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March 28, 2009: Bozos for Eddie Bo
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Listener comments! | |
Sat. 3/28/09 6:21am
steve krinsky:
G'morning. Anybody out there? | |
Sat. 3/28/09 6:22am
ah now i remember youre the dude formerly known as the dude who is just back from austin. part of that show is on a disc and played all the time here. thanks . but what have you done for me lately? well how bout some more info on the sugar magnolia boot? then i sit back, shut up, n listen... ratty | |
Sat. 3/28/09 6:31am
Morning. Almost went back to bed with your first two picks, but it's getting better. | |
Sat. 3/28/09 6:34am
good morning steve. i can successfully confirm that i am here. | |
Sat. 3/28/09 6:36am
Guten Morgen from Germany. - Sgt. Brock | |
Sat. 3/28/09 6:36am
Zach in Philly:
Up and at 'em early today. Nice first set. Went well with making breakfast and coffee. I have to give a second woof for the Sugar Magnolia bootleg. Nice:-) | |
Sat. 3/28/09 6:43am
We've been up a while here in Scotland, on our second cup of coffee. Good to hear you again. Just what I need to set me up for another Saturday. Yee-haw! | |
Sat. 3/28/09 6:47am
hello! just tuning in from mexico great tune, the way young lovers do | |
Sat. 3/28/09 6:50am
steve krinsky:
Gee, nice to hear from you all. I was too frazzled this week to send out an email saying I was on. And it's early: this show will sound better (to me) when I listen to it later on the archive. But I got the groove now. | |
Sat. 3/28/09 6:53am
Zach in Philly:
Thanks for the note about the email. I was hoping I hadn't fallen off the list. Lovin' the title and the tune "Bone Again with Bonerama"! | |
Sat. 3/28/09 7:21am
hi, now i can see where 'yello' got those groovy beats from! | |
Sat. 3/28/09 7:27am
Zach in Philly:
"Wilson Rag" is so where my head is at this morning. | |
Sat. 3/28/09 7:28am
steve k, and if chip's back to bed he's missing an excellent show, his loss! notwithstanding the first two! | |
Sat. 3/28/09 7:33am
har-you and getatchew mekurawere were a bit special in my book. | |
Sat. 3/28/09 7:49am
Zach in Philly:
Paul, given your travels (per comments in Bill Mac's show), I presume you've been to Ethiopia? | |
Sat. 3/28/09 7:51am
Zach in Philly:
Gotta love those vibes on "Mambalypso":-) | |
Sat. 3/28/09 7:59am
hi zach, decorating in other room! no, only cairo i'm afraid. cannot believe etheopian getatchew mekura put an album out in '72 and nothing until 2006! | |
Sat. 3/28/09 8:18am
Zach in Philly:
Paul, that is a stretch. Happy decorating! Krinsky, a little sacharine, sure, but still oh so yummy:-) | |
Sat. 3/28/09 8:27am
Zach in Philly:
Yay for the Flatlanders! Love Joe Ely and the gang! | |
Sat. 3/28/09 8:28am
Are you going to the Flatlander's show at B.B. King's on the 9th? | |
Sat. 3/28/09 8:33am
Zach in Philly:
Chip, I was aloof to the fact that they were playing that close. Don't see myself making up there per work being so crazy these days. Saw them a few years ago... good stuff. Thanks for pointing it out. | |
Sat. 3/28/09 8:35am
steve krinsky:
Probably too expensive but we'll see. | |
Sat. 3/28/09 8:50am
Zach in Philly:
I'm new to the Dexter Romweber Duo but I'm diggin' their sound. That's kewl about Cervenka on vocals on the first track. | |
Sat. 3/28/09 8:52am
rushed back to turn up nick cave, but twas not, turned it up anyway, was nice. | |
Sat. 3/28/09 8:57am
nice show and your comments to your selections nice too, steve | |
Sat. 3/28/09 10:26am
mike tp:
steve a real treat to see you up at bat again. was a reg list of your show on fri rite? good weekend bro steve. mike from the njtpike | |
Sat. 3/28/09 3:08pm
Steve Krinsky rules. That is all. | |
Sat. 3/28/09 4:21pm
James Talley is putting out CDs on his own label, including two albums n 2008. Check out www.jamestalley.com | |
Sat. 3/28/09 7:17pm
The Blackhole of Clifton:
You played the Grateful Dead. Normally, you would have to be sent on a hunting trip with Dick Cheney for that crime against humanity. Fortunately for you, the rest of the show gets an A+. | |
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Does the market discipline banks? New evidence from the regulatory capital mix
Although bank capital regulation permits a bank to choose freely between equity and subordinated debt to meet capital requirements, lenders and investors view debt and equity as imperfect substitutes. It follows that the mix of debt in regulatory capital should isolate the role that the market plays in disciplining banks. I document that since the Federal Deposit Insurance Corporation Improvement Act of 1991 (FDICIA) reduced the ability of the FDIC to absorb losses of subordinated debt investors, the mix of debt has had a positive effect on the future outcomes of distressed banks, as if the presence of debt investors has worked to limit moral hazard. To mitigate concerns about selection, I use the variation across banks in the mix of debt in capital generated by cross-state variation in state corporate income tax rates. Interestingly, instrumental variables (IV) estimates document that selection problems are indeed important, but suggest that the benefits of subordinated debt are even larger. I conclude that the market may play a useful direct role in regulating banks.
