Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1-0.01 mg/kg/day on days P1-P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.

A rat model for orthodontic translational expansive tooth movement

OBJECTIVES To present the development of an experimental model in rats for translational expansive tooth movement. SETTING AND SAMPLE Section of Periodontology at Department of Dentistry Aarhus University. Twenty male Wistar rats in two pilot experimental settings plus seven animals without any intervention serving as controls. MATERIAL AND METHODS The second molar (group P1) or the second and third molar (group P2) in the maxillae of the animals were moved buccally using transpalatal β-titanium springs. In the group P2, two spring types (high force and low force) and two preangulations (0° passive or 30° torsion moment) were tested. The amount and type of tooth movement achieved and the resulting skeletal effect were assessed on microCT images, histological analysis was performed on few selected specimens. RESULTS Expansive translational root movement amounting half a tooth width was achieved. Comparison of the amount of tooth movement at the right and left side of the maxilla showed that the expansion was rather symmetrical in the P2 group. Skeletal widening of the maxilla contributed in the P2 group to approximately one-third of the total root movement, whereas two-thirds were dental movement. CONCLUSION With the model used in the P2 group, further research on translational expansive tooth movement and its effect on the periodontium can be pursued. In models for orthodontic expansion, it is strongly recommended to separately evaluate skeletal and dental effects.

Perceptual and processing differences between physical and dichorhinic odor mixtures

Perceptual integration of sensory input from our two nostrils has received little attention in comparison to lateralized inputs for vision and hearing. Here, we investigated whether a binary odor mixture of eugenol and l-carvone (smells of cloves and caraway) would be perceived differently if presented as a mixture in one nostril (physical mixture), vs. the same two odorants in separate nostrils (dichorhinic mixture). In parallel, we investigated whether the different types of presentation resulted in differences in olfactory event-related potentials (OERP). Psychophysical ratings showed that the dichorhinic mixtures were perceived as more intense than the physical mixtures. A tendency for shift in perceived quality was also observed. In line with these perceptual changes, the OERP showed a shift in latencies and amplitudes for early (more "sensory") peaks P1 and N1 whereas no significant differences were observed for the later (more "cognitive") peak P2. The results altogether suggest that the peripheral level is a site of interaction between odorants. Both psychophysical ratings and, for the first time, electrophysiological measurements converge on this conclusion.

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

Regulatory factors and control of anthrax toxin gene expression

Bacillus anthracis plasmid pXO1 carries genes for three anthrax toxin proteins, pag (protective antigen), cya (edema factor), and lef (lethal factor). Expression of the toxin genes is enhanced by two signals: CO$\sb2$/bicarbonate and temperature. The CO$\sb2$/bicarbonate effect requires the presence of pXO1. I hypothesized that pXO1 harbors a trans-acting regulatory gene(s) required for CO$\sb2$/bicarbonate-enhanced expression of the toxin genes. Characterization of such a gene(s) will lead to increased understanding of the mechanisms by which B. anthracis senses and responds to host environments.^ A regulatory gene (atxA) on pXO1 was identified. Transcription of all three toxin genes is decreased in an atxA-null mutant. There are two transcriptional start sites for pag. Transcription from the major site, P1, is enhanced in elevated CO$\sb2$. Only P1 transcripts are significantly decreased in the atxA mutant. Deletion analysis of the pag upstream region indicates that the 111-bp region upstream of the P1 site is sufficient for atxA-mediated increase of this transcript. The cya and lef genes each have one apparent transcriptional start site. The cya and lef transcripts are significantly decreased in the atxA mutant. The atxA mutant is avirulent in mice. The antibody response to all three toxin proteins is significantly decreased in atxA mutant-infected mice. These data suggest that the atxA gene product activates expression of the toxin genes and is essential for virulence.^ Since expression of the toxin genes is dependent on atxA, whether increased toxin gene expression in response to CO$\sb2$/bicarbonate and temperature is associated with increased atxA expression was investigated. I monitored steady state levels of atxA mRNA and AtxA protein in different growth conditions. The results indicate that expression of atxA is not influenced by CO$\sb2$/bicarbonate. Steady state levels of atxA mRNA and AtxA protein are higher at 37$\sp\circ$C than 28$\sp\circ$C. However, increased pag expression at high temperature can not be attributed directly to increased atxA expression.^ There is evidence that an additional factor(s) may be involved in regulation of pag. Expression of pag in strains overproducing AtxA is significantly decreased compared to the wildtype strain. A specific interaction of tagged-AtxA with the pag upstream DNA has not been demonstrated. Furthermore, four proteins in B. anthracis extract can be co-immunoprecipitated with tagged-AtxA. Amino-terminal sequence of one protein has been determined and found highly homologous to chaperonins of GroEL family. Studies are under way to determine if this GroEL-like protein interactions with AtxA and plays any role in atxA-mediated activation of toxin genes. ^

