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Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell. © 2008 Elsevier B.V. All rights reserved. Chemicals / CAS: dipicolinic acid, 17606-33-6, 499-83-2; Picolinic Acids; dipicolinic acid, 499-83-2

We recently found that the proteins of the proteolytic system of plasminogen activation emerge in late stages of melanocytic tumor progression. A large body of evidence suggests a role for two proteins, the low-density lipoprotein receptor-related protein (LRP)/α2-macroglobulin receptor and its receptor-associated protein (RAP), in the internalization of components of the plasminogen activation system. Here, we present data on the presence of these two proteins in human melanoma cell lines which differ in metastatic capacity, their corresponding xenografts, and in cutaneous melanocytic lesions. With flow cytometry, we found surface expression of LRP to be restricted to urokinase plasminogen activator, producing highly metastatic cell lines. These cell lines also produce higher levels of LRP mRNA, whereas RAP mRNA and protein are expressed at equal levels in all cell lines and not expressed at the cell surface. Xenografts of cell lines producing high levels of LRP remarkably contain only a small fraction of LRP-positive tumor cells. Using immunohistochemistry on frozen sections of 107 human melanocytic lesions comprising the various stages of melanocytic tumor progression, we found that expression of both LRP and RAP decreased in tumor progression. Furthermore, we noted that LRP and RAP are coexpressed within the same lesion. Using immunofluorescence double staining, we found that LRP and RAP colocalize in the same cells in the lesions studied and in the same cell structures in the cell lines studied. In conclusion, our results indicate that LRP and RAP are coordinately expressed in a decreased fashion in melanocytic tumor progression. Based on the staining results in xenografts and in human melanocytic lesions, we conclude that a strong correlation between expression of LRP and urokinase-type plasminogen activator seems not to exist in in vivo melanomas. Chemicals/CAS: LDL-Receptor Related Protein 1; Neoplasm Proteins; Receptors, Immunologic; Receptors, LDL; RNA, Messenger

Recently we developed mouse monocloual antibodies (mAb) against the isolated human 175-kDa mannose receptor. In the present study we tested whether these mAb are suitable for the detection of the mannose receptor on cultured macrophages using flow cytometry and on cells in human tissues using immunohistochemistry. Human monocytes did not react with the mAb in flow cytometry. Mannose receptor expression became detectable on monocytes cultured for 3 days (macrophages), and was maximal from 4 days onward. The mannose receptor was up-regulated on dexamethasone-treated (immunosuppressed) macrophages, and down-regulated on lipopolysaccharide-treated (activated) macrophages. Immunohistochemically the staining pattern of our mAb was compared with the marker of monocytes/macrophages KP1. In a bone marrow smear, only macrophages were stained with our mAb, whereas all myeloid cells were stained with KP1. In the thymus and lymph node, mannose receptor-positive branched cells (macrophages and dendritic cells) were detected in connective tissue, thymus cortex (not medulla), and in the T cell area (not the B cell area) of lymph nodes, whereas KP1 stained branched cells in all areas. It was concluded that the mAb are useful tools in flow cytometry and immunohistochemistry for the specific detection of cells expressing mannose receptor. Chemicals/CAS: Antibodies, Monoclonal; Biological Markers; Dexamethasone, 50-02-2; Glucocorticoids; Lectins, C-Type; Lipopolysaccharides; mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface

CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-γ upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-γ and minute amounts of IL-4 and IL-10, suggesting that these 'naive' cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-γ as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.

To study the role of the mannose receptor in cellular uptake and degradation of tissue-type plasminogen activator (t-PA), a set of five monoclonal antibodies (Moab) was generated against the mannose receptor isolated from human placental tissue. All Moab specifically recognised the 175 kDa mannose receptor in a crude placenta extract, as shown in Western blot analysis. By use of immunohistochemistry, we showed that in human placenta only the Hofbauer cells (fetal macrophages) express the mannose receptor. Epitope competition experiments indicated that the Moab bound to at least two different epitopes on the receptor molecule. Moab 14-3, 14-5, and 15-2, which are directed against one of these epitopes, strongly inhibited the interaction between the purified mannose receptor and t-PA. These Moab also inhibited mannose receptor-mediated degradation of t-PA by cultured human macrophages. The low density lipoprotein receptor-related protein (LRP) mediated t-PA degradation was not affected by the Moab. It is concluded that the Moab are useful for studying the expression of the human mannose receptor in Western blot and in immunohistochemistry, and for studying the interactions between the human mannose receptor and the mannose-containing ligand t-PA. Copyright © 1997 Schattauer Verlag. Chemicals/CAS: Antibodies, Monoclonal; Epitopes; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface; Tissue Plasminogen Activator, EC 3.4.21.68