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Date of creation: | 2006 |
Handle: | RePEc:fip:fednsr:244 |
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References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Cook, Douglas O & Spellman, Lewis J, 1994. "Repudiation Risk and Restitution Costs: Toward Understanding Premiums on Insured Deposits," Journal of Money, Credit and Banking, Blackwell Publishing, vol. 26(3), pages 439-459, August.
- Adam B. Ashcraft, 2008.
"Are Bank Holding Companies a Source of Strength to Their Banking Subsidiaries?,"
Journal of Money, Credit and Banking,
Blackwell Publishing, vol. 40(2-3), pages 273-294, 03.
- Adam B. Ashcraft, 2004. "Are bank holding companies a source of strength to their banking subsidiaries?," Staff Reports 189, Federal Reserve Bank of New York.
- Flannery, Mark J & Sorescu, Sorin M, 1996. " Evidence of Bank Market Discipline in Subordinated Debenture Yields: 1983-1991," Journal of Finance, American Finance Association, vol. 51(4), pages 1347-1377, September.
- Avery, Robert B & Belton, Terrence M & Goldberg, Michael A, 1988. "Market Discipline in Regulating Bank Risk: New Evidence from the Capital Markets," Journal of Money, Credit and Banking, Blackwell Publishing, vol. 20(4), pages 597-610, November.
- Robert DeYoung & Mark J. Flannery & William W. Lang & Sorin M. Sorescu, 1998. "The informational advantage of specialized monitors: the case of bank examiners," Working Paper Series WP-98-4, Federal Reserve Bank of Chicago.
- Douglas D. Evanoff & Larry D. Wall, 2000.
"Subordinated debt and bank capital reform,"
FRB Atlanta Working Paper
2000-24, Federal Reserve Bank of Atlanta.
- Douglas D. Evanoff & Larry D. Wall, 2000. "Subordinated debt and bank capital reform," Working Paper Series WP-00-7, Federal Reserve Bank of Chicago.
- Flannery, Mark J, 1998. "Using Market Information in Prudential Bank Supervision: A Review of the U.S. Empirical Evidence," Journal of Money, Credit and Banking, Blackwell Publishing, vol. 30(3), pages 273-305, August.
- Julapa Jagtiani & George Kaufman & Catharine Lemieux, 1999. "Do markets discipline banks and bank holding companies? evidence from debt pricing," Emerging Issues, Federal Reserve Bank of Chicago, issue Jun.
- Goyal, Vidhan K., 2005. "Market discipline of bank risk: Evidence from subordinated debt contracts," Journal of Financial Intermediation, Elsevier, vol. 14(3), pages 318-350, July.
- Diana Hancock & Myron L. Kwast, 2001.
"Using subordinated debt to monitor bank holding companies: is it feasible?,"
Finance and Economics Discussion Series
2001-22, Board of Governors of the Federal Reserve System (U.S.).
- Diana Hancock & Myron Kwast, 2001. "Using Subordinated Debt to Monitor Bank Holding Companies: Is it Feasible?," Journal of Financial Services Research, Springer;Western Finance Association, vol. 20(2), pages 147-187, October.
- Billett, Matthew T. & Garfinkel, Jon A. & O'Neal, Edward S., 1998. "The cost of market versus regulatory discipline in banking," Journal of Financial Economics, Elsevier, vol. 48(3), pages 333-358, June.
- anonymous, 1999. "Using subordinated debt as an instrument of market discipline," Staff Studies 172, Board of Governors of the Federal Reserve System (U.S.).
- Park, Sangkyun & Peristiani, Stavros, 1998. "Market Discipline by Thrift Depositors," Journal of Money, Credit and Banking, Blackwell Publishing, vol. 30(3), pages 347-364, August.