Role of the B1 short arm in laminin self-assembly

Laminin self-assembles into a basement membrane polymer through specific low-affinity interactions. Recently, it was shown that the terminal short-arm domain (domains VI and V) of the B1 chain (fragment E4) possesses one of the laminin self-interaction sites [Schittny, J.C. & Yurchenco, P.D. (1990) J. Cell Biol. 110, 825-832], but that the binding partner(s) of this domain is unknown. Using affinity retardation chromatography we now investigate the domain(s) fragment E4 binds to. The elution of E4 was clearly retarded on immobilized laminin and fragment E1' (three-chain short-arm complex excluding the distal part of the B1 chain), but not on immobilized E4 in calcium containing buffer and at 37 degrees C. Under the same conditions, E1' strongly interacts with immobilized E4. In addition, E1' is able to non-covalently cross-link soluble E4 to immobilized E4. No further interaction of laminin and E4 with additional fragments (P1', A, B2 and B1 chain short-arm complex without B1-domains VI-IV and without globules; E8, distal long arm and G1-3; E3, long-arm G subdomains 4 and 5) could be demonstrated. These data are interpreted as evidence that (a) the primary laminin-laminin bonds are formed between the short arms of laminin, that (b) the terminal B1 short-arm domain (E4) can interact with the short arm(s) of the A and/or B2 chain(s) (domain E1'), but does not self-interact, and that (c) due to at least three self-binding sites, laminin polymerization behaves co-operatively.

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

Lung cancer screening with CT: evaluation of radiologists and different computer assisted detection software (CAD) as first and second readers for lung nodule detection at different dose levels

OBJECTIVES To find the best pairing of first and second reader at highest sensitivity for detecting lung nodules with CT at various dose levels. MATERIALS AND METHODS An anthropomorphic lung phantom and artificial lung nodules were used to simulate screening CT-examination at standard dose (100 mAs, 120 kVp) and 8 different low dose levels, using 120, 100 and 80 kVp combined with 100, 50 and 25 mAs. At each dose level 40 phantoms were randomly filled with 75 solid and 25 ground glass nodules (5-12 mm). Two radiologists and 3 different computer aided detection softwares (CAD) were paired to find the highest sensitivity. RESULTS Sensitivities at standard dose were 92%, 90%, 84%, 79% and 73% for reader 1, 2, CAD1, CAD2, CAD3, respectively. Combined sensitivity for human readers 1 and 2 improved to 97%, (p1=0.063, p2=0.016). Highest sensitivities--between 97% and 99.0%--were achieved by combining any radiologist with any CAD at any dose level. Combining any two CADs, sensitivities between 85% and 88% were significantly lower than for radiologists combined with CAD (p<0.03). CONCLUSIONS Combination of a human observer with any of the tested CAD systems provide optimal sensitivity for lung nodule detection even at reduced dose at 25 mAs/80 kVp.

Potential role of CYP2D6 in the central nervous system

1. Cytochrome P450 2D6 (CYP2D6) is a pivotal enzyme responsible for a major drug oxidation polymorphism in human populations. Distribution of CYP2D6 in brain and its role in serotonin metabolism suggest that CYP2D6 may have a function in the central nervous system. 2. To establish an efficient and accurate platform for the study of CYP2D6 in vivo, a human CYP2D6 (Tg-2D6) model was generated by transgenesis in wild-type (WT) C57BL/6 mice using a P1 phage artificial chromosome clone containing the complete human CYP2D locus, including the CYP2D6 gene and 5'- and 3'-flanking sequences. 3. Human CYP2D6 was expressed not only in the liver but also in the brain. The abundance of serotonin and 5-hydroxyindoleacetic acid in brain of Tg-2D6 is higher than in WT mice, either basal levels or after harmaline induction. Metabolomics of brain homogenate and cerebrospinal fluid revealed a significant up-regulation of L-carnitine, acetyl-L-carnitine, pantothenic acid, 2'-deoxycytidine diphosphate (dCDP), anandamide, N-acetylglucosaminylamine and a down-regulation of stearoyl-L-carnitine in Tg-2D6 mice compared with WT mice. Anxiety tests indicate Tg-2D6 mice have a higher capability to adapt to anxiety. 4. Overall, these findings indicate that the Tg-2D6 mouse model may serve as a valuable in vivo tool to determine CYP2D6-involved neurophysiological metabolism and function.