Chromosomal defects are frequently present in malignant and premalignant skin disorders; however, it is not known whether ultraviolet radiation from sunlight plays a role in their induction. To obtain information on the ability of ultraviolet A and ultraviolet B to induce chromosomal aberrations, cultured melanocytes and fibroblasts were exposed to physiologic doses of ultraviolet A or ultraviolet B and, for comparison, to γ rays. As a measure of chromosomal aberrations, the formation of micronuclei was determined. To obtain sufficient statistical data on induced micronuclei and cell kinetics, a flow cytometry method has been modified and applied. The flow cytometry method analysis is based on staining the DNA with ethidium bromide and the cell membranes with 1,6-diphenyl-1,3,5,-hexatriene. We observed dose-dependent micronuclei formation after γ or ultraviolet B irradiation in both cell types and also for ultraviolet A in fibroblasts. The yield of micronuclei induced in fibroblasts by ultraviolet A was only a factor 15 smaller than that induced by ultraviolet B (313 nm). The results indicate that 10 kJ per m2 (equivalent to 1 minimal erythema dose) of ultraviolet B and 150 kJ per m2 of ultraviolet A (0.2 minimal erythema dose) can induce 1% of micronuclei in fibroblasts, equivalent to the induction due to 0.6 Gy of γ radiation. In conclusion, physiologic doses of sunlight can induce chromosomal aberrations at a level comparable with that observed after exposure to approximately 1 Gy of ionizing radiation. Therefore, sunlight can be considered a potential inducer of chromosomal aberrations in skin cells, which may contribute to skin carcinogenesis.

In order to study the intestinal mucosal immune cells, with emphasis on single T lymphocytcs, an inventory was made of single and organized lymphocytes in the epithelium and lamina propria of the small intestines of untreated Wistar, Fischer 344, and Lewis rats. The single and organized lymphocytes were examined microscopically. In addition, the single lymphocytes in the epithelium tIEL) and lamina propria (LPL) were analyzed by flow cytometry. Next, the use of flow cytometry analysis was explored to detect changes in the IEL T-lymphocyte population in subacute oral studies with the immunomodulating agents azathioprine and hexachlorobenzene. Untreated random-bred Wistar rats exhibited a large interindividual variability in IEL composition, while the variability was small in inbred Fischer 344 and Lewis rats. The explorative study with the 2 model immunomodulating compounds demonstrated that hexachlorobenzene increased the number of intraepithelial T lymphocytes with CD8+ phenotype at the cost oft cells with CD4+ phenotype in Lewis rats. Azathioprine did not induce distinct effects on the percentages of IEL. The data indicate that the intraepithelial lymphocytes in the intestines are a potential target for orally administered immunomodulating compounds and should therefore receive more attention in toxicologic pathology studies.

The transplantable C57BL/KaLwRij mouse 5T2 multiple myeloma (MM) is a new animal model for studies on MM in man. Histological examination of the 5T2 MM cells revealed their morphological heterogeneity. In this study we investigated whether this heterogeneity reflects subpopulations of 5T2 MM cells with different biological properties. 5T2 MM bone marrow cells were separated according to their sedimentation velocity (s.v.). When intravenously injected into syngeneic recipient mice, cells with s.v. of 8 mm h-1 led to the development of detectable 5T2 MM after 6 weeks; in contrast, 18 weeks elapsed before the same result was achieved with cells of s.v. lower than 5 mm h-1. Flow cytometric analysis revealed that 5T2 MM cells had an aneuploid DNA content and that most cycling 5T2 MM cells were larger, their s.v. rate exceeding 9 mm h-1. It was further demonstrated that about half of all aneuploid cells carried on their membrane the 5T2 MM idiotype. The majority of the idiotype-positive cells had s.v. rate exceeding 6.5 mm h-1 (16%-39%) or lower than 3 mm h-1 (16%-19%). The 5T2 MM was shown to contain subpopulations of cells of different size, proliferation capacity and expression of their membrane 5T2 idiotype; this, most likely reflects cells in different stages of differentiation. The mouse 5T2 MM corresponds also in this respect with MM in man. Chemicals/CAS: DNA, Neoplasm

