Point Absorber Limits to Future Gravitational-Wave Detectors.

High-quality optical resonant cavities require low optical loss, typically on the scale of parts per million. However, unintended micron-scale contaminants on the resonator mirrors that absorb the light circulating in the cavity can deform the surface thermoelastically and thus increase losses by scattering light out of the resonant mode. The point absorber effect is a limiting factor in some high-power cavity experiments, for example, the Advanced LIGO gravitational-wave detector. In this Letter, we present a general approach to the point absorber effect from first principles and simulate its contribution to the increased scattering. The achievable circulating power in current and future gravitational-wave detectors is calculated statistically given different point absorber configurations. Our formulation is further confirmed experimentally in comparison with the scattered power in the arm cavity of Advanced LIGO measured by in situ photodiodes. The understanding presented here provides an important tool in the global effort to design future gravitational-wave detectors that support high optical power and thus reduce quantum noise.

Viscous froth lens.

Microscale models of foam structure traditionally incorporate a balance between bubble pressures and surface tension forces associated with curvature of bubble films. In particular, models for flowing foam microrheology have assumed this balance is maintained under the action of some externally imposed motion. Recently, however, a dynamic model for foam structure has been proposed, the viscous froth model, which balances the net effect of bubble pressures and surface tension to viscous dissipation forces: this permits the description of fast-flowing foam. This contribution examines the behavior of the viscous froth model when applied to a paradigm problem with a particularly simple geometry: namely, a two-dimensional bubble "lens." The lens consists of a channel partly filled by a bubble (known as the "lens bubble") which contacts one channel wall. An additional film (known as the "spanning film") connects to this bubble spanning the distance from the opposite channel wall. This simple structure can be set in motion and deformed out of equilibrium by applying a pressure across the spanning film: a rich dynamical behavior results. Solutions for the lens structure steadily propagating along the channel can be computed by the viscous froth model. Perturbation solutions are obtained in the limit of a lens structure with weak applied pressures, while numerical solutions are available for higher pressures. These steadily propagating solutions suggest that small lenses move faster than large ones, while both small and large lens bubbles are quite resistant to deformation, at least for weak applied back pressures. As the applied back pressure grows, the structure with the small lens bubble remains relatively stiff, while that with the large lens bubble becomes much more compliant. However, with even further increases in the applied back pressure, a critical pressure appears to exist for which the steady-state structure loses stability and unsteady-state numerical simulations show it breaks up by route of a topological transformation.

Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands.

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Effects of respiratory syncytial virus persistence on airway responsiveness and inflammation in guinea-pigs.

Recurrent wheezing and asthma often develop after acute respiratory syncytial virus (RSV) bronchiolitis, but the mechanisms of these sequelae are poorly understood. Using a guinea-pig model of human RSV lung infection, the effects of long-term viral persistence on three hallmarks of asthma: nonspecific airway responsiveness, airway inflammation and airway remodelling were examined. Guinea-pigs were studied 100 days after intranasal instillation of either human RSV or uninfected vehicle, using: 1) acetylcholine challenge to test for airway hyperresponsiveness (AHR); 2) lung histology to quantify the numbers of airway eosinophils and metachromatic cells (mast cells/basophils); 3) airway morphometry of the areas of the airway subepithelial connective tissue, smooth muscle and adventitia, to test for airway remodelling; and 4) immunohistochemistry to identify lung cells containing RSV antigens. The RSV-inoculated group had significantly elevated AHR and airway eosinophils compared to uninfected control animals (p<0.05). There were no significant differences between the two groups in terms of numbers of airway metachromatic cells, or the areas of subepithelial connective tissue, smooth muscle or adventitia. Viral proteins were identified by immunohistochemistry within several types of lung cells. In conclusion, long-term persistence of respiratory syncytial virus in the guinea-pig lung is associated with airway hyperresponsiveness and airway eosinophilia, and these changes may be pertinent to the pathogenesis of postbronchiolitis wheezing and asthma in children.

Effect of respiratory syncytial virus on subsequent allergic sensitization to ovalbumin in guinea-pigs.