- Donald P. Morgan & Kevin J. Stiroh, 1999. "Bond market discipline of banks: is the market tough enough?," Staff Reports 95, Federal Reserve Bank of New York.
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subset: commoncrawl


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Submitted by Editorial Team on October 13, 2011 11:39am
Jack Williams grave at at Terendak Military Cemetery, Malaysia, 2009.
Find out more about this cemetery, including images and cemetery plans (CWGC). Note: New Zealanders wishing to visit this cemetery should contact Margaret Marks at Manatū Taonga Ministry for Culture & Heritage prior to travel.
Margaret Marks, Manatū Taonga Ministry for Culture & Heritage


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Rick has extensive expertise in the automotive industry having worked with both automakers and suppliers with their SEC reporting matters, preparation of financial statements prepared in accordance with international accounting standards, and development of international standard governance policies and practices. He has also assisted clients develop and implement policies and practices related to Section 404 of the Sarbanes-Oxley Act.
Rick spent two years in PwC's London office and later served as the partner-in-charge of the firm's Russian audit and business advisory practice, where he was responsible for people development and the development of the Russian audit practice. Rick also served as the global engagement partner for several major Russian international entities during Russia's privatization period. Upon his return to the U.S. and until his relocation to Detroit, he served as the office managing partner in Peoria, Illinois, USA. He has also served as a member of the firm's strategy committee and extended leadership team in 2006 and 2007.
Rick earned a Master of Arts degree in accounting from Illinois Wesleyan University. His professional affiliations include: American Institute of Certified Accountants, Michigan Association of Certified Public Accountants, and Illinois CPA Society.


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Movies on the Radio Live: A Slate Culture Gabfest Video Archive
Video Archive
Tuesday, May 15, 2012
WQXR's Movies on the Radio meets Slate's Culture Gabfest: On Thursday, May 31 at 7 pm in The Greene Space, host David Garland spoke with Slate's Dana Stevens, Julia Turner and Stephen Metcalf about music and the movies, including a conversation about notable recent and upcoming releases.
Special features included a "Film Scoring 101" segment and a conversation with Academy Award-winning composer Howard Shore, who participated by Skype from New Zealand.
Our discussion is archived below in edited form and will appear on an upcoming installment of the Slate Culture Gabfest.
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Comments [2]
Great interview with Howard Shore! You should also bring his nephew Ryan Shore on the show. He's a tremendously gifted film composer as well, and he worked with Howard earlier in his career. He has a site at http://www.ryanshore.com
On the next show about movie music on the radio....would you consider doing a program on the music of Max Steiner?
Thank you,
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Hi all,
I'm fairly new to concepts of Java Libraries, so forgive me if this is obvious.
I'm developing an application to be distributed to users as a .jar file and would like to use JFreeChart as part the development. Will users need to download/install/configure the JFreeChart library in order to use my .jar? I'd really like to leverage this tool, and would very much like to avoid coding all my charts in swing. Any constructive input is highly appreciated. Thanks!


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After The Air Raids
An insider's account of a startling— and still controversial—investigation of the Allied bombing of Germany
April/may 1981 | Volume 32, Issue 3
In truth, a little earlier we had brought a high official of the Auto Union automobile company to Bad Nauheim for interrogation. As the Russians were about to occupy the part of Germany where he lived, he had asked our colonel for an Army vehicle to move his more valuable possessions into the American zone on the promise of a division of the salvaged goods. The colonel had then shown his loot to a visiting officer who, in civilian life, had worked for Fiorello La Guardia in New York City and was thus a ferocious enemy of graft. It was he who had sent in the military police. We never saw our man again.
As the Big Three meeting approached in Potsdam, couriers came to Bad Nauheim looking for information and guidance on the German economy. We obliged, and George Ball decided that we should attend. When I pointed out that we had not been invited, George said that if we allowed hurt feelings to govern our actions, we would only compound the error. With Paul Nitze we went to Berlin, out to Babelsberg, the site of the conference, were admitted to the compound without question when we explained that we had come to attend the meeting, and began operations with an excellent lunch at the senior officials' mess. I joined Isador Lubin, who was developing the position on reparations and whose people we had previously provided with information on the surviving plant. History was not altered by our brief presence. But it was something to attend a summit on one's own invitation.
George Ball had told me in earlier weeks of the existence of the atomic bomb. Surveying the old-fashioned destruction in Germany, I had deeply hoped it would not work. Now it came and also the end of the Japanese war. The effect on the morale of the men I had sought out from the Army for the survey was immediate. Until then hard, diligent work meant avoiding combat in the Pacific. Now the same hard work could mean going with us to Japan.
