Helga Bárdos

Identification of factor XIII-A as a marker of alternative macrophage activation

Abstract.Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.

Factor XIII subunit A as an intracellular transglutaminase

Abstract: Over the last 2 decades there has been increasing evidence that the role of factor XIII (FXIII) is not restricted to the area of hemostasis and that its subunit A functions as an intracellular enzyme in platelets and monocytes/macrophages. FXIII is already expressed during compartmentalisation of the precursors of megakaryocyte/platelet and monocyte/macrophage cell lines in the bone marrow. FXIII-A, produced by megakaryocytes, is packaged into budding platelets and is present in huge quantity in circulating ones. It seems very likely that it plays an important role in the cytoskeletal remodelling associated with the activation stages of platelets. FXIII-A can also be detected in blood monocytes and in all subsets of monocyte-derived macrophages throughout the body. FXIII-A is mainly localised in the cytoplasm, in association with cytoskeletal filaments, but at a relatively early stage of macrophage differentiation it also appears transiently in the nucleus. Cytoplasmic expression has a very close relationship with phagocytic activities. Further research is needed to understand the biological significance of its nuclear presentation.

Fibrin deposition in primary and metastatic human brain tumours

Extravascular, intratumoral fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumour growth. This is the first report on the presence and distribution of fibrinogen/fibrin in primary (14 glioblastomas) and metastatic (nine samples of lung cancer origin) human brain tumours detected by immunofluorescent techniques. All tissue samples showed specific staining for fibrinogen/fibrin. In glioblastomas fibrin deposits could be detected within and around tumour foci, while in metastatic brain tumours the tumour cell nodules were surrounded by fibrin deposits localized almost exclusively in the connective tissue compartment of tumours. Double-labelling reactions for von Willebrand factor and fibrinogen/fibrin has revealed that fibrin deposition occurred throughout the tumour stroma independently of tumour vasculature. The overlapping reactions for fibrinogen/fibrin and factor XIII subunit A, as well as the urea-insolubility of the deposits indicate the crosslinked, highly stabilized nature of fibrin both within and around tumours. Staining with Ki M7 monoclonal antibody specific for phagocytosing macrophages showed these cells to be scattered in the nonnecrotic areas in glioblastomas and to be accumulated at the interface of tumorous parenchyma and connective tissue in both primary and metastatic tumours. The close association between fibrin deposition and macrophage accumulation strongly suggests the active participation of tumour associated macrophages in the formation of stabilized intratumoral fibrin network in human brain neoplasms.

Obesity-related behaviours and BMI in five urban regions across Europe: sampling design and results from the SPOTLIGHT cross-sectional survey

Objectives To describe the design, methods and first results of a survey on obesity-related behaviours and body mass index (BMI) in adults living in neighbourhoods from five urban regions across Europe. Design A cross-sectional observational study in the framework of an European Union-funded project on obesogenic environments (SPOTLIGHT). Setting 60 urban neighbourhoods (12 per country) were randomly selected in large urban zones in Belgium, France, Hungary, the Netherlands and the UK, based on high or low values for median household income (socioeconomic status, SES) and residential area density. Participants A total of 6037 adults (mean age 52 years, 56% female) participated in the online survey. Outcome measures Self-reported physical activity, sedentary behaviours, dietary habits and BMI. Other measures included general health; barriers and motivations for a healthy lifestyle, perceived social and physical environmental characteristics; the availability of transport modes and their use to specific destinations; self-defined neighbourhood boundaries and items related to residential selection. Results Across five countries, residents from low-SES neighbourhoods ate less fruit and vegetables, drank more sugary drinks and had a consistently higher BMI. SES differences in sedentary behaviours were observed in France, with residents from higher SES neighbourhoods reporting to sit more. Residents from low-density neighbourhoods were less physically active than those from high-density neighbourhoods; during leisure time and (most pronounced) for transport (except for Belgium). BMI differences by residential density were inconsistent across all countries. Conclusions The SPOTLIGHT survey provides an original approach for investigating relations between environmental characteristics, obesity-related behaviours and obesity in Europe. First descriptive results indicate considerable differences in health behaviours and BMI between countries and neighbourhood types.

