patients with Advanced Myelofibrosis Treated within the Multicenter Phase Ib/II MPNSG0212 (POMINC) Trial

Abstract Introduction: Mutations (muts) in JAK2, MPL, and CALR are genetic hallmarks in myeloproliferative neoplasms such as myelofibrosis (MF). Prognostication in MF is predominantly based on clinical parameters according to the Dynamic International Prognostic Scoring System (DIPSS). However, gene mutations become increasingly important allowing for a more precised assessment of prognosis. For instance, CALR mutated MF is associated with favorable prognosis, while mutations in distinct high molecular-risk (HMR) genes are considered adverse. Our multicenter phase-Ib/II MPNSG-0212 trial (NCT01644110) investigating ruxolitinib plus pomalidomide in a total cohort of 92 patients with advanced …

Impaired Mast Cell-Driven Immune Responses in Mice Lacking the Transcription Factor NFATc2

Abstract The three calcium-dependent factors NFATc1, c2, and c3 are expressed in cells of the immune system and play pivotal roles in modulating cellular activation. With regard to NFATc2, it was reported that NFATc2-deficient mice display increased immune responses in several models for infection and allergy in vivo. This led to the assumption that NFATc2 is involved in the maintenance of immune homeostasis. Using the synthetic TLR7 agonist imiquimod as an adjuvant in epicutaneous peptide immunization, we observed that both the inflammatory reaction and the peptide-specific CTL response are severely impaired in NFATc2-deficient mice. Detailed analyses revealed that early production of proi…

A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome.

The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonis…

53 LUSPATERCEPT INCREASES HEMOGLOBIN AND REDUCES TRANSFUSION BURDEN IN PATIENTS WITH LOW OR INTERMEDIATE-1 RISK MYELODYSPLASTIC SYNDROMES (MDS): PRELIMINARY RESULTS FROM A PHASE 2 STUDY

Introduction. ACE-536, a recombinant fusion protein containing modified activin receptor type IIB and IgG Fc, is being developed for the treatment of anemias due to ineffective erythropoiesis, such as myelodysplastic syndromes (MDS). Patients with MDS often have elevated levels of erythropoietin (EPO) and may be non-responsive or refractory to erythropoiesis-stimulating agents (ESAs). MDS patients have also been shown to have increased serum GDF11 levels (Suragani R et al., Nature Medicine 2014) and increased Smad 2/3 signaling in the bone marrow (Zhou L et al., Blood 2008). ACE-536 binds to ligands in the TGF-s superfamily, including GDF11, inhibits Smad 2/3 signaling, and promotes late-st…

Xenograft models for undifferentiated pleomorphic sarcoma not otherwise specified are essential for preclinical testing of therapeutic agents

Undifferentiated pleomorphic sarcoma not otherwise specified belongs to the heterogeneous group of soft tissue tumors. It is preferentially located in the upper and lower extremities of the body, and surgical resection remains the only curative treatment. Preclinical animal models are crucial to improve the development of novel chemotherapeutic agents for the treatment of undifferentiated pleomorphic sarcoma. However, this approach has been hampered by the lack of reproducible animal models. The present study established two xenograft animal models generated from stable non-clonal cell cultures, and investigated the difference in chemotherapeutic effects on tumor growth between undifferenti…

Proteasomes shape the repertoire of T cells participating in antigen-specific immune responses

Differences in the cleavage specificities of constitutive proteasomes and immunoproteasomes significantly affect the generation of MHC class I ligands and therefore the activation of CD8-positive T cells. Based on these findings, we investigated whether proteasomal specificity also influences CD8-positive T cells during thymic selection by peptides derived from self proteins. We find that one of the self peptides responsible for positive selection of ovalbumin-specific OT-1 T cells, which is derived from the f-actin capping protein (Cpalpha1), is efficiently generated only by immunoproteasomes. Furthermore, OT-1 mice backcrossed onto low molecular mass protein 7 (LMP7)-deficient mice show a…