But by now our overall economic report, a large, competent, and literate document, was nearing completion. When it was finished, we closed the headquarters in Bad Nauheim and went to London and, after reviewing it further there, returned home. George Ball slept all the way from Prestwick to Iceland to Newfoundland to New York. This he did by combining a large amount of whisky with a substantial dose of sleeping pills. Contrary to all medical expectation, he arrived greatly refreshed.
Back in Washington in Army Air Forces Annex No. 1 at Gravelly Point, Virginia, not far from National Airport, we found that the secretariat of the survey had written a short summary report which was as the Air Force would have wished it. Our patiently gathered data on the disastrous failures of strategic bombing were extensively ignored. There were no serious failures mentioned. Even the successes were lost in a story of general success. The question had political importance, for part of the claim of the Air Force to independence from the Army was thought to depend on the wartime achievement of the bombers. Ball and I prepared to contest the report and somehow to get it rewritten. It would be dreary work—piecemeal change, word by word.
Then Henry Alexander intervened. He was distressed less by the content of the summary than by its abysmal level of literacy. He asked me to prepare a new and readable draft. I took a typewriter to the Cosmos Club in Washington and wrote exactly what we wanted said with something more for bargaining thrown in.
My manuscript, in turn, led to a war of attrition, one that lasted for days in the dreary buildings by the airport. But now the basic draft was mine; it fell to the opposition to struggle, sentence by sentence, for modification and retraction. General Anderson led the attack. I defended it with, as I only later came to reflect, a maximum of arrogance and a minimum of tact. George Ball, who had resumed his law practice as counsel for Jean Monnet, nonetheless came over to help. Henry Alexander, not concealing his distress at the conflict, presided and sought to mediate. At some point I said to Orvil Andersen, "General, this is just a matter of intellectual honesty."
He replied, "Goddamn it, Ken, you carry intellectual honesty to extremes."
By giving way on nonessentials, we kept the basic case. German war production had, indeed, expanded under the bombing. The greatly heralded efforts, those on the ball-bearing and aircraft plants for example, emerged as costly failures. We had a minor struggle over the B-17, the Flying Fortress itself. A rugged vehicle that brought its occupants home if anything could, it was much respected by all concerned. But it had been designed to fly without escort and to protect itself and its neighbors in the same flight with its own weapons. This meant a heavy load of gunners, guns, and ammunition, and in consequence, it could carry no great weight of bombs. After the Schweinfurt debacle it became clear that the Fortress could not, in fact, defend itself, that escort aircraft would be required. It was still arranged for its great load of men and weaponry; thus it was now a bad design. General Anderson met my efforts to make this point with massive indignation. His exact words are still with me: "You are insulting a great airplane." On this he won.


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The Horse Forum
- English Riding
- - Schooling Exercises?
Schooling Exercises? So I'm looking for some schooling exercises to work with on Murray. Really, I have no clue what to do with him. A normal day is walk and trot, circles and crossing the arena. Sometimes a leg yield here and there. Always in the indoor. Now that it's summer and I have more time to go the the barn (I no longer work at my hotel, just on call) I would like to work outside with him and now that I know he can canter, I would like to bring that into working with him. Any ideas or suggestions? I hate to do the same boring stuff with him. Thank you! |
What kind of "stuff" do you have laying around?? Make an obstacle course with barrells and cavalettis, teach him to cross tarps, open gates, go on trails, walk over logs, make a square with cavalettis and add a pole to each corner to make like a star, thats a good steering formation. |
Maybe serpentines, figure 8's half-voltes, just ride randomly around the arena, and make sure he is always ready to listen to you :) |
Ride squares. Pick up an energetic trot, ride straight, apply a balancing half halt and make a 90 degree turn. (or as much as your horse can do without becoming unbalanced) Repeat until you make a square. If squares are too much, ride octagons instead of circles. Insert 8 distinct points and half halt before each one before changing his line of travel to complete that part of the Octagon. Ride around the ring but stay 2 tracks off the rail. This takes both arms and both legs, really tests your power steering since horses like to be on the rail. This one's great for getting their attention. For canter work, play trot-canter-trot. Sounds like you already have a good working trot going. Pick specifice places in the ring, preferably corners and ask for the canter. If he speeds up instead, come back to a balanced trot before asking again. Once you get the canter, let him canter for as long as he can do it in a balanced fashion. If he breaks, speeds up or flattens out, come back to a good working trot. He may get fussy and try to break into the canter again, but stay with the trot until YOU have the trot you want. Only then, pick another corner and canter again. At first it might help to ask for canter in the same spot until he understands the canter cue. After that, vary the places where you ask or he will quickly begin to anticipate and try to canter whenever you hit that spot. I've got about a zillion more since I ride alone and get bored easily. I'll post more later. |