Multiple Hypersensitivities Including Recurrent Airway Obstruction, Insect Bite Hypersensitivity, and Urticaria in 2 Warmblood Horse Populations.

BACKGROUND Multiple hypersensitivities (MHS) have been described in humans, cats, and dogs, but not horses. HYPOTHESES Horses suffering from recurrent airway obstruction (RAO), insect bite hypersensitivity (IBH), or urticaria (URT) will have an increased risk of also being affected by another one of these hypersensitivities. This predisposition for MHS also will be associated with decreased shedding of strongylid eggs in feces and with a single nucleotide polymorphism (SNP BIEC2-224511), previously shown to be associated with RAO. ANIMALS The first population (P1) included 119 randomly sampled horses representative of the Swiss sporthorse population; the replication population (P2) included 210 RAO-affected Warmblood horses and 264 RAO-unaffected controls. All horses were Warmbloods, 14 years or older. METHODS Associations between disease phenotypes (RAO, IBH, URT, MHS) fecal egg counts, the SNP BIEC2-224511 as well as management and environmental factors were investigated. RESULTS In P1, RAO-affected horses had a 13.1 times higher odds ratio (OR) of also suffering from IBH (P = .004). In P2, the respective OR was 7.4 (P = .002) and IBH-affected horses also showed a 7.1 times increased OR of concomitantly suffering from URT (P < .001). IBH, URT, and MHS phenotypes were significantly associated with the absence of nematode eggs in the feces. CONCLUSIONS AND CLINICAL IMPORTANCE This is the first report of MHS in horses. Specifically, an increased risk for IBH should be expected in RAO-affected horses.

Multi-colour FISH in oesophageal adenocarcinoma-predictors of prognosis independent of stage and grade.

BACKGROUND Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples. METHODS To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data. RESULTS The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months. CONCLUSIONS Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.

Internet-based stress management for women with preterm labour: a case-based experience report

Pregnant women with preterm labour (PTL) in pregnancy often experience increased distress and anxieties regarding both the pregnancy and the child's health. The pathogenesis of PTL is, among other causes, related to the stress-associated activation of the maternal-foetal stress system. In spite of these psychobiological associations, only a few research studies have investigated the potential of psychological stress-reducing interventions. The following paper will present an online anxiety and stress management self-help program for pregnant women with PTL. Structure and content of the program will be illustrated by a case-based experience report. L.B., 32 years (G3, P1), was recruited at gestational week 27 while hospitalized for PTL for 3 weeks. She worked independently through the program for 6 weeks and had regular written contact with a therapist. Processing the program had a positive impact on L.B.'s anxiety and stress levels, as well as on her experienced depressive symptoms and bonding to the foetus. As PTL and the risk of PTB are associated with distress, psychological stress-reducing interventions might be beneficial. This study examines the applicability of an online intervention for pregnant women with PTL. The case report illustrates how adequate low-threshold psychological support could be provided to these women.

The rotation state of 67P/Churyumov-Gerasimenko from approach observations with the OSIRIS cameras on Rosetta

Aims. Approach observations with the Optical, Spectroscopic, and Infrared Remote Imaging System (OSIRIS) experiment onboard Rosetta are used to determine the rotation period, the direction of the spin axis, and the state of rotation of comet 67P’s nucleus. Methods. Photometric time series of 67P have been acquired by OSIRIS since the post wake-up commissioning of the payload in March 2014. Fourier analysis and convex shape inversion methods have been applied to the Rosetta data as well to the available ground-based observations. Results. Evidence is found that the rotation rate of 67P has significantly changed near the time of its 2009 perihelion passage, probably due to sublimation-induced torque. We find that the sidereal rotation periods P1 = 12.76129 ± 0.00005 h and P2 = 12.4043 ± 0.0007 h for the apparitions before and after the 2009 perihelion, respectively, provide the best fit to the observations. No signs of multiple periodicity are found in the light curves down to the noise level, which implies that the comet is presently in a simple rotation state around its axis of largest moment of inertia. We derive a prograde rotation model with spin vector J2000 ecliptic coordinates λ = 65° ± 15°, β = + 59° ± 15°, corresponding to equatorial coordinates RA = 22°, Dec = + 76°. However, we find that the mirror solution, also prograde, at λ = 275° ± 15°, β = + 50° ± 15° (or RA = 274°, Dec = + 27°), is also possible at the same confidence level, due to the intrinsic ambiguity of the photometric problem for observations performed close to the ecliptic plane.