Archival paraffin embedded material was used to examine whether additional quantitative criteria would be helpful to discriminate between histologically benign and malignant rat mammary tumours. To this end nuclear DNA content expressed as DNA ploidy index (DI) was measured using flow cytometry (FCM). A total of 63 benign and malignant mammary tumours were investigated. Thirteen out of 38 (34%) mammary carcinomas were DNA aneuploid against 0 out of 25 benign mammary tumours. Aneuploidy was not significantly increased in tumours showing histological signs of greater malignancy such as cribriform-comedo type or invasive growth. In addition to DI other quantitative criteria indicative for malignancy, such as mitotic count and nuclear morphometric characteristics, were estimated in 24 benign and malignant tubulopapillary tumours, a category where the histological classification may be difficult. It appeared that five out of nine noninvasive tubulopapillary carcinomas and six out of seven invasive carcinomas had abnormal values for either DI, mitotoic count or nuclear area or for a combination of these parameters. Each single parameter however was abnormal only in a minority of the malignant tumours. In this respect our data are in accordance with the fact that rat mammary carcinomas are clinically and histologically less malignant than their human counterparts. Chemicals/CAS: DNA, 9007-49-2; DNA, Neoplasm; Formaldehyde, 50-00-0

Flow cytometry was used to measure the uptake of adriamycin (AM) and daunomycin (DM) by various cell types of the haemopoietic organs and by acute myeloid leukemia (AML) cells. For this purpose, the spontaneous fluorescence of the drugs upon excitation with laser light at 488 nm was measured. The difference in the uptake of these two drugs correlates with their cytotoxicity. Myeloid progenitor cells from mouse bone marrow (CFU-C) are ten times more sensitive for DM than AM [ED10=0.85 ??g/ml (DM); 6.40 ??g/ml (AM)]. The cytotoxicity of DM is correlated with a higher fluorescence intensity of blast cells, lymphocytes and granulocytes when treated with DM as compared to AM. In contrast to this, the fluorescence of AML blast cells is significantly lower after treatment with DM than with AM. The uptake of both drugs seems to occur following active membrane transport, although to a different extent. Chemicals/CAS: daunorubicin, 12707-28-7, 20830-81-3, 23541-50-6; doxorubicin, 23214-92-8, 25316-40-9; Daunorubicin, 20830-81-3; Doxorubicin, 23214-92-8

For in vitro evaluation of functional properties of endothelial cells seeded on synthetic vascular prostheses accurate and reproducible quantification of cells is mandatory. Comparison of these properties with those resulting from other studies requires correlation of the functional parameters to reliably counted cell numbers. The accuracy of methods of quantification currently being used is unknown due to the lack of a "gold standard" method to which these methods can be compared. To determine the accuracy and reproducibility of four widely used methods, we have developed a "gold standard" model, using a flow cytometer (FACS). Endothelial cells, attached to collagen-coated Dacron vascular prostheses, were counted by four conventional methods and a new method of quantification after the attached number of cells had been determined with 99% accuracy by FACS. Subsequently, ratios were computed by dividing the cell numbers determined by the methods under investigation by those determined by FACS (×100%). The four conventional methods investigated were (1) removal and subsequent counting of cells from substrata by trypsin (T), (2) digestion of cells by citric acid and counting of crystal violet-stained cell nuclei (CV), (3) light microscopy after hematoxylin staining (LM), and (4) scanning electron microscopy (SEM). The new method consists of the measurement of cell fluorescence after labeling with fluorescein-diacetate (FDA). T and CV had average accuracy ratios of 127 ± 58% and 96 ± 48%, respectively (± standard deviation). The ratios for LM and SEM were 116 ± 101% and 44 ± 10% (respectively). FDA had a ratio of 99 ± 7%. Reproducibility of cell quantification by T and CV was significantly less than that of quantification by LM, SEM, and FDA, as expressed by data on inter- and intraobserver agreement. Our results indicate that the investigated conventional methods of quantification failed to meet criteria of both high accuracy and reproducibility. Light microscopy and scanning microscopy methods were inaccurate but yielded reproducible countings. We conclude that the FACS method can serve as a "gold standard" to compare the accuracy and reproducibility of cell quantification methods. Moreover, the FDA method results in both accurate and reproducible quantification of endothelial cells attached to vascular prosthetic material. © 1998 Academic Press, Inc. Chemicals/CAS: 3',6'-diacetylfluorescein, 596-09-8; Fluoresceins

