Relating the physicochemical characteristics and dispersion of multiwalled carbon nanotubes in different suspension media to their oxidative reactivity in vitro and inflammation in vivo

Reactive oxygen species (ROS) production is important in the toxicity of pathogenic particles such as fibres. We examined the oxidative potential of straight (50 microm and 10 microm) and tangled carbon nanotubes in a cell free assay, in vitro and in vivo using different dispersants. The cell free oxidative potential of tangled nanotubes was higher than for the straight fibres. In cultured macrophages tangled tubes exhibited significantly more ROS at 30 min, while straight tubes increased ROS at 4 h. ROS was significantly higher in bronchoalveolar lavage cells of animals instilled with tangled and 10 mum straight fibres, whereas the number of neutrophils increased only in animals treated with the long tubes. Addition of dispersants in the suspension media lead to enhanced ROS detection by entangled tubes in the cell-free system. Tangled fibres generated more ROS in a cell-free system and in cultured cells, while straight fibres generated a slower but more prolonged effect in animals.

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Clinical characteristics and causes of pruritus in cats: a multicentre study on feline hypersensitivity-associated dermatoses

Hypersensitivity dermatitides (HD) are often suspected in cats. Cats with HD are reported to present with one or more of the following patterns: miliary dermatitis, eosinophilic dermatitis, self-induced symmetrical alopecia or head and/or neck excoriations. Previous reports on feline HD included small numbers of animals, took place in geographically restricted areas or did not compare these conditions with other causes of pruritus. The goal of the present study was to analyse 72 parameters covering signalment, clinical, laboratory and treatment characteristics from a large group of pruritic cats from different geographical areas. Of the 502 cats, the following diagnoses were made: flea HD (29% of cases), food HD (12%) nonflea/nonfood HD (20%) and other diseases in which pruritus was a feature (24%). Cats with signs consistent with a HD but which did not complete a food trial were not analysed further (15% of cases). Most cats with nonflea HD exhibited signs compatible with one or more of the four typical lesional patterns, but none of these patterns was found to be pathognomonic for any specific diagnosis. Food HD and nonflea/nonfood HD were found to be clinically undistinguishable. Young adult, purebred and female cats appeared predisposed to nonflea/nonfood HD. As many diagnoses presented with similar lesional patterns, a thorough clinical work-up is required for establishment of a specific diagnosis.

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

The surfaces of Bacillus anthracis endospores expose a pentasaccharide containing the monosaccharide anthrose, which has been considered for use as a vaccine or target for specific detection of the spores. In this study B. anthracis strains isolated from cattle carcasses in African countries where anthrax is endemic were tested for their cross-reactivity with monoclonal antibodies (MAbs) specific for anthrose-containing oligosaccharides. Unexpectedly, none of the isolates collected in Chad, Cameroon, and Mali were recognized by the MAbs. Sequencing of the four-gene operon encoding anthrose biosynthetic enzymes revealed the presence of premature stop codons in the aminotransferase and glycosyltransferase genes in all isolates from Chad, Cameroon, and Mali. Both immunological and genetic findings suggest that the West African isolates are unable to produce anthrose. The anthrose-deficient strains from West Africa belong to a particular genetic lineage. Immunization of cattle in Chad with a locally produced vaccine based on anthrose-positive spores of the B. anthracis strain Sterne elicited an anti-carbohydrate IgG response specific for a synthetic anthrose-containing tetrasaccharide as demonstrated by glycan microarray analysis. Competition immunoblots with synthetic pentasaccharide derivatives suggested an immunodominant role of the anthrose-containing carbohydrate in cattle. In West Africa anthrax is highly endemic. Massive vaccination of livestock in this area has taken place over long periods of time using spores of the anthrose-positive vaccine strain Sterne. The spread of anthrose-deficient strains in this region may represent an escape strategy of B. anthracis.

Epidemiological study of pestiviruses in South American camelids in Switzerland

BACKGROUND: In the context of the ongoing eradication campaign for bovine viral diarrhea virus (BVDV) in cattle in Switzerland, the role of South American camelids (SAC) as a possible virus reservoir needed to be evaluated. OBJECTIVE: To assess and characterize the prevalence of pestivirus infections in SAC in Switzerland. ANIMALS: Serum samples collected from 348 animals (40 herds) in 2008 and from 248 animals (39 herds) in 2000 were examined for antibodies against pestiviruses and for the presence of BVDV viral RNA. METHODS: Cross-sectional study using stratified, representative herd sampling. An indirect BVDV-ELISA was used to analyze serum samples for pestivirus antibodies, and positive samples underwent a serum neutralization test (SNT). Real-time RT-PCR to detect pestiviral RNA was carried out in all animals from herds with at least 1 seropositive animal. RESULTS: In 2008, the overall prevalence of animals positive for antibodies (ELISA) and pestiviral RNA or was 5.75 and 0%, respectively. In 2000, the corresponding prevalences were 3.63 and 0%, respectively. The seroprevalences (SNT) for BVDV, border disease virus or undetermined pestiviruses were estimated to be 0, 1.73, and 4.02% in 2008, and 0.40, 1.21, and 2.02% in 2000, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: At the present time, SAC appear to represent a negligible risk of re-infection for the BVDV eradication program in cattle in Switzerland.