Here's a quick list of exercises that I do with the horses I ride. It all depends on how far along the horse is in his/her training though. Shoulder-in/Shoulder-fore Leg yielding 20m, 15m and 10m circles Serpintines/figure-eights Simple changes Collecting and extending the gait Transitions Turns on the forehand Halting Straight lines Loops Counter canter A lot of these have many different ways in which you can do them. For example, may you're working in a rectangle area. You can do shoulder-in down the long side then stretch on the short side and repeat. For leg yilding you and do a teardrop shape back to the wall or you can leg yield to the centerline, then back to the wall, etc. So you can work on the same exercise in different ways. Another thing I also like to do is hillwork, trail rides, endurance rides, and bareback rides. We have a racetrack on the property that I sometimes just hand gallop around. Oh and not to mention lunging/round penning. It's perfect for the days when you don't have time to ride because you don't even need to tack up the horse. I think it's great to mix things up so that your horse doesn't get bored. |
There are some great books about full of schooling exercises. They range from novice to elementary. I used to use them all the time but can't think of the best of hand |
I also struggle with finding stuff to do with Lacey! =) One of my favorite things to do with Lacey is to guide her completely with my legs. I basically drop my reins and have her do stuff at a walk (not comfortable enough to do it at the trot yet) just by telling her with my seat and legs. It seems to have really made her much more sensitive to leg cues and it's pretty fun to be able to not even really have to use the reins ever if I don't want to. =P |
Yes Wallaby, that's a good one. I do that too at the end of my rides to see how much he's really listening to me. We just started doing it at the trot with some success. That's a fun exercise. I like seeing how little I can do with my seat and still have him respond. |
Great ideas everyone! Keep them coming! Thanks a bunch! |
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We are the Valdivia family. We are retired educator and maintence worker from Fresno, Ca. We have been renting our apartment and home to university students from around the world, who have come to Punta Santiago to do research on Monkey Island/Cayo Santiago.
We love to travel, and as travelers, we know the importance of finding a safe, comfortable and affordable place to stay during our travels. Save some money on the hotel fees, and stay with us.


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Engineered silica nanoparticles act as adjuvants to enhance allergic airway disease in mice
© Brandenberger et al.; licensee BioMed Central Ltd. 2013
Received: 24 January 2013
Accepted: 13 June 2013
Published: 1 July 2013
With the increase in production and use of engineered nanoparticles (NP; ≤ 100 nm), safety concerns have risen about the potential health effects of occupational or environmental NP exposure. Results of animal toxicology studies suggest that inhalation of NP may cause pulmonary injury with subsequent acute or chronic inflammation. People with chronic respiratory diseases like asthma or allergic rhinitis may be even more susceptible to toxic effects of inhaled NP. Few studies, however, have investigated adverse effects of inhaled NP that may enhance the development of allergic airway disease.
We investigated the potential of polyethylene glycol coated amorphous silica NP (SNP; 90 nm diameter) to promote allergic airway disease when co-exposed during sensitization with an allergen. BALB/c mice were sensitized by intranasal instillation with 0.02% ovalbumin (OVA; allergen) or saline (control), and co-exposed to 0, 10, 100, or 400 μg of SNP. OVA-sensitized mice were then challenged intranasally with 0.5% OVA 14 and 15 days after sensitization, and all animals were sacrificed a day after the last OVA challenge. Blood and bronchoalveolar lavage fluid (BALF) were collected, and pulmonary tissue was processed for histopathology and biochemical and molecular analyses.
Co-exposure to SNP during OVA sensitization caused a dose-dependent enhancement of allergic airway disease upon challenge with OVA alone. This adjuvant-like effect was manifested by significantly greater OVA-specific serum IgE, airway eosinophil infiltration, mucous cell metaplasia, and Th2 and Th17 cytokine gene and protein expression, as compared to mice that were sensitized to OVA without SNP. In saline controls, SNP exposure did cause a moderate increase in airway neutrophils at the highest doses.