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in an autoimmune disease, multiple sclerosis. Recombinant L. casei which express surface-anchored tetanus toxin fragment C (TTFC) were generated. Quantitative analysis by flow cytometry demonstrated a high level of cell wall-bound expression of TTFC and immunogenicity was demonstrated by parenteral immunization with whole cell extracts of the recombinants. A series of expression vectors was constructed to secrete human myelin basic protein (hMBP) or hMBP as a fusion protein with β-glucuronidase from Escherichia coli. These heterologous products produced by L. casei were detected in the growth medium and parenteral immunization with this medium evoked antibodies against hMBP, confirming that secretion indeed had occurred. Based on the different localization of the heterologous proteins, lactobacilli expressing surface-anchored TTFC are ideally suited for the induction of antibody responses, whereas lactobacilli that secrete myelin proteins can be used for the induction of peripheral T-cell tolerance. In conclusion, the specific technology described here allows the construction of a wide array of safe live recombinant lactobacilli which may prove to be useful in oral intervention strategies for the prevention of infectious diseases or treatment of autoimmune diseases.

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83+ cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model. © Blackwell Munksgaard, 2006.

Objectives: To determine the effect of skin thickness on the percutaneous penetration and distribution of test compounds with varying physicochemical properties using in vitro systems. Studies were carried out in accordance with OECD guidelines on skin absorption tests. Methods: Percutaneous penetration of caffeine (log P -0.01), testosterone (log P 3.32), propoxur (log P 1.52) (finite dose in ethanol to water vehicle ratio) and butoxyethanol (log P 0.83) (undiluted finite dose or as an infinite dose 50% [v/v] aqueous solution) through skin of varying thicknesses under occluded conditions was measured using flow through cells for 8-24 h. Saline (adjusted to pH 7.4) was used as receptor fluid, with BSA added for studies with testosterone and propoxur. Following exposure, the remaining surface dose was removed by swabbing and the skin digested prior to scintillation counting. Results: The maximum flux of caffeine was increased with decreasing skin thickness, although these differences were found to be non-significant. The presence of caffeine in the skin membrane was not altered by skin thickness. Maximum flux and cumulative dose absorbed of testosterone and butoxyethanol (in both finite and infinite doses) were markedly reduced with full thickness (about 1 mm thick) skin compared with split thickness skin (about 0.5 mm). Maximum flux of propoxur (dissolved in 60% ethanol) was clearly higher through skin of 0.71 mm than through skin of 1.36 mm, but no difference was found between 0.56 and 0.71 mm. The proportion of propoxur present in the membrane after 24 h increased significantly over the complete range of thicknesses tested (0.56-1.36 mm). Conclusions: A complex relationship exists between skin thickness, lipophilicity and percutaneous penetration and distribution. This has implications for risk assessment studies and for the validation of models with data from different sources. © Springer-Verlag 2006.

Studies of UV-induced skin cancers show that malignisation of skin cells, as well as alterations in anti-tumor immune control, are triggered by UV-induced lesions in cellular DNA. Such lesions can probably appear in the human mononuclear leukocytes (lymphocytes) during exposure of skin to sunlight. With the aim of studying the processing of UV-induced DNA lesions in these cells, we used flow cytometry and labelling of their partially denatured nuclei with the monoclonal antibody (H3) that binds cyclobutane pyrimidine dimers in single-stranded DNA. After the first few hours of cultivation of the irradiated cells, we found an increase in H3-specific fluorescence from cellular nuclei, while there was a decrease in the number of H3-positive sites in isolated DNA from these cells. We examined cells cultured under different conditions and concluded that the effect of enhancement of H3 labelling of nuclei did not result from changes in temperature and culture medium. Furthermore, we have found that this effect, as well as the decrease in H3 labelling in isolated DNA, are both prevented by pretreatment of the cells with Novobiocin, which we used as an inhibitor for the topoisomerase II-induced relaxation of supercoiled DNA prior to repair-specific incision. The inhibition by Novobiocin of the above-mentioned changes in H3 labelling in cellular nuclei and isolated DNA of the irradiated cells clearly indicate the association of both effects with an excision repair-related DNA modification. While the partial loss of H3-binding sites from isolated DNA is obviously a result of excision of some fraction of pyrimidine dimers, the enhancement of the H3 labelling of nuclei might be due to the formation of open structures at dipyrimidine-containing DNA fragments in preparation for incision. We suggest that formation of open structures predominates quantitatively over dual incision and excision of these fragments, and leads to enhanced exposure of the pyrimidine dimers in nuclei to H3 binding. Thus, unstimulated human lymphocytes appear to be capable of performing pre-incision steps for removal of these DNA lesions. © The Royal Society of Chemistry and Owner Societies 2004.

