Publications by authors named "Michael B Fischer"

71 Publications

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

Acta Derm Venereol 2021 Jan 5;101(1):adv00367. Epub 2021 Jan 5.

Department of Dermatology, Medical University of Vienna, Vienna, Austria.

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.
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http://dx.doi.org/10.2340/00015555-3733DOI Listing
January 2021

Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures.

Front Bioeng Biotechnol 2020 16;8:602210. Epub 2020 Nov 16.

Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria.

Background: The generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized culture set-ups mimicking the perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential.

Aim: We established a fibrin-based xeno-free human 3D vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation.

Materials And Methods: Bone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels.

Results: 3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31/CD34 vascular structures, surrounded by basement membrane collagen type-IV cells and matrix, in association with increased VEGF secretion. PBMC contained CD31CD34CD45CD14 progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45 cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a pro-angiogenic paracrine environment. MSC co-cultured with enriched T cells formed "cell-in-cell" structures generated through internalization of T cells by CD31CD45 cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells.

Conclusion: Our xeno-free 3D vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine.
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http://dx.doi.org/10.3389/fbioe.2020.602210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718010PMC
November 2020

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

Am J Transplant 2021 02 3;21(2):525-539. Epub 2020 Nov 3.

Department of Dermatology, Medical University of Vienna, Vienna, Austria.

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.
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http://dx.doi.org/10.1111/ajt.16358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894140PMC
February 2021

Neutrophil Extracellular Trap Degradation by Differently Polarized Macrophage Subsets.

Arterioscler Thromb Vasc Biol 2020 09 16;40(9):2265-2278. Epub 2020 Jul 16.

From the Division of Cardiology, Department of Medicine II (P. Haider, J.B.K.-P., J.M., M.R., C.K., W.S.S., C.H., J.W., P. Hohensinner), Medical University of Vienna, Austria.

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.
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http://dx.doi.org/10.1161/ATVBAHA.120.314883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447175PMC
September 2020

Extracellular Vesicles Derived From Platelets, Red Blood Cells, and Monocyte-Like Cells Differ Regarding Their Ability to Induce Factor XII-Dependent Thrombin Generation.

Front Cell Dev Biol 2020 5;8:298. Epub 2020 May 5.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Biomedical Research, Danube University Krems, Krems, Austria.

As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.
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http://dx.doi.org/10.3389/fcell.2020.00298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232549PMC
May 2020

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

Haematologica 2021 Feb 1;106(2):454-463. Epub 2021 Feb 1.

Medical University of Vienna.

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.
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http://dx.doi.org/10.3324/haematol.2019.220210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849567PMC
February 2021

Influence of Platelet Lysate on 2D and 3D Amniotic Mesenchymal Stem Cell Cultures.

Front Bioeng Biotechnol 2019 15;7:338. Epub 2019 Nov 15.

Department for Biomedical Research, Center of Experimental Medicine, Danube University Krems, Krems an der Donau, Austria.

The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-γ and TNF-α with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-γ and TNF-α. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.
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http://dx.doi.org/10.3389/fbioe.2019.00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873824PMC
November 2019

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

Biochem Biophys Res Commun 2019 10 3;517(4):709-714. Epub 2019 Aug 3.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Biomedical Research, Danube University Krems, Krems, Austria. Electronic address:

Human monocytes include CD14CD16 (classical), CD14CD16 (intermediate), and CD14CD16 (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 CD14CD16 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.
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http://dx.doi.org/10.1016/j.bbrc.2019.07.120DOI Listing
October 2019

A 3-Dimensional Model of Zonally Organized Extracellular Matrix.

Cartilage 2019 Aug 2:1947603519865320. Epub 2019 Aug 2.

1 Karl Chiari Lab for Orthopaedic Biology, Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Vienna, Austria.

Objective: Functional cartilage repair requires the new formation of organized hyaline cartilaginous matrix to avoid the generation of fibrous repair tissue. The potential of mesenchymal progenitors was used to assemble a 3-dimensional structure in vitro, reflecting the zonation of collagen matrix in hyaline articular cartilage.