The SPOTLIGHT virtual audit tool: a valid and reliable tool to assess obesogenic characteristics of the built environment

BackgroundA lack of physical activity and overconsumption of energy dense food is associated with overweight and obesity. The neighbourhood environment may stimulate or hinder the development and/or maintenance of a healthy lifestyle. To improve research on the obesogenicity of neighbourhood environments, reliable, valid and convenient assessment methods of potential obesogenic characteristics of neighbourhood environments are needed. This study examines the reliability and validity of the SPOTLIGHT-Virtual Audit Tool (S-VAT), which uses remote sensing techniques (Street View feature in Google Earth) for desk-based assessment of environmental obesogenicity.MethodsA total of 128 street segments in four Dutch urban neighbourhoods – heterogeneous in socio-economic status and residential density – were assessed using the S-VAT. Environmental characteristics were categorised as walking related items, cycling related items, public transport, aesthetics, land use-mix, grocery stores, food outlets and physical activity facilities. To assess concordance of inter- and intra-observer reliability of the Street View feature in Google Earth, and validity scores with real life audits, percentage agreement and Cohens Kappa (k) were calculated.ResultsIntra-observer reliability was high and ranged from 91.7% agreement (k = 0.654) to 100% agreement (k = 1.000) with an overall agreement of 96.4% (k = 0.848). Inter-observer reliability results ranged from substantial agreement 78.6% (k = 0.440) to high agreement, 99.2% (k = 0.579), with an overall agreement of 91.5% (k = 0.595). Criterion validity was substantial to high for most of the categories ranging from 87.3% agreement (k = 0.539) to 99.9% agreement (k = 0.887) with an overall score of 95.6% agreement (k = 0.747).ConclusionThese study results suggest that the S-VAT is a highly reliable and valid remote sensing tool to assess potential obesogenic environmental characteristics.

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Factor XIIIa as a nerve-associated transglutaminase

Recent findings have led to changes in the traditional concept of nerve recovery, including the realization that injured nerves, like any other injured tissue, need the assistance of blood‐derived cells and factors in order to heal. We show that factor XIIIa (FXIIIa, the potentially active a2‐subunit of factor XIII), an enzyme that participates in blood coagulation by stabilizing the fibrin clot, is also active in the nervous system where it may play a key role in the healing of injured tissue. We demonstrate that the plasma, macrophages and nerves of fish contain a 55 kDa form of transglutaminase that cross‐reacts immunologically with the a‐subunit of FXIII in mammals (80 kDa). The fish enzyme in the plasma, unlike its mammalian counterpart, is active, pointing to a difference in control of the coagulation pathway in the two species. Analysis of FXIIIa expression in mammalian neural tissues and their response to injury revealed high levels of the enzyme in media conditioned by peripheral nerves as compared with medium conditioned by nerves of the central nervous system. Furthermore, similarity was observed in the postinjury behavior of FXIIIa in regenerating nerve tissues (peripheral nervous system of mammals and the central nervous system of fish). We suggest that the postinjury level of factor XIIIa in the nervous system may be related to the tissues regenerative capacity, and that FXIIIa may therefore be a link underlying a possible association between the processes of blood coagulation and nerve healing.—Monsonego, A., Mizrahi, T., Eitan, S., Moalem, G., Bárdos, H., Ádány, Róza, Schwartz, M. Factor XIIIa as a nerve‐associated transglutaminase. FASEB J. 12, 1163–1171 (1998)

Factor XIII-A is involved in the regulation of gene expression in alternatively activated human macrophages

Factor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.

Characteristic distribution patterns of tenascin in laryngeal and hypopharyngeal cancers

Objectives: Progression of malignant neoplasias is accompanied by alteration of the extracellular matrix (ECM) composition. Tenascin is known as a member of the adhesion‐modulating family of ECM macromolecules; thus its expression and distribution may have significant influence on tumor cell proliferation and invasiveness.

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H- thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that pro- tein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal or-ganisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.