Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells

The aim of this study was to evaluate adverse effects of multiwalled carbon nanotubes (MWCNT), produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNT were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15-35 μm2) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 μg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines. [Authors]

Decreased acute vascular remodeling, anaphylaxis and delayed type hypersensitivity in PPARβ/δ-deficient mice

Endothelial cells form a semi-permeable barrier that participates in the exchange of plasma fluids, proteins and cells, and helps to maintain the physiological functions of organs as well as circulatory homeostasis. Vascular permeability and vasodilatation are increased during acute and chronic inflammation, cancer and wound healing. This is mediated by exposure to certain vascular permeability increasing factors, such as vascular endothelial growth factor (VEGF). The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor (NHRs) family of ligand-activated transcription factors. Three isotypes, PPARa, PPARp/5 and PPARy have been identified. They are all expressed in endothelial cells (ECs). Recent data have demonstrated their involvement in important mechanisms for vasculogenesis and angiogenesis, such as cell proliferation/differentiation, directional sensing/migration, and survival. PPARs were reported to modulate the expression of pro-angiogenic soluble factors, such as VEGF-A and may also participate in the regulation of expression of VEGF receptors. The aim of the present work was to elucidate the role of PPARp/δ in endothelial cell functions important for angiogenesis as well as in vascular permeability and vasodilatation. Using organ culture models of mouse aorta expiants, cultures of human umbilical vein endothelial cells (HUVECs) and genetically modified mouse models, we studied the consequences of loss and gain of PPARp/5 activity on endothelial cell functions. In the first part of this study, we show that the activation of PPARp/δ promotes EC outgrowth in murine aorta expiants. In vivo we observed that dermal vessel acute permeability in response to VEGF-A stimulation is strongly impaired in PPARfi/δ -I- animals. Additionally, observation of the dermal vessel morphology showed a clear enlargement of the wild-type dermal vessels upon VEGF-A injection, whereas vessels of PPARp/5 -/- animals showed almost no enlargement. The impaired response to VEGF stimulation in the knock-out animals was not due to structural or morphological abnormalities. Based on this data, we suggest that PPARp/5 may act on intracellular signaling cascades in ECs, downstream of the VEGF-A receptor. In the second part of this study, we address the relevance of PPARβ/δ vascular functions in pathophysiological inflammatory conditions, such as delayed- type hypersensitivity (DTH) reaction and anaphylaxis in mice. The DTH reaction is a cell-mediated immune reaction to protein, bacterial and viral antigens, whereas anaphylaxis is the most severe form of allergic reaction. In these in vivo models, we demonstrated that the absence of PPARβ/δ in ECs prevents the formation of severe edema in the DTH reaction, and that Ρ PARβ/δ accelerates recovery following systemic anaphylaxis, at least partially through the control of vascular permeability. Our data not only describe a novel function of PPARβ/δ in vessel permeability and vasodilatation, but also open new routes of research for the development of vessel permeability/vasodilatation regulating agents. - Les cellules endothéliales qui bordent la face interne des vaisseaux sanguins forment l'endothélium, une barrière semi-perméable qui régule les échanges de fluides, de protéines et de cellules immunes entre la circulation et les organes. L'endothélium participe également au maintien de la fonction des organes et de l'homéostasie circulatoire. La perméabilité vasculaire augmente dans des situations inflammatoires aigties ou chroniques, dans les tumeurs, et pendant la réparation de blessures. Cette augmentation de perméabilité est due à la production de facteurs sécrétés, tels que le Vascular Endothelial Growth Factor (VEGF-A), la thrombine ou I'histamine. Lès récepteurs nucléaires Peroxisome Proliferator-Activated Receptors (PPAR) sont des facteurs de transcription mis en activité par des ligands. Trois isotypes de PPARs, PPARa, ΡΡΑΡβ/δ and PPARy ont été caractérisés. Ils sont exprimés dans les cellules endothéliales, et des travaux récents ont montré qu'ils régulent des comportements cellulaires importants pour la vasculogenèse et l'angiogenèse, tels que la prolifération, la différenciation, la migration, et la survie des cellules. Ils régulent également la production de VEGF-A par divers types cellulaires. Le but de ce travail était d'élucider le rôle de PPARβ/δ dans la régulation de la perméabilité vasculaire, plus particulièrement dans les cellules endothéliales. Grâce à des cultures d'expiants d'aortes de souris, à la culture d'une lignée endothéliale humaine (HUVECs) et de souris génétiquement modifiées, nous avons étudié le rôle de PPARβ/δ dans les cellules endothéliales, dans des situations gain et perte de fonction du récepteur. Dans la première partie de ce travail, nous avons montré les propriétés pro-angiogéniques de PPARβ/δ dans des explants d'aortes. In vivo, nous avons observé l'absence d'hyperperméabilité aiguë induite par le VEGF-A, la thrombine et I'histamine chez les souris PPARβ/δ -/-. De plus, l'analyse morphologique des vaisseaux dans le derme des souris après stimulation par VEGF- A a confirmé l'absence de réponse à la stimulation. Ces analyses morphologiques nous ont également permis de montrer que l'absence de réponse aiguë n'était pas due à un défaut de structure des vaisseaux dermiques chez les souris PPARp/δ -/-. Sur la base de ces résultats, nous proposons que PPARp/δ régule des voies de signalisation intracellulaires dans les cellules endothéliales, voie de signalisation impliquées dans la régulation de la perméabilité vasculaire: Dans la seconde partie du travail, nous avons étudié l'importance de la régulation de la perméabilité vasculaire par PPARβ/δ dans des situations pathophysiologiques impliquant une hyperperméabilité aiguë des vaisseaux : une réaction d'hypersensibilité cutanée retardée d'une part (delayed-type hypersensitivity, DTH), et un choc anaphylactique d'autre part. Dans ces deux modèles induits expérimentalement chez la souris, l'absence de PPARβ/δ prévient en partie la formation de l'oedème inflammatoire local (DTH), et accélère la récupération (anaphylaxie), au moins partiellement en réglant la perméabilité vasculaire. Ces résultats ouvrent un nouveau champs d'étude quant au rôle de PPARβ/δ dans les vaisseaux et à d'éventuelles applications thérapeutiques dans des pathologies inflammatoires.