Background. Curcumin is a spice and a coloring food compound with a promising role in colon cancer prevention. Curcumin protects against development of colon tumors in rats treated with a colon carcinogen, in colon cancer cells curcumin can inhibit cell proliferation and induce apoptosis, it is an anti-oxidant and it can act as an anti-inflammatory agent. The aim of this study was to elucidate mechanisms and effect of curcumin in colon cancer cells using gene expression profiling. Methods. Gene expression changes in response to curcumin exposure were studied in two human colon cancer cell lines, using cDNA microarrays with four thousand human genes. HT29 cells were exposed to two different concentrations of curcumin and gene expression changes were followed in time (3, 6, 12, 24 and 48 hours). Gene expression changes after short-term exposure (3 or 6 hours) to curcumin were also studied in a second cell type, Caco-2 cells. Results. Gene expression changes (>1.5-fold) were found at all time points. HT29 cells were more sensitive to curcumin than Caco-2 cells. Early response genes were involved in cell cycle, signal transduction, DNA repair, gene transcription, cell adhesion and xenobiotic metabolism. In HT29 cells curcumin modulated a number of cell cycle genes of which several have a role in transition through the G2/M phase. This corresponded to a cell cycle arrest in the G2/M phase as was observed by flow cytometry. Functional groups with a similar expression profile included genes involved in phase-II metabolism that were induced by curcumin after 12 and 24 hours. Expression of some cytochrome P450 genes was downregulated by curcumin in HT29 and Caco-2 cells. In addition, curcumin affected expression of metallothionein genes, tubulin genes, p53 and other genes involved in colon carcinogenesis. Conclusions. This study has extended knowledge on pathways or processes already reported to be affected by curcumin (cell cycle arrest, phase-II genes). Moreover, potential new leads to genes and pathways that could play a role in colon cancer prevention by curcumin were identified. © 2004 van Erk et al; licensee BioMed Central Ltd.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

Type 1 diabetes mellitus (T1DM) is an autoimmune destruction of insulin producing pancreatic beta-cells due to a genetic predisposition and can be triggered by environmental factors. We have previously shown that bisphenol A (BPA) accelerates the spontaneous development of diabetes in non-obese diabetic (NOD) mice. Here, we hypothesized that oral exposure to a mixture of the endocrine disruptors BPA and phthalates, relevant for human exposure, would accelerate diabetes development compared to BPA alone. NOD mice were exposed to BPA (1. mg/l), a mixture of phthalates (DEHP 1. mg/l, DBP 0.2. mg/l, BBP 10. mg/l and DiBP 20. mg/l) or a combination of BPA and the phthalate mixture through drinking water from conception and throughout life. Previous observations that BPA exposure increased the prevalence of diabetes and insulitis and decreased the number of tissue resident macrophages in pancreas were confirmed, and extended by demonstrating that BPA exposure also impaired the phagocytic activity of peritoneal macrophages. None of these effects were observed after phthalate exposure alone. The phthalate exposure in combination with BPA seemed to dampen the BPA effects on macrophage number and function as well as diabetes development, but not insulitis development. Exposure to BPA alone or in combination with phthalates decreased cytokine release (TNFα, IL-6, IL-10, IFNγ, IL-4) from in vitro stimulated splenocytes and lymph node cells, indicating systemic changes in immune function. In conclusion, exposure to BPA, but not to phthalates or mixed exposure to BPA and phthalates, accelerated diabetes development in NOD mice, apparently in part via systemic immune alterations including decreased macrophage function. © 2015 The Authors.

