Exploring absolute and relative measures of exposure to food environments in relation to dietary patterns among European adults.

OBJECTIVE: To explore the associations of absolute and relative measures of exposure to food retailers with dietary patterns, using simpler and more complex measures. DESIGN: Cross-sectional survey. SETTING: Urban regions in Belgium, France, Hungary, the Netherlands and the UK.ParticipantsEuropean adults (n 4942). Supermarkets and local food shops were classified as 'food retailers providing healthier options'; fast-food/takeaway restaurants, cafés/bars and convenience/liquor stores as 'food retailers providing less healthy options'. Simpler exposure measures used were density of healthy and density of less healthy food retailers. More complex exposure measures used were: spatial access (combination of density and proximity) to healthy and less healthy food retailers; density of healthier food retailers relative to all food retailers; and a ratio of spatial access scores to healthier and less healthy food retailers. Outcome measures were a healthy or less healthy dietary pattern derived from a principal component analysis (based on consumption of fruits, vegetables, fish, fast foods, sweets and sweetened beverages). RESULTS: Only the highest density of less healthy food retailers was significantly associated with the less healthy dietary pattern (ß = -129·6; 95 % CI -224·3, -34·8). None of the other absolute density measures nor any of the relative measures of exposures were associated with dietary patterns. CONCLUSIONS: More complex measures of exposure to food retailers did not produce stronger associations with dietary patterns. We had some indication that absolute and relative measures of exposure assess different aspects of the food environment. However, given the lack of significant findings, this needs to be further explored.

Exploring the relationship between perceived barriers to healthy eating and dietary behaviours in European adults.

PURPOSE: Dietary behaviours may be influenced by perceptions of barriers to healthy eating. Using data from a large cross-European study (N = 5900), we explored associations between various perceived barriers to healthy eating and dietary behaviours among adults from urban regions in five European countries and examined whether associations differed across regions and socio-demographic backgrounds. METHODS: Frequency of consumption of fruit, vegetables, fish, fast food, sugar-sweetened beverages, sweets, breakfast and home-cooked meals were split by the median into higher and lower consumption. We tested associations between barriers (irregular working hours; giving up preferred foods; busy lifestyle; lack of willpower; price of healthy food; taste preferences of family and friends; lack of healthy options and unappealing foods) and dietary variables using multilevel logistic regression models. We explored whether associations differed by age, sex, education, urban region, weight status, household composition or employment. RESULTS: Respondents who perceived any barrier were less likely to report higher consumption of healthier foods and more likely to report higher consumption of fast food. 'Lack of willpower', 'time constraints' and 'taste preferences' were most consistently associated with consumption. For example, those perceiving lack of willpower ate less fruit [odds ratio (OR) 0.57; 95% confidence interval (CI) 0.50-0.64], and those with a busy lifestyle ate less vegetables (OR 0.54; 95% CI 0.47-0.62). Many associations differed in size, but not in direction, by region, sex, age and household composition. CONCLUSION: Perceived 'lack of willpower', 'time constraints' and 'taste preferences' were barriers most strongly related to dietary behaviours, but the association between various barriers and lower intake of fruit and vegetables was somewhat more pronounced among younger participants and women.

Self-defined residential neighbourhoods: size variations and correlates across five European urban regions.

The neighbourhood is recognized as an important unit of analysis in research on the relation between obesogenic environments and development of obesity. One important challenge is to define the limits of the residential neighbourhood, as perceived by study participants themselves, in order to improve our understanding of the interaction between contextual features and patterns of obesity. An innovative tool was developed in the framework of the SPOTLIGHT project to identify the boundaries of neighbourhoods as defined by participants in five European urban regions. The aims of this study were (i) to describe self-defined neighbourhood (size and overlap with predefined residential area) according to the characteristics of the sampling administrative neighbourhoods (residential density and socioeconomic status) within the five study regions and (ii) to determine which individual or/and environmental factors are associated with variations in size of self-defined neighbourhoods. Self-defined neighbourhood size varies according to both individual factors (age, educational level, length of residence and attachment to neighbourhood) and contextual factors. These findings have consequences for how residential neighbourhoods are defined and operationalized and can inform how self-defined neighbourhoods may be used in research on associations between contextual characteristics and health outcomes such as obesity.

Neighbourhood typology based on virtual audit of environmental obesogenic characteristics.