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA

Cells 9(9), 2034 (1-19) (2020). doi:10.3390/cells9092034

Bone Marrow Derived Mesenchymal Cells Secrete Granulopoietic Cytokines upon Danger Signaling

Abstract Granulopoietic homeostasis is regulated at steady-state to supply sufficient numbers of pooled and circulating neutrophils to maintain barrier function against commensal flora. In addition, upon pathogenic microbial challenge, an increased formation of neutrophils is induced, termed ‘emergency granulopoiesis’. Antibody-mediated reduction of neutrophil numbers in steady-state induces a feedback loop leading to an increase of bone marrow granulopoiesis with expansion of hematopoetic stem and progenitor cells. This feedback loop was demonstrated to depend on TLR4 and TRIF, but not MyD88 signaling (Bugl et al. Blood 2013). In contrast, emergency granulopoiesis was shown to be dependent…

The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Abstract Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as…

Combined immunotherapy: CTLA-4 blockade potentiates anti-tumor response induced by transcutaneous immunization.

Abstract Background The epidermal application of the Toll Like Receptor 7 agonist imiquimod and a T-cell peptide epitope (transcutaneous immunization, TCI) mediates systemic peptide-specific cytotoxic T-cell (CTL) responses and leads to tumor protection in a prophylactic tumor setting. However, it does not accomplish memory formation or permanent defiance of tumors in a therapeutic set-up. As a distinct immunologic approach, CTLA-4 blockade augments systemic immune responses and has shown long-lasting effects in preclinical experiments as well as in clinical trials. Objective The study investigates the vaccination capacity of TCI in combination with the checkpoint inhibitor CTLA-4 in matter…

T cell avidity determines the level of CTL activation

To investigate the influence of avidity on T cell activation in vitro and in vivo, we analyzed T cells from St40 and St42 mice, which express the same transgenic TCR specific for an E1a-derived epitope of adenovirus type 5 with different expression levels and therefore different avidities. Splenocytes from both strains showed comparable cytolytic activities and required identical peptide concentrations for efficient target cell lysis and up-regulation of activation markers. However, the kinetics of CD25 up-regulation were strikingly different: whereas the majority of the high-avidity St42 T cells up-regulated the IL-2Ralpha chain within a few hours, low-avidity St40 T cells expressed only 5…

Transcutaneous immunization with imiquimod is amplified by CD40 ligation and results in sustained cytotoxic T-lymphocyte activation and tumor protection.

Transcutaneous immunization (TCI) using ligands of Toll-like receptors (TLRs) and cytotoxic T-lymphocyte (CTL) epitopes lead to the induction of potent T-cell responses. To characterize the efficacy of TCI-mediated CTL activation, we monitored the frequency and functional activity of specific CTL induced with TCI using the ovalbumin-derived epitope SIINFEKL composed in creme containing the synthetic TLR7 ligand R-837. We found that the frequency and activity decayed rapidly 10 d post-TCI. Consistently, no significant memory T-cell formation was detectable. In a prophylactic vaccination setting, TCI was protective against a lethal challenge with ovalbumin expressing EG.7 thymoma cells when t…

Challenges of patients with myeloproliferative neoplasms (MPN) in times of COVID: first results from a patient survey by the German Study Group for MPN

Luspatercept Treatment Leads to Long Term Increases in Hemoglobin and Reductions in Transfusion Burden in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes (MDS): Preliminary Results from the Phase 2 PACE-MDS Extension Study

Background: Luspatercept is a fusion protein (modified activin receptor IIB-IgG Fc) being investigated for the treatment of anemias with ineffective erythropoiesis. MDS patients have increased Smad2/3 signaling in the bone marrow, leading to ineffective erythropoiesis. Luspatercept inhibits Smad2/3 signaling and promotes late-stage erythroid differentiation, thereby correcting the ineffective erythropoiesis. Aims: This is an ongoing, phase 2, multicenter, open-label, 24-month extension study (following a 3-month base study) to evaluate the longer-term effects of luspatercept on anemia in patients (pts) with low/int-1 risk MDS (IPSS classification). Study outcomes include erythroid response…

Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity.