Fracture imaging within a granitic rock aquifer using multiple-offset single-hole and cross-hole GPR reflection data

The sparsely spaced highly permeable fractures of the granitic rock aquifer at Stang-er-Brune (Brittany, France) form a well-connected fracture network of high permeability but unknown geometry. Previous work based on optical and acoustic logging together with single-hole and cross-hole flowmeter data acquired in 3 neighbouring boreholes (70-100 m deep) has identified the most important permeable fractures crossing the boreholes and their hydraulic connections. To constrain possible flow paths by estimating the geometries of known and previously unknown fractures, we have acquired, processed and interpreted multifold, single- and cross-hole GPR data using 100 and 250 MHz antennas. The GPR data processing scheme consisting of timezero corrections, scaling, bandpass filtering and F-X deconvolution, eigenvector filtering, muting, pre-stack Kirchhoff depth migration and stacking was used to differentiate fluid-filled fracture reflections from source generated noise. The final stacked and pre-stack depth-migrated GPR sections provide high-resolution images of individual fractures (dipping 30-90°) in the surroundings (2-20 m for the 100 MHz antennas; 2-12 m for the 250 MHz antennas) of each borehole in a 2D plane projection that are of superior quality to those obtained from single-offset sections. Most fractures previously identified from hydraulic testing can be correlated to reflections in the single-hole data. Several previously unknown major near vertical fractures have also been identified away from the boreholes.

Specific inhibition of the JNK pathway promotes locomotor recovery and neuroprotection after mouse spinal cord injury.

Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation.

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature.

BACKGROUND: In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo. RESULTS: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development. CONCLUSION: We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

The outer limiting membrane (OLM) revisited: clinical implications.

PURPOSE: The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. MATERIAL AND METHODS: Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. RESULTS: In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. CONCLUSIONS: In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.

Investigation into Improved Pavement Curing Materials and Techniques: Part 2 - Phase III, March 2003

Appropriate curing is important for concrete to obtain the designed properties. This research was conducted to evaluate the curing effects of different curing materials and methods on pavement properties. At present the sprayed curing compound is a common used method for pavement and other concrete structure construction. Three curing compounds were selected for testing. Two different application rates were employed for the white-pigmented liquid curing compounds. The concrete properties of temperature, moisture content, conductivity, and permeability were examined at several test locations. It was found, in this project, that the concrete properties varied with the depth. Of the tests conducted (maturity, sorptivity, permeability, and conductivity), conductivity appears to be the best method to evaluate the curing effects in the field and bears potential for field application. The results indicated that currently approved curing materials in Iowa, when spread uniformly in a single or double application, provide adequate curing protection and meet the goals of the Iowa Department of Transportation. Experimental curing methods can be compared to this method through the use of conductivity testing to determine their application in the field.