Virtual audit (using tools such as Google Street View) can help assess multiple characteristics of the physical environment. This exposure assessment can then be associated with health outcomes such as obesity. Strengths of virtual audit include collection of large amount of data, from various geographical contexts, following standard protocols. Using data from a virtual audit of obesity-related features carried out in five urban European regions, the current study aimed to (i) describe this international virtual audit dataset and (ii) identify neighbourhood patterns that can synthesize the complexity of such data and compare patterns across regions. Data were obtained from 4,486 street segments across urban regions in Belgium, France, Hungary, the Netherlands and the UK. We used multiple factor analysis and hierarchical clustering on principal components to build a typology of neighbourhoods and to identify similar/dissimilar neighbourhoods, regardless of region. Four neighbourhood clusters emerged, which differed in terms of food environment, recreational facilities and active mobility features, i.e. the three indicators derived from factor analysis. Clusters were unequally distributed across urban regions. Neighbourhoods mostly characterized by a high level of outdoor recreational facilities were predominantly located in Greater London, whereas neighbourhoods characterized by high urban density and large amounts of food outlets were mostly located in Paris. Neighbourhoods in the Randstad conurbation, Ghent and Budapest appeared to be very similar, characterized by relatively lower residential densities, greener areas and a very low percentage of streets offering food and recreational facility items. These results provide multidimensional constructs of obesogenic characteristics that may help target at-risk neighbourhoods more effectively than isolated features.

Mismatch between perceived and objectively measured environmental obesogenic features in European neighbourhoods.

Findings from research on the association between the built environment and obesity remain equivocal but may be partly explained by differences in approaches used to characterize the built environment. Findings obtained using subjective measures may differ substantially from those measured objectively. We investigated the agreement between perceived and objectively measured obesogenic environmental features to assess (1) the extent of agreement between individual perceptions and observable characteristics of the environment and (2) the agreement between aggregated perceptions and observable characteristics, and whether this varied by type of characteristic, region or neighbourhood. Cross-sectional data from the SPOTLIGHT project (n = 6037 participants from 60 neighbourhoods in five European urban regions) were used. Residents' perceptions were self-reported, and objectively measured environmental features were obtained by a virtual audit using Google Street View. Percent agreement and Kappa statistics were calculated. The mismatch was quantified at neighbourhood level by a distance metric derived from a factor map. The extent to which the mismatch metric varied by region and neighbourhood was examined using linear regression models. Overall, agreement was moderate (agreement < 82%, kappa < 0.3) and varied by obesogenic environmental feature, region and neighbourhood. Highest agreement was found for food outlets and outdoor recreational facilities, and lowest agreement was obtained for aesthetics. In general, a better match was observed in high-residential density neighbourhoods characterized by a high density of food outlets and recreational facilities. Future studies should combine perceived and objectively measured built environment qualities to better understand the potential impact of the built environment on health, particularly in low residential density neighbourhoods.

Exploring why residents of socioeconomically deprived neighbourhoods have less favourable perceptions of their neighbourhood environment than residents of wealthy neighbourhoods.

Residents of socioeconomically deprived areas perceive their neighbourhood as less conducive to healthy behaviours than residents of more affluent areas. Whether these unfavourable perceptions are based on objective neighbourhood features or other factors is poorly understood. We examined individual and contextual correlates of socioeconomic inequalities in neighbourhood perceptions across five urban regions in Europe. Data were analysed from 5205 participants of the SPOTLIGHT survey. Participants reported perceptions of their neighbourhood environment with regard to aesthetics, safety, the presence of destinations and functionality of the neighbourhood, which were summed into an overall neighbourhood perceptions score. Multivariable multilevel regression analyses were conducted to investigate whether the following factors were associated with socioeconomic inequalities in neighbourhood perceptions: objectively observed neighbourhood features, neighbourhood social capital, exposure to the neighbourhood, self-rated health and lifestyle behaviours. Objectively observed traffic safety, aesthetics and the presence of destinations in the neighbourhood explained around 15% of differences in neighbourhood perceptions between residents of high and low neighbourhoods; levels of neighbourhood social cohesion explained around 52%. Exposure to the neighbourhood, self-rated health and lifestyle behaviours were significant correlates of neighbourhood perceptions but did not contribute to socioeconomic differences. This cross-European study provided evidence that socioeconomic differences in neighbourhood perceptions are not only associated with objective neighbourhood features but also with social cohesion. Levels of physical activity, sleep duration, self-rated health, happiness and neighbourhood preference were also associated with neighbourhood perceptions.