Design: The 3-dimensional architecture of collagen alignment in pellet cultures of chondroprogenitors (CPs) was assessed with Picrosirius red staining analyzed under polarized light. In parallel assays, the trilineage capability was confirmed by calcium deposition during osteogenesis by alizarin S staining and alkaline phosphatase staining. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), mRNA levels of ALP, RUNX2, and BGLAP were assessed after 21 days of osteoinduction. Lipid droplets were stained with oil red O and adipogenic differentiation was confirmed by RT-qPCR analysis of PPARG and LPL gene expression.

Results: Under conditions promoting the chondrogenic signature in self-assembling constructs, CPs formed an aligned extracellular matrix, positive for glycosaminoglycans and collagen type II, showing developing zonation of birefringent collagen fibers along the cross section of pellets, which reflect the distribution of collagen fibers in hyaline cartilage. Induced osteogenic and adipogenic differentiation confirmed the trilineage potential of CPs.

Conclusion: This model promotes the differentiation and self-organization of postnatal chondroprogenitors, resulting in the formation of zonally organized engineered hyaline cartilage comparable to the 3 zones of native cartilage.
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http://dx.doi.org/10.1177/1947603519865320DOI Listing
August 2019

Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo.

Thromb Haemost 2019 Sep 10;119(9):1394-1402. Epub 2019 Jul 10.

Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria.

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16 monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16 monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16 monocytes and nonclassical CD16 monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16 monocytes and nonclassical CD16 monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16 monocytes and increased slightly albeit not significantly in CD16 monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.
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http://dx.doi.org/10.1055/s-0039-1692219DOI Listing
September 2019

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

Blood 2019 08 20;134(6):561-567. Epub 2019 Jun 20.

Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria.

Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-R 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-R by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14CD16 human monocytes and Ly6C mouse monocytes expressed the highest levels of Plg-R 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-R 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-R monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6C monocyte recruitment was observed in Plg-R compared with Plg-R mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-R in vivo. Our data demonstrate higher expression of Plg-R on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity.
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http://dx.doi.org/10.1182/blood.2018850420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688429PMC
August 2019

Hypomorphic Mutations in the BCR Signalosome Lead to Selective Immunoglobulin M Deficiency and Impaired B-cell Homeostasis.

Front Immunol 2018 18;9:2984. Epub 2018 Dec 18.

Immunology Outpatient Clinic, Vienna, Austria.

B cell activation via the B cell receptor (BCR) signalosome involves participation of signaling molecules such as BTK and BLNK. Genetic defects in these molecules are known to impair B cell differentiation and subsequently lead to agammaglobulinemia. Here we identified novel mutations in BTK and BLNK in two unrelated patients that perturb the intrinsic B-cell receptor signaling pathway and lead to selective IgM deficiency, whereas production of other immunoglobulin isotypes and IgG antibody response remain intact. Currently it is unknown how BCR signaling strength affects mature B cell development in humans. Both patients show reduced levels of BCR signalosome phosphorylation as well as impaired BCR-dependent Ca influx, which was accompanied by a marked decrease in IgDIgMCD27 MZ-like B-cells. We further describe reduced expression of essential B cell differentiation factors such as BAFF-R and T-Bet in the patients' B-cells, which might contribute to the observed deficiency of MZ-like B cells. MZ-like B cells are known to produce natural IgM antibodies that play an essential role in immune homeostasis. By using surface plasmon resonance (SPR) technology and a synthetic blood group A trisaccharide as antigen we were able to show that both patients lack the presence of anti-blood group A IgM considered to be prototypical natural antibodies whereas IgG levels were normal. Antibody binding dynamics and binding affinity of anti-blood group A IgG were comparable between patients and healthy controls. These results indicate that human IgM deficiency can be associated with signaling defects in the BCR signalosome, defective production of natural IgM antibodies in the blood group A/B/0 system and abnormalities in B cell development.
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http://dx.doi.org/10.3389/fimmu.2018.02984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305442PMC
November 2019

Differential Interaction of Platelet-Derived Extracellular Vesicles With Circulating Immune Cells: Roles of TAM Receptors, CD11b, and Phosphatidylserine.

Front Immunol 2018 11;9:2797. Epub 2018 Dec 11.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Biomedical Research, Danube University Krems, Krems, Austria.

Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding.
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http://dx.doi.org/10.3389/fimmu.2018.02797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297748PMC
October 2019

Pattern of myogenesis and vascular repair in early and advanced lesions of juvenile dermatomyositis.

Neuromuscul Disord 2018 12 19;28(12):973-985. Epub 2018 Sep 19.

Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

Regenerative processes that counteract perifascicular muscle atrophy and capillary loss in juvenile dermatomyositis (JDM) are not well characterized. We aimed to analyze the pattern of myo-regeneration in relation to vascular damage and repair in muscle specimens from JDM patients. Myogenic regulatory factors that are sequentially expressed during myogenesis were studied by immunohistochemistry. Capillary density, numbers of CD34 endothelial progenitor cells within the endomysium and molecules implicated in angiogenesis were evaluated by double-immunofluorescence techniques. Myogenic regulatory factors were significantly up-regulated in JDM muscle exhibiting a different pattern in early and advanced lesions. In early lesions Pax7 satellite cells and both MyoD and Myogenin myogenic cells were moderately increased. In lesions with advanced perifascicular atrophy Pax7 satellite cells were numerous, but absence of MyoD in the context of increased Myogenin expression suggested a dysregulation of the myogenic regenerative pathway. The overall capillary density in JDM was decreased, but regions of capillary loss in advanced lesions alternated with focal increase of hyperplastic endothelial cells in early lesions. Up-regulation of endoglin in hyperplastic endothelial cells in conjunction with overexpression of TGF-β1 and VEGF suggested activation of neovascularization. Conversely, CD34 endothelial progenitor cells were not increased arguing against relevant contribution to vascular repair. Our results demonstrate substantial induction of myogenesis in JDM. While the early phase of myogenesis appears to be associated with endothelial cell activation, an altered expression of MRFs in perifascicular regions with capillary depletion suggests an impairment of myogenic differentiation that may contribute to perifascicular muscle fiber atrophy in JDM.
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http://dx.doi.org/10.1016/j.nmd.2018.09.002DOI Listing
December 2018

Audencel Immunotherapy Based on Dendritic Cells Has No Effect on Overall and Progression-Free Survival in Newly Diagnosed Glioblastoma: A Phase II Randomized Trial.

Cancers (Basel) 2018 Oct 5;10(10). Epub 2018 Oct 5.

Clinical Division of Medical Oncology, Department for Internal Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

Dendritic cells (DCs) are antigen-presenting cells that are capable of priming anti-tumor immune responses, thus serving as attractive tools to generate tumor vaccines. In this multicentric randomized open-label phase II study, we investigated the efficacy of vaccination with tumor lysate-charged autologous DCs (Audencel) in newly diagnosed glioblastoma multiforme (GBM). Patients aged 18 to 70 years with histologically proven primary GBM and resection of at least 70% were randomized 1:1 to standard of care (SOC) or SOC plus vaccination (weekly intranodal application in weeks seven to 10, followed by monthly intervals). The primary endpoint was progression-free survival at 12 months. Secondary endpoints were overall survival, safety, and toxicity. Seventy-six adult patients were analyzed in this study. Vaccinations were given for seven (3⁻20) months on average. No severe toxicity was attributable to vaccination. Seven patients showed flu-like symptoms, and six patients developed local skin reactions. Progression-free survival at 12 months did not differ significantly between the control and vaccine groups (28.4% versus 24.5%, = 0.9975). Median overall survival was similar with 18.3 months (vaccine: 564 days, 95% CI: 436⁻671 versus control: 568 days, 95% CI: 349⁻680; = 0.89, harzard ratio (HR) 0.99). Hence, in this trial, the clinical outcomes of patients with primary GBM could not be improved by the addition of Audencel to SOC.
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http://dx.doi.org/10.3390/cancers10100372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210090PMC
October 2018

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

Front Physiol 2018 5;9:679. Epub 2018 Jun 5.

Department of Vascular Biology and Thrombosis Research, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Here we investigated the potential of small fragments of synovial tissue to form vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices , and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45 leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures. Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.
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http://dx.doi.org/10.3389/fphys.2018.00679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996074PMC
June 2018

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

Front Immunol 2018 27;9:852. Epub 2018 Apr 27.

Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβCD39CD73 Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.
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http://dx.doi.org/10.3389/fimmu.2018.00852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946032PMC
June 2019

Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood.

Sci Rep 2018 04 26;8(1):6598. Epub 2018 Apr 26.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500, Krems, Austria.

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14CD16) monocytes in whole blood.
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http://dx.doi.org/10.1038/s41598-018-25047-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920058PMC
April 2018

Canine macrophages can like human macrophages be in vitro activated toward the M2a subtype relevant in allergy.

Dev Comp Immunol 2018 05 10;82:118-127. Epub 2018 Jan 10.

Comparative Medicine, The Interuniversity Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Veterinärplatz 1, 1210, Vienna, Austria; Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. Electronic address:

The M2a subtype of macrophages plays an important role in human immunoglobulin E (IgE-mediated allergies) and other Th2 type immune reactions. In contrast, very little is known about these cells in the dog. Here we describe an in vitro method to activate canine histiocytic DH82 cells and primary canine monocyte-derived macrophages (MDMs) toward the M2a macrophages using human cytokines. For a side-by-side comparison, we compared the canine cells to human MDMs, and the human monocytic cell line U937 activated towards M1 and M2a cells on the cellular and molecular level. In analogy to activated human M2a cells, canine M2a, differentiated from both DH82 and MDMs, showed an increase in CD206 surface receptor expression compared to M1. Interestingly, canine M2a, but not M1 derived from MDM, upregulated the high-affinity IgE receptor (FcεRI). Transcription levels of M2a-associated genes (IL10, CCL22, TGFβ, CD163) showed a diverse pattern between the human and dog species, whereas M1 genes (IDO1, CXCL11, IL6, TNF-α) were similarly upregulated in canine and human M1 cells (cell lines and MDMs). We suggest that our novel in vitro method will be suitable in comparative allergology studies focussing on macrophages.
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http://dx.doi.org/10.1016/j.dci.2018.01.005DOI Listing
May 2018

Development of a Multifunctional Nanobiointerface Based on Self-Assembled Fusion-Protein rSbpA/ZZ for Blood Cell Enrichment and Phenotyping.

ACS Appl Mater Interfaces 2017 Oct 25;9(39):34423-34434. Epub 2017 Sep 25.

Vienna University of Technology , Faculty of Technical Chemistry, Institute of Applied Synthetic Chemistry & Institute of Chemical Technologies and Analytics, Getreidemarkt 9, 1060 Vienna, Austria.

We present a multifunctional nanobiointerface for blood cell capture and phenotyping applications that features both excellent antifouling properties and high antibody activity. Multifunctionality is accomplished by modifying polymeric materials using self-assembled S-layer fusion-protein rSbpA/ZZ to immobilize high density antibodies at the two protein A binding sites of the rSbpA/ZZ nanolattice structure. Controlled orientation and alignment of the antibodies reduced antibody consumption 100-fold and increased cell capture efficiency 4-fold over standard methodologies. Cell analysis in complex samples was made possible by the remarkable antifouling properties of the rSbpA domain, while at the same time reducing unspecific binding and forgoing tedious blocking procedures. An automated microfluidic in situ cell analysis platform for isolation and phenotyping of primary peripheral blood mononuclear cells was developed as practical application. Results obtained using our automated microfluidic cell analysis platform showed that the multifunctional nanobiointerface can discriminate among T helper and cytotoxic T cells, and thymocytes. Additionally, on-chip cell capture under flow conditions using a high affinity CD 3 selective nanobiointerface preferentially isolated cells with strong surface marker expression. This means that our dynamic microfluidic cell purification method allows the enrichment of 773 CD 8 positive cytotoxic T cells out of a total blood cell population of 7728 PBMCs, which is an increase in cell enrichment of 8-fold with a purity of 85%.
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http://dx.doi.org/10.1021/acsami.7b09041DOI Listing
October 2017

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

Arterioscler Thromb Vasc Biol 2017 10 17;37(10):1913-1922. Epub 2017 Aug 17.