The Role of Antennapedia in Anteroposterior Patterning of the Drosophila Melanogaster Ventral Nervous System

A major unresolved question in developmental neurobiology is how the nervous system is adapted to the specific needs of the organism at different life stages. In the holometabolous insect Drosophila melanogaster, the larval ventral nervous system (VNS) is comprised of similar repeating segments, as opposed to the adult VNS, which varies greatly from segment to segment both in number and types of neurons. The adult-specific neurons of each segment are generated by 25 distinct types of neuronal progenitor cells called neuroblasts (NBs) that appear in a stereotyped array (Truman et al., 2004). Each NB divides repeatedly to produce a distinct set of daughter cells termed a lineage, which is bilaterally symmetric but present to varying degrees in each segment. These daughter cells can be distinguished by their position within the nervous system as well as by their axonal projections. Each of the 25 NBs produces neurons; if both daughter cells are present in a lineage then both sibling populations survived, whereas if only one projection is seen cell death occurred, leaving a hemilineage (half lineage). In some lineages, the same sibling type survives in all segments in which the lineage appears, but in others, the surviving sibling type varies across segments, resulting in a different morphology for the same lineage in different segments. How are these differences in survival and morphology controlled? The Hox genes provide positional information for developing structures along the anterior-posterior (AP) axis of animals. They encode transcription factors, thereby controlling the activity of genes down stream. In the postembryonic VNS, each NB lineage features its own characteristic expression pattern of Hox genes Antp and Ubx, which can vary from segment-to-segment, and can thereby cause variation in the number of neural cells and axonal projections that survive. This study defines the wild-type expression pattern of Antp and elucidates the role of Antp in gain of function studies. These studies are possible due to the MARCM (Mosaic Analysis with a Repressible Cell Marker) method, which allows the genetically manipulated cells to be specifically labeled in an otherwise normal, unlabeled organism. The results indicate that Antp is expressed in a segment-, lineage-, and hemilineage-specific manner. Antp is sufficient for both anterior and posterior transformations of particular lineages, including promotion of cell death and/or survival as well as axon guidance.

Financial analysis of various strategies for the control of Neospora caninum in dairy cattle in Switzerland

The present study was conducted to estimate the direct losses due to Neospora caninum in Swiss dairy cattle and to assess the costs and benefits of different potential control strategies. A Monte Carlo simulation spreadsheet module was developed to estimate the direct costs caused by N. caninum, with and without control strategies, and to estimate the costs of these control strategies in a financial analysis. The control strategies considered were "testing and culling of seropositive female cattle", "discontinued breeding with offspring from seropositive cows", "chemotherapeutical treatment of female offspring" and "vaccination of all female cattle". Each parameter in the module that was considered to be uncertain, was described using probability distributions. The simulations were run with 20,000 iterations over a time period of 25 years. The median annual losses due to N. caninum in the Swiss dairy cow population were estimated to be euro 9.7 million euros. All control strategies that required yearly serological testing of all cattle in the population produced high costs and thus were not financially profitable. Among the other control strategies, two showed benefit-cost ratios (BCR) >1 and positive net present values (NPV): "Discontinued breeding with offspring from seropositive cows" (BCR=1.29, NPV=25 million euros ) and "chemotherapeutical treatment of all female offspring" (BCR=2.95, NPV=59 million euros). In economic terms, the best control strategy currently available would therefore be "discontinued breeding with offspring from seropositive cows".

Mechanical Properties of Plant Underground Storage Organs and Implications for Dietary Models of Early Hominins