Perceived environmental correlates of cycling for transport among adults in five regions of Europe.

Regular cycling for transport is an important potential contributor to daily physical activity among adults. Characteristics of the physical environment are likely to influence cycling for transport. The current study investigated associations between perceived physical environmental neighbourhood factors and adults' cycling for transport across five urban regions across Europe, and whether such associations were moderated by age, gender, education and urban region. A total of 4,612 adults from five European regions provided information about their transport-related cycling and their neighbourhood physical environmental perceptions in an online survey. Hurdle models adjusted for the clustering within neighbourhoods were performed to estimate associations between perceived physical environmental neighbourhood factors and odds of engaging in cycling for transport and minutes of cycling for transport per week. Inhabitants of neighbourhoods that were perceived to be polluted, having better street connectivity, having lower traffic speed levels and being less pleasant to walk or cycle in had higher levels of cycling for transport. Moderation analyses revealed only one interaction effect by gender. This study indicates that cycling for transport is associated with a number of perceived physical environmental neighbourhood factors across five urban regions across Europe. Our results indicated that the majority of the outcomes identified were valid for all subgroups of age, gender, education and across regions in the countries included in the study.

The relation between sleep duration and sedentary behaviours in European adults.

Too much sitting, and both short and long sleep duration are associated with obesity, but little is known on the nature of the relations between these behaviours. We therefore examined the associations between sleep duration and time spent sitting in adults across five urban regions in Europe. We used cross-sectional survey data from 6,037 adults (mean age 51.9 years (SD 16.4), 44.0% men) to assess the association between self-reported short (<6 h per night), normal (6-8 h per night) and long (>8 h per night) sleep duration with self-report total time spent sitting, time spent sitting at work, during transport, during leisure and while watching screens. The multivariable multilevel linear regression models were tested for moderation by urban region, age, gender, education and weight status. Because short sleepers have more awake time to be sedentary, we also used the percentage of awake time spent sedentary as an outcome. Short sleepers had 26.5 min day(-1) more sedentary screen time, compared with normal sleepers (CI 5.2; 47.8). No statistically significant associations were found with total or other domains of sedentary behaviour, and there was no evidence for effect modification. Long sleepers spent 3.2% higher proportion of their awake time sedentary compared with normal sleepers. Shorter sleep was associated with increased screen time in a sample of European adults, irrespective of urban region, gender, age, educational level and weight status. Experimental studies are needed to assess the prospective relation between sedentary (screen) time and sleep duration.

Interactions of individual perceived barriers and neighbourhood destinations with obesity-related behaviours in Europe.

Perceived barriers towards physical activity and healthy eating as well as local availability of opportunities (destinations in the neighbourhood) are important determinants of obesity-related behaviours in adults. Little is known, however, about how these factors interact with the behaviours. Data were analysed from 5,205 participants of the SPOTLIGHT survey, conducted in 60 neighbourhoods in urban regions of five different countries across Europe. A virtual audit was conducted to collect data on the presence of destinations in each neighbourhood. Direct associations of, and interactions between, the number of individual perceived barriers and presence of destinations with obesity-related behaviours (physical activity and dietary behaviours) were analysed using multilevel regression analyses, adjusted for key covariates. Perceiving more individual barriers towards physical activity and healthy eating was associated with lower odds of physical activity and healthy eating. The presence of destinations such as bicycle lanes, parks and supermarkets was associated with higher levels of physical activity and healthier dietary behaviours. Analyses of additive interaction terms suggested that the interaction of destinations and barriers was competitive, such that the presence of destinations influenced obesity-related behaviours most among those perceiving more barriers. These explorative findings emphasize the interest and importance of combining objective (e.g. virtual neighbourhood audit) methods and subjective (e.g. individual perceived barriers collected in a survey) to better understand how the characteristics of the residential built environment can shape obesity-related behaviours depending on individual characteristics.

Psychosocial environment: definitions, measures and associations with weight status--a systematic review.