BACKGROUND AIMS Triggering receptor expressed on myeloid cells 1 (TREM 1) is a potent amplifier of pro inflammatory responses. We have previously demonstrated a substantial increase in TREM 1 expressing macrophages in the inflamed intestinal mucosa of patients with inflammatory bowel diseases (IBD). TREM 1 is also produced as a soluble receptor (sTREM 1). Here we aimed to determine whether serum sTREM 1 could be used as a surrogate marker of disease activity in patients with IBD. METHODS Intestinal biopsies and concurrently collected sera from patients with Crohn's disease (CD) and Ulcerative colitis (UC) enrolled in the Swiss IBD cohort study were analyzed for intestinal TREM 1 mRNA and se…

Mast cell-derived mediators promote murine neutrophil effector functions

Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neu…

Erythropoietic cellular analyses in luspatercept-treated lower-risk myelodysplastic syndromes (MDS): Phase 2 PACE-MDS study.

7018Background: Luspatercept (ACE-536) is a TGF-β family ligand trap promoting late-stage erythroid (E) differentiation and increases in hemoglobin. Endpoints of the ongoing, phase 2, open-label st...

Neuroendocrine Modulation of IL-27 in Macrophages

Abstract Heterodimeric IL-27 (p28/EBV-induced gene 3) is an important member of the IL-6/IL-12 cytokine family. IL-27 is predominantly synthesized by mononuclear phagocytes and exerts immunoregulatory functional activities on lymphocytic and nonlymphocytic cells during infection, autoimmunity or neoplasms. There is a great body of evidence on the bidirectional interplay between the autonomic nervous system and immune responses during inflammatory disorders, but so far IL-27 has not been defined as a part of these multifaceted neuroendocrine networks. In this study, we describe the role of catecholamines (as mediators of the sympathetic nervous system) related to IL-27 production in primary …

Transcutaneous Immunization with a Solid Nanoscopic Imiquimod Suspension Enhances Tumor Rejection

Abstract Introduction: Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod that partial tumor protection in experimental rodent models. In our present work, we describe a novel optimized formulation of imiquimod in a solid suspension of crystalline imiq…

Signaling pathways of the TREM-1- and TLR4-mediated neutrophil oxidative burst.

The triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in the innate inflammatory response to microbial infections. Activation and expression of TREM-1 by polymorphonuclear neutrophils (PMN) occurs in concert with Toll-like receptors (TLR) such as TLR4 for bacterial lipopolysaccharide. However, it is currently unclear how this is mediated on a molecular level. Using pharmacological inhibitors and Western blot analysis we demonstrate that phosphatidyl inositide 3-kinase, phospholipase C and the mitogen-activated kinase p38MAPK are essential for the TREM-1- and TLR4-induced oxidative burst of human PMN. The activation of protein kinase B and extracellular signal-related kin…

Interruption of Macrophage-Derived IL-27(p28) Production by IL-10 during Sepsis Requires STAT3 but Not SOCS3

Abstract Severe sepsis and septic shock are leading causes of morbidity and mortality worldwide. Infection-associated inflammation promotes the development and progression of adverse outcomes in sepsis. The effects of heterodimeric IL-27 (p28/EBI3) have been implicated in the natural course of sepsis, whereas the molecular mechanisms underlying the regulation of gene expression and release of IL-27 in sepsis are poorly understood. We studied the events regulating the p28 subunit of IL-27 in endotoxic shock and polymicrobial sepsis following cecal ligation and puncture. Neutralizing Abs to IL-27(p28) improved survival rates, restricted cytokine release, and reduced bacterial burden in C57BL/…

Cutting edge: priming of CTL by transcutaneous peptide immunization with imiquimod.