Children with acute respiratory syncytial virus (RSV) bronchiolitis often develop recurrent wheezing, asthma and allergic sensitization, but the role of RSV in the pathogenesis of these sequelae is unclear. This study examined whether RSV infection potentiates subsequent allergic sensitization, airway hyperresponsiveness (AHR) and airway inflammation induced by repeated exposures to aerosolized ovalbumin (OA) in guinea-pigs. Guinea-pigs received either RSV or sham inoculum, followed by exposures to OA- or saline-containing aerosols to form the following groups: 1) noninfected, nonsensitized controls (sham/saline group); 2) RSV-infected, nonsensitized animals (RSV/ saline group); 3) noninfected, OA-sensitized animals (sham/OA group); 4) RSV infection and first OA exposure on the same day (RSV/OA group), and 5) RSV infection six days prior to first OA exposure (RSV6/OA group). Three days after the final aerosol exposure, circulating OA-specific immunoglobulin (Ig)G1 antibody titres and AHR to inhalation acetylcholine challenge were measured and morphometry performed to evaluate allergic inflammation of the airways. OA-exposed animals developed OA-specific IgG1 antibodies, AHR and airway eosinophilia (sham/OA, RSV/OA and RSV6/OA groups. RSV infection alone induced significant AHR and airway eosinophilia (RSV/saline group). RSV infection, and concomitant exposure to OA (RSV/OA group) enhanced OA-specific IgG1 antibodies, but not airway eosinophilia or AHR. Such increases were not observed in the RSV6/OA group. In conclusion, respiratory syncytial virus potentiates the production of ovalbumin-specific immunoglobulin G1 antibodies in guinea-pigs, but circulating titres of these antibodies do not reflect the extent of airway hyperresponsiveness or airway inflammation. In addition, respiratory syncytial virus infection alone can produce slight increases in airway hyperresponsiveness that are associated with increased numbers of eosinophils in the airways.

Cell cycle-dependent fluctuation of urokinase-type plasminogen activator, its receptor, and inhibitors in cultured bovine mammary epithelial and myoepithelial cells.

Bovine mammary epithelial (BME-UV1, clone E-T and BME-UV, clone E-T2) and myoepithelial (BMM-UV, clone m-T2) cell lines were used to study the modulation of cell-associated activity of urokinase-type plasminogen activator (u-PA), as well as mRNA transcripts of u-PA, its receptor (u-PAR), and inhibitors (PAI-1 and PAI-2) during the cell cycle. After release from a growth arrest accomplished by growth factor deprivation, the length of the cell cycle was determined as 19-21 h, with G1, S, and G2+M phases of 6-7, 7-9, and 5-6 h respectively. As the cell cycle progressed, accumulated cell-associated u-PA activity increased. Maximal activity occurred at the S/G2 boundary and decreased during the G2/M phases. All cell lines tested produced plasmin-specific inhibitor(s). Accumulation of u-PA mRNA peaked 3 h after stimulation into the growth cycle for m-T2 and E-T and during 3-6 h for E-T2 cells. Maximum levels of u-PAR mRNA were observed at 3 h for the E-T cell line, 6-9 h for E-T2 cells, and 3-9 h for m-T2 cells. The cell cycle distribution of the PAI-1 mRNA was similar to that of u-PA for both epithelial cell lines, while for m-T2 cells maximal accumulation of PAI-1 mRNA was detected at 3-9 h after growth initiation. The increase of PAI-2 mRNA transcription for m-T2 and E-T cells was detected at 3-6 h. The PAI-2 mRNA in E-T2 cells was under detectable levels. The data indicate that the expression of the constituents of the PA system in bovine mammary epithelial and myoepithelial cells is not cell type-dependent but is tightly connected to the phase of the cell cycle.

Usefulness of bronchoalveolar lavage for diagnosis of acute and persistent respiratory syncytial virus lung infections in guinea pigs.