These results suggest that airway exposure to engineered SNP could enhance allergen sensitization and foster greater manifestation of allergic airway disease upon secondary allergen exposures. Whereas SNP caused innate immune responses at high doses in non-allergic mice, the adjuvant effects of SNP were found at lower doses in allergic mice and were Th2/Th17 related. In conclusion, these findings in mice suggest that individuals exposed to SNP might be more prone to manifest allergic airway disease, due to adjuvant-like properties of SNP.
Silica nanoparticles Adjuvant potential Allergic airway disease Th2/Th17 response Murine ovalbumin modelBackground
Engineered nanoparticles (NP) have unique and desirable functional properties, due to their extremely small size (≤ 100 nm). As a result, NP have an enormous economic potential and are rapidly being introduced into commercial products, such as textiles, cosmetics and food packaging [1]. Recent reviews [2, 3], however, have presented compelling evidence that occupational exposure during the production of NP might have significant health risks. Due to their small size and potential for airborne dispersion, inhalation of NP may be a plausible route of human exposure in the workplace. Under certain conditions, inhalation of NP could potentially exacerbate or contribute to the onset of common respiratory diseases, such as chronic bronchitis, asthma or allergic rhinitis [3, 4].
Asthma and other allergic airway diseases are a worldwide health problem. In the United States approximately 26 million people suffer from asthma (National Health Interview Survey, National Center for Health Statistics, CDC, 1980–2009). Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness (AHR), increased production of immunoglobulin isotype IgE, airway inflammatory cell infiltrates of eosinophils and CD4+ T helper type 2 (Th2) lymphocytes, mucus hypersecretion, and airway remodeling (e.g., epithelial mucous cell metaplasia, intramural interstitial fibrosis).
Various factors have been recognized to exacerbate asthma such as indoor and outdoor allergens, tobacco smoke and air pollution [5]. In urban air pollution, particulate matter (PM) is a well-recognized risk factor, causing exacerbation of asthmatic symptoms (e.g., dyspnea, airway constriction, airway mucus hypersecretion). In addition, results of recent animal toxicology studies have demonstrated that exposure to intranasally aspirated ultrafine PM (UFP; < 100 nm) during sensitization with ovalbumin (OVA), a commonly used experimental allergen, can enhance the development and severity of allergic airway disease in mice [6, 7]. UFP acted as adjuvants to boost the secondary immune response upon subsequent OVA challenge. In these studies, the adjuvant potential of UFP was closely associated with their oxidant potential [7]. These laboratory findings 1) suggest a plausible biological mechanism for epidemiological reports that PM exposure caused enhancement of morbidity in humans with allergic airway disease [8] and 2) provide a reproducible in vivo murine model to test the potentially adverse adjuvant effects of other NP, such as engineered NP that have distinctive physical and chemical characteristics.
In the present study, we used an OVA-induced murine model of asthma to test the hypothesis that engineered amorphous silica nanoparticles (SNP) may act as inhaled adjuvants to enhance allergic airway disease. SNP are used as additives to cosmetics, drugs, printer toners, varnishes and food [9]. It is well known that chronic inhalation exposure of coarse-sized (2.5 to 10 μm), crystalline silica particles can lead to a debilitating fibrotic condition known as pulmonary silicosis [10]. In contrast, synthetic amorphous silica particles are thought to be much less toxic to the lung. Inhalation of engineered amorphous silica causes only minimal and transient pulmonary inflammation in laboratory rodents [11, 12] and no fibrosis of the lungs [13, 14] as compared to crystalline silica particles. Few toxicology studies have been conducted to examine the adverse effects of inhaled amorphous SNP and, to the best of our knowledge, no studies have been designed to investigate the potential of these NP to act as adjuvants to enhance the development or exacerbation of allergic airway disease.
Grafting amount and size of SNP after different steps of synthesis: plain SNP (SNP) to amine-modified SNP (aSNP), to alkyne-modified (aaSNP) and to final PEG-coated SNP
Sample | Weight Loss (%) a | Grafting amount (t, mmol/g) a | Grafting density (group/nm 2) a | Size (nm) b |
plain SNP | 1.7g | 1.9h | 8.1h | 30c |
aSNP | 3.6 | 0.63 | 2.7 | 103d |
aaSNP | 2.4 | 0.29 | 1.2 | 126e |
PEG-coated SNP | 3.6 | 0.15 | 0.7 | 90c |
OVA induced allergic airway disease
Adjuvant effects of SNP
The exposure of SNP during OVA sensitization (SNP/OVA-mice) resulted in an exacerbation of allergic airway disease after OVA challenge, hence implying an adjuvant effect of SNP in the development of asthma. The adjuvant effect observed in SNP/OVA-mice increased with SNP dose (0, 10, 100, 400 μg SNP/animal).