Abstract CTL are important in combating cancer and viruses. Therefore, triggering the complete potential of CTL effector functions by new vaccination strategies will not only improve prophylaxis of tumor or virus-related diseases, but also open opportunities for effective therapeutic immunizations. Using transcutaneous immunization, we show that epicutaneous (e.c.)4 application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activ…

CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Abstract Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked …

Leukocyte–platelet aggregates—a phenotypic characterization of different stages of peripheral arterial disease

The formation of monocyte-platelet aggregates and neutrophil-platelet aggregates (MPA and NPA, respectively) is influenced by inflammation, but also might contribute to an exacerbation of inflammatory responses in atherosclerotic plaque. The purpose of this study was to analyze MPA and NPA proportions in regard to different stages of peripheral arterial disease (PAD). Forty-five patients with intermittent claudication (IC) (3 groups: Rutherford (R)-1, R-2, and R-3; each n = 15), 20 patients with critical limb ischemia (CLI) (Rutherford 5 (40%) and 6 (60%)), and 20 healthy controls were studied. Analyses of monocyte (Mon) subpopulations (CD14++CD16- (classical) Mon1, CD14++CD16+ (intermediat…

Real Life Experience with ATRA-Arsenic Trioxide Based Regimen in Acute Promyelocytic Leukemia - Updated Results of the Prospective German Intergroup Napoleon Registry

Abstract Background: Standard therapy of acute promyelocytic leukemia has long relied on the combination of All-trans-retinoic acid (ATRA) and chemotherapy. The introduction of arsenic trioxide (ATO) in APL treatment has allowed achievement of similarly high remission and survival rates coupled with significantly reduced myelosuppression. Recent results of the APL0406 trial by the GIMEMA-AMLSG-SAL study groups showed that the combination of ATRA and arsenic trioxide (ATO) is superior to standard ATRA and chemotherapy (CHT) in front-line therapy of low/intermediate risk acute promyelocytic leukemia (APL). The implications of these results for the clinical practice of APL patients in Germany …

Lipid presentation by the protein C receptor links coagulation with autoimmunity.

A lipid-protein autoimmunity target Several autoimmune diseases, including systemic lupus erythematosus and primary antiphospholipid syndrome, are characterized by the presence of antiphospholipid antibodies (aPLs). These molecules can activate the complement and coagulation cascades, which contributes to pathologies such as thrombosis, stroke, and pregnancy complications. Müller-Calleja et al. found that endothelial protein C receptor (EPCR) in complex with lysobisphosphatidic acid (LBPA) is the cell-surface target for aPL and mediates its internalization (see the Perspective by Kaplan). aPL binding to EPCR-LBPA resulted in the activation of tissue factor–mediated coagulation and interfero…

Mechanisms of Synergy Between Toll-Like Receptor 4 and Triggering Receptor Expressed on Myeloid Cells-1 in Human Neutrophils

Abstract The triggering receptor expressed on myeloid cells 1 (TREM-1) is an important player in the innate inflammatory response to microbial infections. Activation and expression of TREM-1 by polymorphonuclear neutrophils (PMN) occurs in concert with Toll-like receptors (TLR) such as TLR4 for bacterial lipopolysaccharide. However, it is currently unclear how this is mediated on a molecular level. Using pharmacologic inhibitors and western blot analysis we demonstrate that phosphatidyl inositide 3-kinase, phospholipase C and the mitogen activated kinase p38 are essential for the TREM-1 and TLR4 mediated respiratory burst of human PMN. The down stream phosphorylation of protein kinase B and…

Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection

Abstract Background Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. Objective Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination …

Luspatercept for the treatment of anaemia in patients with lower-risk myelodysplastic syndromes (PACE-MDS): a multicentre, open-label phase 2 dose-finding study with long-term extension study