From the Department of Internal Medicine II, Division of Cardiology (P.J.H., J.B., B.E., B.T., K.D., S.S., S.D., W.S.S., J.W.), Center for Physiology and Pharmacology, Institute of Vascular Biology and Thrombosis Research (J.B.K.-P., P.U., G.S.), Department of Laboratory Medicine (S.D.), Clinic for Blood Group Serology and Transfusion Medicine (M.B.F.), and Core Facilities (J.W.), Medical University of Vienna, Austria; Department for Health Science and Biomedicine, Danube University Krems, Austria (M.B.F.); 3rd Medical Department, Wilhelminenspital, Vienna, Austria (K.H.); and Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna, Austria (K.H., J.W.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.117.309383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627534PMC
October 2017

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

Transfusion 2017 06 10;57(6):1459-1469. Epub 2017 Apr 10.

Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, Regensburg, Germany.

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.
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http://dx.doi.org/10.1111/trf.14109DOI Listing
June 2017

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.

BMC Oral Health 2017 Mar 21;17(1):66. Epub 2017 Mar 21.

Department of Conservative Dentistry and Periodontology, School of Dentistry, Medical University of Vienna, Sensengasse 2a, 1090, Vienna, Austria.

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) β1 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, TGFβ1 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.
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http://dx.doi.org/10.1186/s12903-017-0357-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361806PMC
March 2017

Mechanisms of endothelial activation in sepsis and cell culture models to study the heterogeneous host response.

Int J Artif Organs 2017 Mar 13;40(1):9-14. Epub 2017 Feb 13.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Krems - Austria.

Sepsis is currently viewed as a fundamental disintegration of control functions from intracellular signalling to immunoregulatory and neuroendocrine mechanisms. The immediate threat in sepsis is invasive infection, and the need to activate immune defense mechanisms to clear the pathogen before irreparable damage occurs. In the process of pathogen elimination, however, the systemic host response to infection may cause collateral damage to the endothelium and may lead to the destruction of host tissues.A number of experimental models have been developed to monitor endothelial activation and to study endothelial dysfunction under septic conditions. Here, we review the application of these models to assess the highly variable host response in sepsis and to investigate the efficacy of adsorbent-based extracorporeal therapies. We also highlight the need for efficient diagnostic tools, which are indispensable to select patients who are likely to benefit from distinct adjunctive therapies.
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http://dx.doi.org/10.5301/ijao.5000560DOI Listing
March 2017

Biomimetic principles to develop blood compatible surfaces.

Int J Artif Organs 2017 Mar 13;40(1):22-30. Epub 2017 Feb 13.

 Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Danube University Krems, Krems - Austria.

Functionalized biomaterial surface patterns capable of resisting nonspecific adsorption while retaining their bioactivity are crucial in the advancement of biomedical technologies, but currently available biomaterials intended for use in whole blood frequently suffer from nonspecific adsorption of proteins and cells, leading to a loss of activity over time. In this review, we address two concepts for the design and modification of blood compatible biomaterial surfaces, zwitterionic modification and surface functionalization with glycans - both of which are inspired by the membrane structure of mammalian cells - and discuss their potential for biomedical applications.
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http://dx.doi.org/10.5301/ijao.5000559DOI Listing
March 2017

Pneumococcal Polysaccharide Vaccination Elicits IgG Anti-A/B Blood Group Antibodies in Healthy Individuals and Patients with Type I Diabetes Mellitus.

Front Immunol 2016 14;7:493. Epub 2016 Nov 14.

Clinic for Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria; Department for Health Science and Biomedicine, Danube University Krems, Krems, Austria.

Hypothesis: Blood group antibodies are natural antibodies that develop early in life in response to cross-reactive environmental antigens in the absence of antigen encounter. Even later in life structural similarities in saccharide composition between environmental antigens such as bacterial polysaccharides and blood group A/B antigens could lead to changes in serum levels, IgM/IgG isotype, and affinity maturation of blood group anti-A/B antibodies. We addressed the question whether immunization with pneumococcal polysaccharide (PnP) vaccine Pneumo 23 Vaccine "Pasteur Merieux" (Pn23) could have such an effect in patients with type I diabetes mellitus (DM I), an autoimmune disease where an aberrant immune response to microbial antigens likely plays a role.