Development of Performance Properties of Ternary Mixes: Scoping Study, June 2005

Pozzolans and slag extend the market for concrete products by improving specific properties of the products, which allows the products to be constructed with materials or placed in environments that would have precluded the use of portland cement alone. In properly formulated concrete mixes, pozzolans and slag have been shown to enhance long-term strength, decrease permeability, increase durability, reduce thermal cracking of mass concrete, minimize or eliminate cracking related to alkali-silica reaction (ASR), and minimize or eliminate cracking related to sulfate attack. The purpose of this research project was to conduct a scoping study that could be used to evaluate the need for additional research in the area of supplementary cementitious materials (SCMs) that are used in concrete for highway applications. Special emphasis was given to the concept of using two or more SCMs in a single concrete mixture. The scope of the study was limited to a literature survey and panel discussions concerning issues relevant to the project. No laboratory work was conducted for this project. A problem statement with research plan was created that could be used to guide a pooled fund project.

Neuroretinitis: review of the literature and new observations.

Neuroretinitis (NR) is an inflammatory disorder characterized by optic disc edema and subsequent formation of a macular star figure. The underlying pathophysiology involves increased permeability of disc vasculature, but the etiology is not fully defined. In some cases, NR is probably due to an infectious process involving the disc; in others, a postviral or autoimmune mechanism is more likely. Cases can be divided into those in which a specific infectious agent has been identified, those considered idiopathic, and those with recurrent attacks. Some reports have not distinguished among these subgroups, and it is unclear if their clinical features vary. We reviewed the literature and our own patients looking particularly at features that might better distinguish these subtypes. Features common to all 3 groups included age, absence of pain, and fundus appearance. Preceding systemic symptoms were more common in patients with cat scratch disease (CSD) and uncommon in those with recurrence. The pattern and magnitude of visual field loss differed, more commonly confined to the central field in CSD cases and more severe in recurrent cases. Recovery of visual acuity and field was less substantial in recurrent cases even after the initial episode. MRI was usually normal in all 3 groups. Enhancement confined to the optic disc was found in all 3 groups, but enhancement of the retrobulbar optic nerve was seen only in recurrent cases. Findings that are strongly suggestive of CSD include very young age, preceding systemic symptoms, and poor visual acuity but with a small or absent relative afferent pupil defect (RAPD). In contrast, the following are suggestive of idiopathic NR with a high risk of recurrence: absence of systemic symptoms, visual field defect outside the central field, preserved visual acuity with a large RAPD, and poor recovery of vision. Decisions regarding evaluation and treatment should be made with these features in mind.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Human skin in vitro permeation of bentazon and isoproturon formulations with or without protective clothing suit.

Skin exposures to chemicals may lead, through percutaneous permeation, to a significant increase in systemic circulation. Skin is the primary route of entry during some occupational activities, especially in agriculture. To reduce skin exposures, the use of personal protective equipment (PPE) is recommended. PPE efficiency is characterized as the time until products permeate through material (lag time, Tlag). Both skin and PPE permeations are assessed using similar in vitro methods; the diffusion cell system. Flow-through diffusion cells were used in this study to assess the permeation of two herbicides, bentazon and isoproturon, as well as four related commercial formulations (Basagran(®), Basamais(®), Arelon(®) and Matara(®)). Permeation was measured through fresh excised human skin, protective clothing suits (suits) (Microchem(®) 3000, AgriSafe Pro(®), Proshield(®) and Microgard(®) 2000 Plus Green), and a combination of skin and suits. Both herbicides, tested by itself or as an active ingredient in formulations, permeated readily through human skin and tested suits (Tlag < 2 h). High permeation coefficients were obtained regardless of formulations or tested membranes, except for Microchem(®) 3000. Short Tlag, were observed even when skin was covered with suits, except for Microchem(®) 3000. Kp values tended to decrease when suits covered the skin (except when Arelon(®) was applied to skin covered with AgriSafe Pro and Microgard(®) 2000), suggesting that Tlag alone is insufficient in characterizing suits. To better estimate human skin permeations, in vitro experiments should not only use human skin but also consider the intended use of the suit, i.e., the active ingredient concentrations and type of formulations, which significantly affect skin permeation.

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells.

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated whether or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M(r) 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0 degrees C or 37 degrees C. The matrix fraction retained 20-40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37 degrees C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.