Myelodysplastic syndromes are characterised by ineffective erythropoiesis. Luspatercept (ACE-536) is a novel fusion protein that blocks transforming growth factor beta (TGF β) superfamily inhibitors of erythropoiesis, giving rise to a promising new investigative therapy. We aimed to assess the safety and efficacy of luspatercept in patients with anaemia due to lower-risk myelodysplastic syndromes.In this phase 2, multicentre, open-label, dose-finding study (PACE-MDS), with long-term extension, eligible patients were aged 18 years or older, had International Prognostic Scoring System-defined low or intermediate 1 risk myelodysplastic syndromes or non-proliferative chronic myelomonocytic leuk…

Genetic Variation Determines Mast Cell Functions in Experimental Asthma

Abstract Mast cell-deficient mice are a key for investigating the function of mast cells in health and disease. Allergic airway disease induced as a Th2-type immune response in mice is employed as a model to unravel the mechanisms underlying inception and progression of human allergic asthma. Previous work done in mast cell-deficient mouse strains that otherwise typically mount Th1-dominated immune responses revealed contradictory results as to whether mast cells contribute to the development of airway hyperresponsiveness and airway inflammation. However, a major contribution of mast cells was shown using adjuvant-free protocols to achieve sensitization. The identification of a traceable ge…

Current insights into neutrophil homeostasis

Neutrophil granulocytes represent the first immunologic barrier against invading pathogens, and neutropenia predisposes to infection. However, neutrophils may also cause significant collateral inflammatory damage. Therefore, neutrophil numbers are tightly regulated by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Granulocyte colony-stimulating factor (G-CSF) is accepted to be the major determinant of neutrophil production, and G-CSF levels have, soon after its discovery, been described to be inversely correlated with neutrophil counts. A neutrophil sensor, or "neutrostat," has, therefore, been postulated. The prevailing feedback hypo…

Precursor frequency can compensate for lower TCR expression in T cell competition during priming in vivo.

The factors controlling clonal dominance of cytotoxic T lymphocyte (CTL) responses are currently not well understood. To study the functional impact of the strength of the interaction of a T cell with an antigen-presenting cell in this context, we established a new mouse model comprised of two T cell receptor (TCR)-transgenic strains expressing the identical TCR in differing amounts, hence providing two CTL clones with different avidities but identical specificity and affinity. Utilizing this new model, we show that upon antigen challenge higher-avidity CTL expand at the expense of moderate-avidity CTL in vivo if present in equal numbers. Beyond this, moderate-avidity T cells can also contr…

Efficacy and Safety of Single-Agent Quizartinib (Q), a Potent and Selective FLT3 Inhibitor (FLT3i), in Patients (pts) with FLT3-Internal Tandem Duplication (FLT3-ITD)-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Enrolled in the Global, Phase 3, Randomized Controlled Quantum-R Trial

Abstract Introduction: FLT3-ITD mutations are among the most common molecular abnormalities in AML, occurring in ≈ 25% of pts. These driver mutations are associated with high leukemic burden and poor prognosis, eg, high risk of relapse, decreased response to salvage therapy, and shorter overall survival (OS). Pts with R/R FLT3-ITD AML have a worse prognosis and represent a population with high unmet medical need. Q is a once-daily, oral, highly potent and selective FLT3i shown in phase 2 trials to have promising single-agent antileukemic activity and a manageable safety profile. QuANTUM-R was the first global, phase 3, randomized controlled trial (NCT02039726) to show that an FLT3i prolonge…

Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

Abstract Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival. Methods: IPA was…

Improved Outcome with ATRA-Arsenic Trioxide Compared to ATRA-Chemotherapy in Non-High Risk Acute Promyelocytic Leukemia - Updated Results of the Italian-German APL0406 Trial on the Extended Final Series