The diet of early human ancestors has received renewed theoretical interest since the discovery of elevated d13C values in the enamel of Australopithecus africanus and Paranthropus robustus. As a result, the hominin diet is hypothesized to have included C4 grass or the tissues of animals which themselves consumed C4 grass. On mechanical grounds, such a diet is incompatible with the dental morphology and dental microwear of early hominins. Most inferences, particularly for Paranthropus, favor a diet of hard or mechanically resistant foods. This discrepancy has invigorated the longstanding hypothesis that hominins consumed plant underground storage organs (USOs). Plant USOs are attractive candidate foods because many bulbous grasses and cormous sedges use C4 photosynthesis. Yet mechanical data for USOs—or any putative hominin food—are scarcely known. To fill this empirical void we measured the mechanical properties of USOs from 98 plant species from across sub-Saharan Africa. We found that rhizomes were the most resistant to deformation and fracture, followed by tubers, corms, and bulbs. An important result of this study is that corms exhibited low toughness values (mean = 265.0 J m-2) and relatively high Young’s modulus values (mean = 4.9 MPa). This combination of properties fits many descriptions of the hominin diet as consisting of hard-brittle objects. When compared to corms, bulbs are tougher (mean = 325.0 J m-2) and less stiff (mean = 2.5 MPa). Again, this combination of traits resembles dietary inferences, especially for Australopithecus, which is predicted to have consumed soft-tough foods. Lastly, we observed the roasting behavior of Hadza hunter-gatherers and measured the effects of roasting on the toughness on undomesticated tubers. Our results support assumptions that roasting lessens the work of mastication, and, by inference, the cost of digestion. Together these findings provide the first mechanical basis for discussing the adaptive advantages of roasting tubers and the plausibility of USOs in the diet of early hominins.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Prospects for microbeam radiation therapy of brain tumours in children to reduce neurological sequelae

Microbeam radiation therapy (MRT), a form of experimental radiosurgery of tumours using multiple parallel, planar, micrometres-wide, synchrotron-generated X-ray beams ('microbeams'), can safely deliver radiation doses to contiguous normal animal tissues that are much higher than the maximum doses tolerated by the same normal tissues of animals or patients from any standard millimetres-wide radiosurgical beam. An array of parallel microbeams, even in doses that cause little damage to radiosensitive developing tissues, for example, the chick chorioallantoic membrane, can inhibit growth or ablate some transplanted malignant tumours in rodents. The cerebella of 100 normal 20 to 38g suckling Sprague-Dawley rat pups and of 13 normal 5 to 12kg weanling Yorkshire piglets were irradiated with an array of parallel, synchrotron-wiggler-generated X-ray microbeams in doses overlapping the MRT-relevant range (about 50-600Gy) using the ID17 wiggler beamline tangential to the 6GeV electron synchrotron ring at the European Synchrotron Radiation Facility in Grenoble, France. Subsequent favourable development of most animals over at least 1 year suggests that MRT might be used to treat children's brain tumours with less risk to the development of the central nervous system than is presently the case when using wider beams.

A model of cerebral aspergillosis in non-immunosuppressed nursing rats

Central nervous system aspergillosis is an often fatal complication of invasive Aspergillus infection. Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop novel therapeutic approaches. This study presents a model of central nervous system aspergillosis that mimics important aspects of human disease. Eleven-day-old non-immunosuppressed male Wistar rats were infected by an intracisternal injection of 10 mul of a conidial suspension of Aspergillus fumigatus. An inoculum of 7.18 log(10) colony-forming units (CFU) consistently produced cerebral infection and resulted in death of all animals (n = 25) within 3-10 days. Median survival time was 3 days. Histomorphologically, all animals developed intracerebral abscesses (2-26 per brain) containing abundant fungal hyphae and neutrophils. Fungal culture of cortical homogenates yielded maximal growth on day 3 after infection (5.4 log(10) CFU/g, n = 15) that declined over time. Galactomannan concentrations in cortical homogenates, assessed as an index for hyphal burden, peaked on days 3-5. Fungal infection spread to peripheral organs in 83% of animals. Fungal burden in lung, liver, spleen and kidney was two orders of magnitude lower than in the brain. The successful establishment of a model of cerebral aspergillosis in a non-immunosuppressed host provides the opportunity to investigate mechanisms of disease and to develop novel treatment regimens for this commonly fatal infection.

Neuropathological survey of fallen stock: Active surveillance reveals high prevalence of encephalitic listeriosis in small ruminants

This paper describes the prevalence of brain lesions in the Swiss fallen stock population of small ruminants. 3075 whole brains (75% sheep, 25% goats) were collected as part of a year-long active survey of transmissible spongiform encephalopathies (TSEs) in small ruminants conducted by the Swiss authorities between July 2004 and July 2005. All fallen stock brains were systematically examined by histopathology to obtain reliable data on histologically identifiable brain lesions. Lesions were found in an unexpectedly high number of animals (8.1% of all examined brains). A wide spectrum of diseases was detected showing that this approach provides an excellent opportunity to screen for the prevalence of neurological diseases. Encephalitic listeriosis was by far the most frequent cause of CNS lesions in both species and its prevalence was unexpectedly high when compared to notified confirmed cases. In conclusion, the prevalence of listeriosis as estimated by passive surveillance based on the notification of clinical suspects has been underestimated in the past.