To investigate whether bronchoalveolar lavage (BAL) fluid specimens can be used to diagnose acute and persistent respiratory syncytial virus (RSV) lung infections in guinea pigs, we tested BAL fluid and lung tissue specimens for evidence of viral infection, and compared BAL cytology between infected and uninfected animals. RSV-inoculated guinea pigs were studied during acute bronchiolitis (days 3 and 7 postinoculation), convalescence (Day 14 postinoculation), and persistent infection (Days 28 and 60 postinoculation), and were compared to the sham-infected control animals. BAL and lung tissue specimens were cultured for virus and tested by immunocytochemistry for viral protein. A reverse transcription-polymerase chain reaction (RT-PCR) method was used to test for viral nucleic acid. Total and differential BAL cell counts were compared between RSV-inoculated and control animals on each study day. In BAL specimens, replicating RSV was isolated by culture in one out of four of the animals on Day 3 postinoculation; immunocytochemistry for RSV antigens was positive in all virus-exposed animals from Days 3-14 postinoculation, and viral nucleic acid was detected by RT-PCR in one-fourth of the animals on Day 3 postinoculation. In contrast, replicating virus, viral antigens, and viral nucleic acid were documented in lung tissues obtained from the same RSV-infected animals on all study days. BAL specimens of RSV-inoculated animals contained more eosinophils on all study days (two-tailed P value < 0.01) compared to the controls. The results of this animal study demonstrate that BAL fluid is not useful for diagnosis of persistent RSV infection. However, BAL fluid may be helpful for the documentation of acute RSV lung infection when immunocytochemistry may provide a more accurate test for virus detection than RT-PCR or viral culture.

Iralukast Novartis AG.

Iralukast is an LTD4 and LTE4 antagonist under development by Novartis and in phase II clinical trials as a potential treatment for asthma [244117], [177071]. In a double-blind, placebo-controlled trial in 16 patients with mild to moderate asthma, a single 1.5 mg inhaled dose of iralukast reduced the incidence of exercise-induced bronchiospasm and was well-tolerated [272161]. Novartis has an agreement with Rhone-Poulenc Rorer to use its Ultrahaler delivery system for iralukast and phase II trials are underway [220836]. The agreement also involves the development of further related compounds and delivery systems.

Staphylococcus aureus stimulates urokinase-type plasminogen activator expression by bovine mammary cells.

Staphylococcus aureus commonly causes bovine mastitis, but bovine strains, unlike human isolates of S. aureus, do not produce the bacterial plasminogen activator, staphylokinase. By use of bovine mammary epithelial and myoepithelial cell lines, it was found that bovine S. aureus M60 and its culture filtrates induce a 3- to 10-fold increase in urokinase-type plasminogen activator activity in mammary cell-conditioned media and cellular lysates. Furthermore, transcytosis of S. aureus M60 across a mammary epithelial cell monolayer was significantly enhanced by the addition of bovine plasminogen and inhibited by aprotinin. These findings provide evidence that S. aureus M60 can trigger superactivation of host plasminogen activator production and may then utilize the plasminogen activator-plasmin(ogen) system to facilitate tissue invasion without producing staphylokinase.

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7.

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.

An in vitro approach to ruminant mammary gland biology.

This review discusses both fundamental and applied in vitro studies on ruminant mammary gland biology and summarizes progress made over the last decade in development of in vitro techniques to study growth, function and pathology of the mammary gland. The advantages and limitations of different in vitro systems are considered including explant cultures, primary cell cultures and immortalized lines of mammary-derived cells from cow, sheep and goat. The cell growth, differentiation and response to lactogenic hormones and growth factors are discussed as well as the relevance of the cell behavior in different culture conditions.

Cell cycle regulation of mammary epithelial cell detachment by Staphylococcus aureus.

The effect of Staphylococcus aureus on detachment of bovine mammary epithelial cells in culture was examined. Mammary epithelial cells became detached from fresh monolayers following a 3 h incubation in the presence of Staph. aureus M60. Two different procedures indicated that cell detachment coincided with the S-phase of the cell cycle. The roles of proteinases, toxins and Ca availability in inducing cell detachment were examined. Addition of the proteinase inhibitor phenylmethylsulphonyl fluoride (1 mM) to the culture medium prevented cell detachment. Addition of a combination of purified staphylococcal proteinases XVI and XVII-B to the culture medium of mammary epithelial cells induced cell detachment in the absence of Staph. aureus. Cell detachment may be caused by a staphylococcal proteinase. However, addition of Ca (10 mM) to the culture medium abolished Staph. aureus-induced cell detachment, despite the fact that proteinase activity was still apparently present. Isogenic mutants of Staph. aureus M60, expressing either alpha or beta toxins but not both, induced cell detachment, but to a lesser extent than the wild type. Thus, Ca and toxins play some role during cell detachment. Clones established from detached cells that were washed and replated showed the same susceptibility to Staph. aureus-induced cell detachment as the parental cells. This indicated that there is no subclone of mammary epithelial cells more sensitive to this effect.