Abstract Background: We recently showed that the combination of ATRA and arsenic trioxide (ATO) is at least not inferior and possibly superior to standard ATRA and chemotherapy (CHT) in the front-line management of low/intermediate risk APL (Italian-German APL 0406 trial; Lo-Coco et al., NEJM 2013). We report herein on the extended and final series of 276 patients (162 were in the previous report) with the last case being enrolled into the study in January 2013. Methods: The APL0406 study was a prospective, open-label, randomized intergroup trial conducted by the Italian GIMEMA and the German SAL and AMLSG study groups. Eligible patients were adults aged 18-<71 years with newly diagnosed…

Investigation of charge ratio variation in mRNA – DEAE-dextran polyplex delivery systems

Biomaterials 192, 612 - 620 (2019). doi:10.1016/j.biomaterials.2018.10.020

Additional file 2 of Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

Additional file 2. Primer table.

Comparative transcutaneous immunization with imiquimod-containing ointments and potential of in vitro methods to predict effects

This work evaluates the transcutaneous in vitro and in vivo immunization efficacy of five commercially available 5% imiquimod containing formulations. The parameters included micro- scopic analysis, rheological properties, drug permeation across synthetic membranes of molecular weight cutoff 10kDa and ablated murine skin with both 0.1 M HCl and a phthalate buffer pH 3.6 Ph.Eur./methanol 3/7 (v/v) as receiver solutions in a Franz-diffusion cell model. For in vivo formu- lation characterization, the cytotoxic T-cell activity and interferon-g production in C57BL/6 mice was determined ex vivo 24h after transcutaneous administration. OVA257264 (SIINFEKL) from chicken al- bumin served as a target…

Molecular Characterization of Relapsed Core-Binding Factor (CBF) Acute Myeloid Leukemia (AML)

Abstract Background: CBF-AML is defined by recurrent genetic abnormalities which encompass t(8;21)(q22;q22), inv(16)(p13.1q22) or less frequently t(16;16)(p13.1;q22). Most frequent secondary chromosome aberrations in t(8;21) AML are del(9q) or loss of a sex chromosome, and in inv(16)/t(16;16) AML trisomy 22 or trisomy 8. At the molecular level mutations involving KIT, FLT3, or NRAS were identified as recurrent lesions in CBF-AML. However, the underlying genetic alterations which might trigger relapse in CBF-AML are not well delineated. Thus, the aim of our study was to characterize the clonal architecture of relapsed CBF-AML. Methods: We performed mutational profiling (KIT, FLT3-ITD, FLT3-T…

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

Clinical epigenetics 14, 29 (2022). doi:10.1186/s13148-022-01245-3

TREM-1 ligand expression on platelets enhances neutrophil activation

Abstract The triggering receptor expressed on myeloid cells 1 (TREM-1) plays an important role in the innate immune response related to severe infections and sepsis. Modulation of TREM-1–associated activation improves the outcome in rodent models for pneumonia and sepsis. However, the identity and occurrence of the natural TREM-1 ligands are so far unknown, impairing the further understanding of the biology of this receptor. Here, we report the presence of a ligand for TREM-1 on human platelets. Using a recombinant TREM-1 fusion protein, we demonstrate specific binding of TREM-1 to platelets. TREM-1–specific signals are required for the platelet-induced augmentation of polymorphonuclear leu…

A New Algorithm and Panel Construction for Pediatric Leukemia Immunophenotyping Using 10-Color Flow Cytometry

Abstract Abstract 4799 Background: Rapid identification and quantification of abnormal cell populations in minimal specimen are crucial for diagnosis and longitudinal minimal residual disease (MRD) testing of childhood leukemia. So far, most standard immunophenotypic analyses are performed using antibody panels with up to five-colors and require high cell numbers. For infant and pediatric specimen, high-level multicolor analyses is highly desirable to gather sufficient data for initial diagnostic and follow up monitoring of pathologic populations. Objective: In this study, we aimed to establish a newly defined pediatric multicolor flow cytometric panel algorithm with high reliability yet mi…