Background: Probiotic bacteria may render mice resistant to the development of various inflammatory and infectious diseases. Objective: This study aimed to identify mechanisms by which probiotic bacteria may influence intestinal immune homeostasis in noninflammatory conditions. Methods: The effect of VSL#3, a mixture of 8 probiotic bacteria, on intestinal gene expression was studied in healthy female BALB/c and C57BL/6 mice after prolonged oral treatment (28 d, triweekly) with 3 × 108 colony-forming units of VSL#3. In a separate experiment in BALB/c mice, the effects of prolonged administration of VSL#3 and of phosphatebuffered saline (PBS), followed by 1 single dose of VSL#3, on innate and adaptive immune cells were evaluated. Results: Microarray analysis of the intestines ofmice treatedwith PBS confirmedwell-established differences in the expression of immune-related genes between C57BL/6 and BALB/c mice. Prolonged administration of VSL#3 was associated with downregulation of Il13 [fold change (FC) = 0.46] and Eosinophil peroxidase (Epx) (FC = 0.44) and upregulation of Il12rb1 (FC = 2.1), C-C chemokine receptor type 5 (Ccr5) (FC = 2.6), chemokine (C-X-C motif) receptor 3 (Cxcr3) (FC = 1.6), and C-X-C motif chemokine 10 (Cxcl10) (FC = 2.8) in BALB/cmice but not in C57BL/6mice. In BALB/c mice, it was shown that 28 d of treatment with VSL#3 affected the Peyer's patches (PPs) and mesenteric lymph nodes (MLNs), which was evident from an increase in B cells (26% and 8%, respectively), a decrease in T cells (21% and 8%, respectively), and an increase in cluster of differentiation (CD) 11c+ cells (57%in PPs) comparedwith PBS-treated mice. This treatment was also associated with increased frequencies of T helper 17 (13%) and regulatory T cells (11%) in the MLNs. Treatment with PBS followed by 1 single dose of VSL#3, 18 h before killing, was associated with a 2-fold increase in CD103+CD11c+ dendritic cells in MLNs and PPs. Conclusion: VSL#3 treatmentmediatesmouse strain-specific alterations in immunologic phenotype in conditions of homeostasis, suggesting that the effects of probiotic bacteria depend on the genetic background of the host. © 2015 American Society for Nutrition.

Objective-The beneficial effect of dietary fish oil, rich in omega-3 polyunsaturated fatty acids (PUFAs), on cardiovascular disease is multifactorial and may partly rely on their anticoagulant action. We studied how fish oil intake influenced thrombin generation in plasma and which factors were involved herein. Methods and Results-Twenty-five healthy males with borderline overweight received 3.0 g omega-3 PUFAs daily for 4 weeks. Fish oil intake reduced plasma triglycerides and lowered platelet integrin activation, as well as plasma levels of fibrinogen and factor V, but had no effect on vitamin K-dependent coagulation factors. Before fish oil intake, thrombin generation (reflecting the coagulant potential) considerably varied between plasmas from individual subjects, which were partly explained by variation in prothrombin, antithrombin, fibrinogen, and factor V levels. Fish oil intake reduced thrombin generation in the presence and absence of platelets. This reduction correlated with the fish oil effect on fibrinogen and factor V levels. Interestingly, the lowering effect of fish oil on thrombin generation and fibrinogen clustered around subjects with high fibrinogen carrying a structural fibrinogen α-chain polymorphism. Conclusions-Dietary omega-3 PUFAs provoke a hypocoagulant, vitamin K-independent effect in humans, the degree of which may depend on fibrinogen level. Chemicals / CAS: antithrombin, 9000-94-6; blood clotting factor 5, 9001-24-5, 9013-23-4; cholesterol, 57-88-5; fibrinogen, 9001-32-5; fish oil, 8016-13-5; protein C, 60202-16-6; prothrombin, 9001-26-7; thrombin, 9002-04-4; vitamin K group, 12001-79-5; Cholesterol, LDL; Factor V, 9001-24-5; Fatty Acids, Omega-3; Fibrinogen, 9001-32-5; Fish Oils; Peptide Fragments; Thrombin, 3.4.21.5; Triglycerides; thrombin receptor peptide (42-47)