Total BALF cells were significantly increased up to 2.5-fold in SNP/OVA-mice compared to OVA-mice (135,833 ± 21,337, 257,917 ± 48,958, 323,333 ± 40,284 and 345,833 ± 66,476 for OVA- and SNP/OVA-mice with SNP doses of 10, 100 and 400 μg, respectively). The adjuvant effect was consistently demonstrated in the number of BALF eosinophils. Significant, dose-dependent increases in lavaged eosinophils were present in SNP/OVA-mice compared to OVA-mice (11-, 14- and 25-fold increases for 10, 100 and 400 μg SNP/OVA co-exposures respectively; Figure 2C). A significant increase was also observed for BALF lymphocytes in SNP/OVA-mice relative to OVA-mice (2.9-, 5.0- and 4.4-fold for 10, 100 and 400 μg SNP/OVA, respectively; Figure 2D). No adjuvant-related increases were found for macrophages and neutrophils (Figure 2A and B).
Serum OVA-specific IgG1 antibody levels (Figure 3A) were significantly elevated (1.6-fold) in 100 and 400 μg SNP/OVA-mice compared to OVA-mice. A significant increase in OVA-specific IgE antibodies levels was also detected in 400 μg SNP/OVA-mice (Figure 3B), which was not present in any other exposure group. A systemic allergic response was observed only in mice exposed to SNP during OVA sensitization at the highest SNP dose.
Intracellular mucosubstances in epithelial cells lining the proximal axial airway (G5) increased with increasing SNP dose in the SNP/OVA-mice (Figure 5A-E). There was a significant 6 to 7-fold increase in 100 and 400 μg SNP/OVA-mice compared to OVA-mice. Gene expression analysis of Muc5ac and Clca3 revealed 5.5-fold (p < 0.001) overexpression of Muc5ac in 400 μg SNP/OVA as compared to OVA-mice (Figure 5F).
SNP co-sensitization induces a Th2/Th17 cytokine response in OVA-mice
To investigate the mechanisms of SNP adjuvancy in the development of allergic airway disease, a gene expression array of lung tissue and BALF cytokine expression analysis were performed. For gene expression analysis, 96 genes were chosen as part of different gene clusters (i.e. chemokines, cytokines and other immune responsive genes, mucus/surfactant production and secretion, growth factors and cell cycle, oxidative stress and redox response as well as transcription factors). A detailed list of all genes is available in the supplemental information (Additional file 1: Table S1) as well as an expression heat map of all analyzed genes (Additional file 1: Figure S5). For BALF cytokine analysis, a panel of acute phase, Th1, Th2 and Th17 cytokines and chemokines was used, including interleukin-1β (IL1β), IL2, IL4, IL5, IL6, IL 13 and IL17, keratinocyte chemoattractant (KC; CXCL1), macrophage inflammatory protein 1α (MIP-1α; CXCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), tumor necrosis factor α (TNFα), and interferon γ (IFNγ).
Detailed gene expression analysis of responsive genes
Gene symbol | Gene alias | Gene function | Saline/0 μg NP | Saline/400 μg NP | OVA/0 μg NP | OVA/400 μg NP |
Cxcl1 | KC | Neutrophil chemokine | 1.00+/-0.05 | 2.80+/-0.78a | 2.48+/-0.28b | 6.29+/-0.38a,b |
Cxcl2 | MIP-2 | PMN chemokine | 1.00+/-0.08 | 2.85+/-0.75a | 4.22+/-0.76b | 8.77+/-0.99a,b |