Cyclic adenosine monophosphate and IL-10 coordinately contribute to nTreg cell-mediated suppression of dendritic cell activation

In humans and mice naturally occurring regulatory T cells (nTregs) are crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Here we show that co-culture of murine dendritic cells (DC) and nTregs results in an immediate increase of cAMP in DC, responsible for a rapid down-regulation of co-stimulatory molecules (CD80, CD86). In addition, the inhibitory surface molecule B7-H3 on DC is up-regulated. Subsequently, nTreg-derived IL-10 inhibits the cytokine production (IL-6, IL-12) of suppressed DC therewith preserving their silent phenotype. Hence, our data indicate that nTreg…

Phenotypic characterisation of pro-inflammatory monocytes and dendritic cells in peripheral arterial disease

SummaryAtherosclerosis is a chronic inflammatory process involving antigen-presenting cells like monocytes and dendritic cells (DC). The aim of this study was to perform a phenotypic characterisation of these cell types in patients with different degrees of peripheral arterial disease (PAD). Sixty patients with PAD [N= 30 intermittent claudication (IC), N= 30 critical limb ischemia (CLI)] and 30 controls were included. Peripheral blood leucocytes were analysed from peripheral blood by flow cytometry using different gating strategies to directly identify and analyse monocytes, myeloid DC, (mDC) and plasmacytoid DC (pDC). PAD patients showed a significantly higher proportion of proinflammator…

Effects of Regulatory T Cell–Dendritic Cell Interactions on Adaptive Immune Responses

The limited efficacy of chemo- or radiotherapy against neoplasias necessitates the development of complementary therapeutic strategies. Tumor immunotherapy represents a promising approach as it harnesses the potential of the host immune system to recognize and eradicate transformed cells. So far, T cell-based immunotherapy still suffers from a striking discrepancy between the induction of tumor-specific immune responses in experimental settings and therapeutic immunity in clinically relevant conditions. However, therapeutic approaches targeting immune regulatory mechanisms have lately shown encouraging results and have initiated long-lasting tumor control in patients. Therefore, a deeper un…

Ibrutinib Abrogates TREM-1 Mediated Neutrophil Activation

Abstract Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor on neutrophils (PMN) and important in the innate host defence against microbial pathogens. Here we examined the influence of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib on TREM-1 dependent activation of human PMNs. Firstly, ibrutinib specifically inhibited TREM-1 mediated PMN activation of the oxidative burst and CD62L shedding, whereas TLR mediated activation remained unaffected. Correspondingly, ibrutinib suppressed ERK phosphorylation after TREM-1, but not after TLR ligation. To clarify whether this TREM-1 specific effect of ibrutinib was also relevant in vivo, we treated mice with ibrut…

Oxidative burst and neutrophil elastase contribute to clearance of Aspergillus fumigatus pneumonia in mice.

Polymorphonuclear neutrophils (PMN) are important for the control of invasive aspergillosis (IA), a major threat to immunocompromised individuals. For clearance of Aspergillus fumigatus infections, PMN employ their potent oxidative and non-oxidative mechanisms. To clarify the relative contribution of these mechanisms, we analyzed p47(phox-/-), gp91(phox-/-) and elastase (ELA) deficient mice (ELANE) after intratracheal infection with A. fumigatus. Infected p47(phox-/-) and gp91(phox-/-) mice died within 4 days and had a significant higher fungal burden in the lungs compared to wild-type controls. Interestingly, the survival of ELANE mice after infection was unimpaired suggesting that ELA is …

Quizartinib in FLT3-ITD-Mutated Relapsed/Refractory Acute Myeloid Leukemia: QuANTUM-R Trial Results