Socio-ecological models suggest that many elements of the social environment act as upstream determinants of obesity. This systematic review examined definitions, measures and strength of associations between the psychosocial environment and adult weight status. Studies were included if they were conducted on adults, the outcome was weight status, carried out in any developed country and investigated at least one psychosocial environmental construct. Six databases for primary studies were searched: EMBASE, MEDLINE, PsycINFO, Scopus, Web of Science and the Cochrane Library. We restricted our search to studies published in English between January 1995 and February 2015. An adapted 'Quality Assessment Tool for Quantitative Studies' was used to evaluate risk of bias of included studies. Out of 14,784 screened records, 42 articles were assessed using full text. A total of 19 studies were included. The strongest associations with weight status were found for social capital and collective efficacy, although few studies found significant associations. There was heterogeneity in the definitions and metrics of psychosocial environmental constructs. There is limited evidence that greater social capital and collective efficacy are associated with healthier weight status. The research conducted to date has not robustly identified relations. We highlight challenges to undertaking research and establishing causality in this field and provide recommendations for further research.

Detection of factor XIII-A is a valuable tool for distinguishing dendritic cells and tissue macrophages in granuloma annulare and necrobiosis lipoidica.

BACKGROUND: Factor XIII subunit A (FXIII-A) is used as a diagnostic marker in a wide range of dermatological diseases ranging from inflammatory lesions to malignancies, although neither the cell types responsible for its expression nor the mechanism(s) resulting in its local accumulation in pathological conditions have been characterized. OBJECTIVE: In this study, we aimed to gain information on the cells showing an immunohistochemical reaction for FXIII-A and answer the question whether macrophages and/or dendritic cells are labelled for FXIII-A. METHODS: We carried out our studies on samples of granuloma annulare (GA) and necrobiosis lipoidica (NL), the prime examples for granulomatous skin lesions with a non-infectious background in which extracellular matrix remodelling is a key feature without any sign of malignant transformation. We used markers for macrophages and dendritic cells in combination with the detection of FXIII-A in double labelling immunohistochemical reactions. RESULTS: We demonstrated that FXIII-A positivity clearly distinguishes macrophages (CD163+/FXIII-A+) from dendritic cells (CD11c+/FXIII-A-) not only in the normal dermis as previously described by Zaba et al. (J Clin Invest 2007; 117: 2517-2525) but also in the pathological conditions of GA and NL. Detecting the expression of DC-SIGN/CD209 and mannose receptor molecules on FXIII-A+ macrophages we confirmed that FXIII-A is expressed in the alternatively activated macrophages. However, while DC-SIGN/CD209 was invariably expressed on FXIII-A+ cells both in normal and pathological conditions of GA/NL (98.7% vs. 93.5/96%), mannose receptor was only partially coexpressed with FXIII-A (94.8% vs. 74.7/52.2%), suggesting that FXIII-A+ macrophages do not represent a homogenous population. CONCLUSIONS: FXIII-A selectively marks macrophages and distinguishes them from dendritic cells. The presence of FXIII-A is not a disease-specific marker but indicates a possible common mechanism of macrophage activation in various dermatological diseases.

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

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-gamma, 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.

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.

Phorbol ester treatment inhibits proliferation and differentiation of cultured human skeletal muscle satellite cells by differentially acting on protein kinase C isoforms.

We have previously shown that cultured human skeletal muscle cells express five protein kinase C (PKC) isoforms (PKCalpha, -gamma, -eta, -theta, and -zeta) and that expression levels of various PKC isozymes differentially change during differentiation. In this study we investigated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on differentiation and on PKC isozymes of human skeletal muscle satellite cells. PMA inhibited the growth and fusion of cultured human myoblasts in a dose-dependent manner. In addition, prolonged treatment of cells with PMA suppressed the expression of the myogenic differentiation marker desmin showing similar dose-response characteristics. Furthermore, PMA also induced the intracellular translocation of PKCgamma, -eta, and -theta, whereas cellular localization of PKCalpha and -zeta were not altered. These changes in subcellular localization patterns were of great importance since only those PKC isoforms were translocated that possessed alterations in their expression levels during differentiation. Our findings, therefore, suggest that the PMA-induced inhibition of differentiation of human skeletal muscle cells is mediated by certain PKC isoforms. Moreover, these data strongly argue for differential and isozyme-specific roles of various PKC isoforms in these processes.

Factor XIII of blood coagulation as a nuclear crosslinking enzyme.