The postantibiotic effect in the treatment of experimental meningitis caused by Streptococcus pneumoniae in rabbits

The relevance of a postantibiotic effect in the treatment of pneumococcal meningitis was evaluated in a rabbit model. After administration of a single intravenous bolus of ampicillin at various dosages, such an effect was observed in all animals. The duration of this effect in vivo (2.5-18 hr) was consistently longer than that in vitro (1-4.3 hr); however, in rabbits the postantibiotic effect was eliminated by the administration of intravenous plus intracisternal beta-lactamase. In an assessment of the potential therapeutic benefit of the postantibiotic effect, the efficacy to two regimens of treatment with different intervals between doses was compared. One group of animals received ampicillin every 4 hr and another every 12 hr. With sufficiently high doses, drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration for most of the 4-hr interval but for only about one-third of the 12-hr interval. The rate of cure was similar for the two regimens and approximated 100% when peak drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration by at least 10-fold.

Animal model for percutaneous creation of traumatic thoracic aortic transection

PURPOSE: This study was conducted to create an animal model for thoracic aortic transection that is suitable for thoracic endograft research. MATERIALS AND METHODS: Percutaneous aortic transection creation was attempted in 12 sheep. A custom collapsible circumferential cutting device was inserted into the proximal descending thoracic aorta via a femoral approach with an 11-F delivery catheter. The device was deployed 2 cm distal to the left subclavian artery origin and rotated 20 times to create aortic transection. Aortic diameters, mean aortic pressures, and heart rates were tested for degrees of difference between measurements before and after the creation of transection. On necropsy, the extent of aortic damage was classified as none, nontransmural, or transmural, and aortic transection was classified as none, partial, or circumferential. RESULTS: On angiography, creation of transmural thoracic aortic transection was successful in 91.7% (11/12) of animals. Aortic transection was circumferential in 54.4% (6/11) of animals and partial in 45.6% (5/11) of animals. Mean aortic diameter was 19.6 +/- 3.4 mm (range 12-24 mm) pre-transection and 25.8 +/- 4.5 mm (range 17.8-33 mm) post-transection (P = .0003). Pre-transection, mean aortic pressure was 79 +/- 13.8 mmHg, and 64.6 +/- 15.8 mmHg 15 min post-transection (P = .041). Pre-transection, mean heart rate was 94.5 +/- 17.2 beats per minute (bpm), and 105.8 +/- 17.2 bpm 15 min post-transection (P = .0057). CONCLUSIONS: Thoracic aortic transection was successfully created percutaneously in most animals. The animals remained in hemodynamically stable condition for as long as 240 minutes after the creation of aortic injury. This percutaneous animal model is straightforward and may be of potential value for future thoracic endograft research.

Bronchial circulation after experimental lung transplantation: the effect of direct revascularization of a bronchial artery

Direct revascularization of a bronchial artery has been proposed as a measure to alleviate the problem of bronchial ischemia after lung transplantation. To assess the effect of restoration of arterial blood flow to the transplanted bronchus, bronchial mucosal blood flow was measured in a model of modified unilateral lung transplantation in pigs. Laser Doppler velocimetry (LDV) and radioisotope studies using radio-labeled erythrocytes (RI) were used to measure blood flow at the donor main carina (DC) and upper lobe carina (DUC) after 3 h of reperfusion. The recipient carina was used as a reference point; values obtained by LDV and RI were expressed as percentage of blood flow at the recipient carina. Two groups of animals were studied. In group 1 (n = 6) standard unilateral transplantation was performed; in group 2 (n = 6) a left bronchial artery was reimplanted into the descending thoracic aorta of the recipient. No differences were observed between the two groups with respect to preoperative or postoperative gas exchange or hemodynamics. In group 1, bronchial blood flow at the DC was 37.6 +/- 2.2% (LDV) and 44.1 +/- 14.8% (RI) of reference blood flow. At the DUC, blood flow was 54.9 +/- 7.7% (LDV) and 61.6 +/- 25.7% (RI) of normal flow. In group 2, blood flow was increased at the DC as measured by LDV (55.3 +/- 17.1%; p less than 0.05) and by RI (60.8 +/- 25.3%; p less than 0.2). A similar increase was found at the DUC (LDV: 81.8 +/- 19.3%; p less than 0.05; RI: 88.6 +/- 31.0%; p less than 0.2). It is concluded that there is a significant gradient of blood flow from intra- to extrapulmonary airways after lung transplantation. Reimplantation of a bronchial artery results in significant improvement of graft bronchial blood flow. Restoration of bronchial perfusion to normal levels, however, cannot be achieved, suggesting a possible defect in the microcirculation of the donor airways.