Abstract Background FLT3-ITD mutations occur in about 25% of patients (pts) with acute myeloid leukemia (AML) and are associated with poor outcomes. Pts with relapsed/refractory (R/R) FLT3-ITD AML have worse prognosis and high unmet medical need. Quizartinib (Q) is a potent and selective FLT3i with promising activity and a manageable safety profile. QuANTUM-R was a global, phase 3, randomized trial of Q vs chemotherapy (SC) in pts with R/R FLT3-ITD AML (NCT02039726). Methods Pts with R/R FLT3-ITD AML w/wo hematopoietic stem cell transplant (HSCT) were randomized to receive Q or a preselected investigator choice SC: low-dose cytarabine; mitoxantrone, etoposide, and intermediate-dose cytarabi…

Granulocyte functions are independent of arginine availability.

Abstract Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti-tumor metabolic strategy to deprive arginine-auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation-induced IL-8 synthesis, chemotaxis, phagocytosis, generation of RO…

Critical limb ischaemia is characterised by an increased production of whole blood reactive oxygen species and expression of TREM-1 on neutrophils

Atherosclerosis is a chronic inflammatory process involving polymorphonuclear neutrophils (PMN) and formation of reactive oxygen species (ROS). The aim of the present study was to investigate the phenotype of inflammatory cells in regard to the expression of triggering receptor expressed on myeloid cells (TREM)-1 and its soluble form (sTREM-1) as well as its relationship with oxidative stress in peripheral artery disease (PAD) patients.In total 90 patients with PAD (N = 30 intermittent claudication (IC)300 m absolute walking distance, N = 30 IC300 m absolute walking distance, N = 30 critical limb ischaemia (CLI)) and 30 control persons were included. ROS formation was measured at basal or s…

Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses

AbstractThe tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell responses. These inhib…

Mast Cell–deficient KitW-sh “Sash” Mutant Mice Display Aberrant Myelopoiesis Leading to the Accumulation of Splenocytes That Act as Myeloid-Derived Suppressor Cells

Abstract Mast cell-deficient KitW-sh “sash” mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that KitW-sh causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are …

Solid nanoemulsion as antigen and immunopotentiator carrier for transcutaneous immunization

Imiquimod, a toll-like receptor 7 (TLR7) agonist, is an active pharmaceutical ingredient (API) established for the topical treatment of several dermal cancerous and precancerous skin lesions. Within this work, the immunostimulatory effect of imiquimod is further exploited in a transcutaneous immunization (TCI) approach based on a solid nanoemulsion (SN) formulation. SN contains a combination of imiquimod with the model peptide antigen SIINFEKL as a novel approach to omit needle and syringe and optimize dermal antigen administration. Excipients including sucrose fatty acid esters and the pharmaceutically acceptable oils MCT (middle chain triglycerides), avocado oil, jojoba wax and squalene a…

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice.

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in th…

Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses.

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (0.5 …

Synergistic activation of dendritic cells by combined Toll-like receptor ligation induces superior CTL responses in vivo.

Toll-like receptors (TLRs) are able to interact with pathogen-derived products and their signals induce the coordinated activation of innate and adaptive immune mechanisms. Dendritic cells (DCs) play a central role in these events. As the different TLRs are able to trigger MyD88/TRIF-dependent and -independent signaling pathways, we wondered if the simultaneous activation of these signaling cascades would synergize with respect to DC activation and induce superior cytotoxic T-lymphocyte (CTL) activity in vivo. We observed that indeed the combined activation of MyD88-dependent and -independent signaling induced by TLR7 and TLR3 ligands provoked a more rapid and more sustained bone marrow–der…

The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Abstract Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation. Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absen…

9-Phenanthrol enhances the generation of an CD8 + T cell response following transcutaneous immunization with imiquimod in mice

Abstract Background Transcutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses. Objective Based on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system. Methods We investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells i…

Author response: ERK3/MAPK6 controls IL-8 production and chemotaxis

Additional file 1 of Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

Additional file 1. NNLS deconvolution table.