Airway muscle stereology: implications for increased shortening in asthma.

Increased airway smooth muscle, resulting from either hyperplasia or hypertrophy, has been implicated as a cause of excessive bronchoconstriction in asthma despite the many methodologic limitations of studies to date. Our recent failure to demonstrate increased muscle volume in an asthmatic airway preparation having 3-fold greater shortening than nonasthmatic controls prompted us to reassess the quantity of muscle in asthmatic versus nonasthmatic airways. Smooth muscle was quantified in axially sectioned, 2nd- to 4th-generation bronchi, using standardized stereologic methods on high-magnification images of cross-sectional airway smooth muscle profiles in tissues from five asthmatic subjects and five nonasthmatic smokers. When data were normalized by total cross-sectional tissue area, no differences between the two groups (asthmatic versus nonasthmatic) were detected for the proportion of smooth muscle (3.45 +/- 0.81% versus 2.74 +/- 0.76%), extracellular matrix between muscle cells (1.65 +/- 0.46% versus 1.06 +/- 0.25%), or connective tissue within smooth muscle bundles (1.65 +/- 0.34% versus 1.53 +/- 0.59%). These methodologies for evaluating cross-sectional airway muscle in axial airway sections at high resolution provide no evidence of increased airway smooth muscle in asthmatic large airways, and suggest that differences in mechanical responses of asthmatic airways cannot be explained solely by the amount of smooth muscle.

Effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of Staphylococcus aureus to bovine mammary epithelial cells.

The effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of S. aureus. to bovine mammary epithelial cells was studied. Bovine erythrocytes and mammary epithelial cells were incubated with purified staphylococcal alpha and beta toxins and with culture supernatants from S. aureus M60 and two mutant strain that are negative for either the production of alpha (DU5789 alpha-) or beta (DU5846 beta-) toxin. Lysis of bovine erythrocytes was due primarily to beta toxin. Alpha toxin increased the lysis of bovine erythrocytes by purified beta toxin, but the presence of alpha toxin in culture supernatants from S. aureus did not increase the lysis of bovine erythrocytes. Purified beta toxin was cytotoxic to mammary secretory epithelial cells, but to a lesser extent than alpha toxin. Together they exhibited an additive effect on mammary epithelial cells. Inactivation of the alpha toxin-gene of S. aureus M60 decreased the cytotoxic effect on mammary epithelial cells to a greater extent than the inactivation of the beta toxin-gene. Also, the relative percentages of DU5789 alpha- and DU5846 beta- adhering to mammary cell monolayers, the number and size of colonies and the number of infected epithelial cells decreased. This in vitro study showed that beta toxin damages bovine mammary secretory epithelial cells, increased the damaging effects of alpha toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of S. aureus.

Collagenase increases shortening of human bronchial smooth muscle in vitro.

The recent demonstration of increased shortening of asthmatic airway smooth muscle could result from increased contractility of the muscle itself or from a decreased load that must be overcome by the smooth muscle to shorten. To evaluate the role of smooth muscle-associated extracellular matrix in limiting smooth muscle responses, we investigated the effect of collagenase on the mechanical responses of human bronchial smooth muscle strips. Contractile responses of second- to fourth-generation bronchi were evoked by electrical field stimulation, and measurements of length and tension were made at preloads between 0 and 2.5 g. The passive tension, active isometric, and isotonic responses were obtained at each preload before and after 90 min of incubation with 20 U/ml collagenase. Shortening to 10(-4) M histamine was also measured. Collagenase treatment caused a significant decrease in passive tension, with the most pronounced change occurring below Lmax (optimal length for force generation). At optimal lengths for shortening, the degree of shortening, expressed as a percentage of starting length, increased significantly from 8.9 +/- 1.4% before to 13.8 +/- 2.9% after collagenase treatment (n = 7) (p < 0.02). Shortening to histamine also increased from 14.3 +/- 2.5% before to 23.5 +/- 5.3% after collagenase treatment (n = 7) (p < 0.02). These results suggest that degradation of the collagenous matrix surrounding muscle in the airway wall reduces the load on the muscle, allowing increased smooth muscle shortening.