Methods: Anti-PnP IgM and IgG responses were determined by ELISA, and the DiaMed-ID Micro Typing System was used to screen anti-A/B antibody titer before and after Pn23 immunization in 28 healthy individuals and 16 patients with DM I. In addition, surface plasmon resonance (SPR) technology using the Biacore device and a synthetic blood group A/B trisaccharide as the antigen was applied to investigate IgM and IgG anti-A/B antibodies and to measure antibody binding dynamics.

Results: All healthy individuals and DM I patients responded with anti-PnP IgM and IgG antibody production 4-6 weeks after Pn23 immunization, while no increase in blood group anti-A/B antibody titer was observed when measured by the DiaMed-ID Micro Typing System. Interestingly, isotype-specific testing by SPR technology revealed an increase in blood group anti-A/B IgG, but not IgM, following Pn23 immunization in both patients and controls. No change in binding characteristics of blood group anti-A/B antibodies could be detected following Pn23 vaccination, supporting the assumption of an increase in IgG antibody titer with no or very little affinity maturation.

Conclusion: The study provides evidence for epitope sharing between pneumococcal polysaccharides and blood group ABO antigens, which leads to a booster of blood group anti-A/B antibodies of the IgG isotype after Pn23 immunization in healthy individuals. Manifest autoimmunity such as present in DM I patients has no additional effect on the cross-reactive antibody response against pneumococcal polysaccharides and blood group antigens.
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http://dx.doi.org/10.3389/fimmu.2016.00493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108245PMC
November 2016

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

J Clin Apher 2017 Dec 17;32(6):375-383. Epub 2016 Nov 17.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria.

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.
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http://dx.doi.org/10.1002/jca.21519DOI Listing
December 2017

Polystyrene-Divinylbenzene-Based Adsorbents Reduce Endothelial Activation and Monocyte Adhesion Under Septic Conditions in a Pore Size-Dependent Manner.

Inflammation 2016 Oct;39(5):1737-46

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500, Krems, Austria.

Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators.
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http://dx.doi.org/10.1007/s10753-016-0408-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023745PMC
October 2016

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

Biochem Biophys Res Commun 2016 09 19;478(1):168-173. Epub 2016 Jul 19.

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2016.07.073DOI Listing
September 2016

A New Method Using Autogenous Impacted Third Molars for Sinus Augmentation to Enhance Implant Treatment: Case Series with Preliminary Results of an Open, Prospective Longitudinal Study.

Int J Oral Maxillofac Implants 2016 May-Jun;31(3):622-30

Purpose: This prospective longitudinal study reports on the results in patients given autologous tooth material for augmentation in a sinus elevation procedure.

Materials And Methods: Six patients with inadequate bone supply for augmentation in the maxillary posterior tooth region and at least one impacted maxillary third molar underwent sinus elevation surgery with lateral access using the particulate tooth material. One of the patients received four implants during the same session, while the other patients had a total of 15 implants placed after a healing phase of an average 5.5 months. Drill cylinders collected from the implant bed during the procedure were subjected to histologic/immunohistochemical evaluation.

Results: All six patients showed normal and unobtrusive postoperative healing, having undergone prosthetic restoration up to 5 years before. The average peri-implant probing pocket depth after a period of up to 5 years ranged between 1.86 mm (mesial and lingual) and 2.07 mm (distal and buccal). No bleeding could be triggered with any of the peri-implant probes. The average peri-implant bone resorption measured during the first year was up to 0.63 mm, with the lowest being 0 mm and the maximum 2.9 mm. Peri-implant bone remained stable for the follow-up time of up to 5 years. Histologically, six biopsy specimens collected from five patients showed osteoconductive osteogenesis with encapsulation of tooth enamel and dentin portions and partial resorption of the tooth components. Cementum shares were no longer discernible. Immunohistochemical assessment showed intense new vessel formation that could be observed in the area of loose stroma of reorganized tissue in the augmented area.

Conclusion: Within the limits of these preliminary results and with adequate consideration of the small number of patients included, the use of autogenous crushed tooth material from impacted third molars may represent an alternative augmentation material for use in sinus elevation procedures.
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http://dx.doi.org/10.11607/jomi.4172DOI Listing
April 2017