Intracellular localization and distribution of Factor XIII subunit A (FXIIIA) was investigated in association with monocyte-macrophage differentiation in a long term culture of human monocytes by light- and electron microscopical as well as biochemical and immunobiochemical techniques. To allow the detection of FXIIIA in cells with well-preserved ultrustructure, immunosera against glutaraldehyde-derivatized recombinant FXIIIA were developed in rabbits, then characterized and used in this study. In the early phase of macrophage differentiation intranuclear accumulation of FXIIIA was detected as a transient phenomenon in cells of the 2nd day culture by optical sectioning with 0,7 microm steps in laser scanning confocal microscopy and immunoblotting technique. FXIIIA could be detected by immunoelectron microscopic postembedding staining over electrodense DNA-containing areas. Fluoresceinated monodansylcadaverine incorporation assay was used to demonstrate that FXIIIA is not only present in the nuclei, but also expresses its transglutaminase activity. Our finding of the nuclear accumulation of FXIIIA in differentiating human macrophages is also unique in that a blood clotting factor has, for the first time, been localized in nuclei and has been shown to be an intracellular crosslinking enzyme. The possible role of nuclear FXIIIA in association with cellular processes involving chromatin structure remodeling, such as cell death, cell differentiation or cellular proliferation requires further in-depth investigation.

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 protein 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 organisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

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. STUDY DESIGN: The present study was carried out to determine the distribution pattern of tenascin in laryngeal and hypopharyngeal cancer samples. METHODS: In double and triple immunofluorescent staining reactions the detection of tenascin was combined with labelings for cytokeratin (marker protein of epithelial cells), for CD-34 (endothelial cell surface glycoprotein), and for a reaction with Ki-67 monoclonal antibody (nuclear antigen in proliferating cells). RESULTS: In laryngeal cancers, in early stages of tumor growth a markedly enhanced production of tenascin at the tumor host interphase was observed. In the later stages of tumor progression, a high number of blood vessels located in the tumorous tissues were also strongly labeled for tenascin. Around these vessels a significant number of proliferating tumor cells could be detected. In contrast, in hypopharyngeal cancers this vasculature-associated staining pattern could be observed from the very early stage of tumor development. In laryngeal and in hypopharyngeal cancers, tenascin upregulation strongly correlated with metastasis formation, early tumor recurrence, and lethal outcome of the disease. CONCLUSIONS: Clinical and immunohistologic data indicate that the accumulation of tenascin in the tumor blood vessels is an unfavorable prognostic indicator in laryngeal and hypopharyngeal cancers.

Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx.

Extravascular fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumor growth. The distribution of fibrin deposits was investigated in squamous cell carcinomas representing different stages of tumor progression of the larynx (n = 25) and hypopharynx (n = 9) by immunofluorescent techniques. Double and treble labelings were used to detect fibrinogen and fibrin in combination with marker antigens for tumor cells (cytokeratin), endothelial cells (von Willebrand factor), macrophages (recognized by KiM7), as well as factor XIII subunit A (FXIIIA) and tenascin (an embryonic extracellular matrix protein newly expressed during tumorigenesis). All tissue samples showed specific staining for fibrinogen/fibrin. Fibrin deposition was localized almost exclusively in the connective tissue compartment of tumors with characteristic accumulation at the interface of connective tissue and the tumorous parenchyma. In certain tumor samples showing highly invasive characteristics, fibrin deposits were observed in close association with tumor blood vessels in the tumor cell nodules. The overlapping reactions with polyclonal antibody to fibrinogen/fibrin and monoclonal antibody to fibrin indicate the activation of the coagulation cascade resulting in in situ thrombin activation and fibrin formation. Fibrin was crosslinked and stabilized by FXIIIA as revealed by urea insolubility test. Accumulation of phagocytozing macrophages detected by Ki M7 monoclonal antibody could be seen in areas of fibrin deposition. The blood coagulation factor XIIIA was detected in and around the cells labeled with Ki M7 antibody. Tenascin and fibrin deposits were found in the same localization in the tumor stroma and in association with tumor blood vessels within the tumor cell nodules. Neither fibrin nor tenascin were detected in the histologically normal tissue adjacent to tumors. The close association between fibrin deposits and macrophage accumulation strongly suggests the active participation of tumor-associated macrophages in the formation of stabilized intratumoral fibrin that facilitates tumor matrix generation and tumor angiogenesis.

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 a2subunit 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 tissue's regenerative capacity, and that FXIIIa may therefore be a link underlying a possible association between the processes of blood coagulation and nerve healing.

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.