Cxcl10 | IP10 | T-cell chemokine | 1.00+/-0.05 | 1.17+/-0.05 | 14.32+/-3.95b | 44.84+/-4.29a,b |
Ccl2 | MCP-1 | Monocyte and T-cell chemokine | 1.00+/-0.08 | 1.50+/-0.28 | 3.66+/-0.86b | 13.84+/-1.71a,b |
Ccl8 | MCP-2 | Monocyte chemokine | 1.00+/-0.08 | 1.56+/-0.25 | 15.48+/-5.22b | 105.12+/-32.47a,b |
Ccl11 | Eotaxin | Eosinophil chemokine | 1.00+/-0.05 | 1.32+/-0.11 | 2.50+/-0.71 | 11.47+/-2.84a,b |
Itln1 | Itlna | Pathogen-associated molecular pattern recognition | 1.00+/-0.25 | 4.95+/-1.78a | 6.14+/-0.95b | 38.65+/-13.92a,b |
Irg1 | Irg1 | Toll like receptor response | 1.00+/-0.14 | 4.70+/-1.15a | 15.45+/-4.36b | 154.18+/-16.97a,b |
Chi3l3 | YM1 | Chitinase-like protein | 1.00+/-0.07 | 1.01+/-0.12 | 1.30+/-0.26 | 1.94+/-0.28b |
Chi3l4 | YM2 | Chitinase-like protein | 1.00+/-0.35 | 4.61+/-2.98 | 9.13+/-3.96 | 33.10+/-12.56b |
Saa3 | Saa3 | Acute phase protein | 1.00+/-0.09 | 4.51+/-1.85 | 65.9+/-12.7b | 157.88+/-19.38a,b |
Tnfα | TNFα | Acute phase response cytokine | 1.00+/-0.12 | 1.69+/-0.17a | 2.20+/-0.23b | 3.36+/-0.30b |
Il6 | IL6 | Acute phase response cytokine | 1.00+/-0.13 | -1.14+/-0.13 | 2.00+/-0.40b | 5.60+/-0.96a,b |
Il1β | IL1β | Acute phase response cytokine | 1.00+/-0.13 | 1.01+/-0.05 | 1.26+/-0.03 | 1.71+/-0.13b |
Il1rn | IL1rn | IL1 receptor antagonist | 1.00+/-0.04 | 1.13+/-0.07 | 1.70+/-0.23 | 4.62+/-0.71a,b |
Il2 | IL2 | Th1 cytokine | 1.00+/-0.06 | 1.06+/-0.07 | 1.57+/-0.24b | 1.37+/-0.09 |
Ifnγ | IFNγ | Th1 cytokine | 1.00+/-0.08 | -1.06+/-0.14 | 1.67+/-0.28b | 2.31+/-0.30b |
Il4 | IL4 | Th2 cytokine | 1.00+/-0.15 | 1.01+/-0.12 | 1.40+/-0.26 | 3.77+/-0.77a,b |
Il5 | IL5 | Th2 cytokine | 1.00+/-0.10 | -1.20+/-0.18 | 2.39+/-0.59 | 7.17+/-1.44a,b |
Il13 | IL13 | Th2 cytokine | 1.00+/-0.10 | 1.34+/-0.39 | 6.80+/-2.96b | 44.23+/-12.67a,b |
Retnla | Fizz 1, HIMF | Th2 suppressor | 1.00+/-0.12 | 1.32+/-0.37 | 6.88+/-1.78b | 21.90+/-3.98a,b |
Il17a | IL17A | Th17 cytokine | 1.00+/-0.16 | 2.52+/-0.69 | 8.34+/-2.32b | 46.24+/-6.02a,b |
Foxp3 | Foxp3 | T-reg transcription factor | 1.00+/-0.08 | 1.49+/-0.18 | 2.08+/-0.19b | 3.98+/-0.33a,b |
Il10 | IL10 | T-reg cytokine | 1.00+/-0.09 | 1.09+/-0.17 | 2.97+/-0.76b | 8.22+/-2.01a,b |
Muc5ac | Muc5ac | Mucin production | 1.00+/-0.19 | 1.61+/-0.61 | 4.72+/-1.44b | 25.97+/-4.70a,b |
Clca3 | Gob5 | Ion channel, mucus secretion | 1.00+/-0.39 | 2.52+/-1.42 | 6.16+/-2.26 | 16.72+/-3.42b |
Clca2 | Clca2 | Ion channel, fibrosis development | 1.00+/-0.12 | 1.40+/-0.16 | 3.25+/-0.78b | 7.96+/-1.20a,b |
Mmp12 | Mmp12 | Macrophage elastase | 1.00+/-0.08 | 3.06+/-0.81 | 1.83+/-0.22 | 7.91+/-0.59 |
Timp1 | Timp1 | Mmp inhibitor | 1.00+/-0.05 | -1.00+/-0.10 | 3.73+/-0.87b | 12.40+/-1.68a,b |
Effects of SNP in SAL-mice
In animals that were exposed to SNP only (SNP-mice), there was a significant dose-dependent increase in neutrophils at 100 and 400 μg SNP with 3,210 ± 1,583 and 5,400 ± 1,999 neutrophils/mL BALF respectively, compared to non at 0 μg SNP (Figure 2B). No increases in BALF eosinophils and lymphocytes were detected (Figure 2C and D) and SNP exposure was not associated mucus airway remodeling (Figure 5E and F).
SNP exposure by itself, without OVA, resulted in significant gene expression of neutrophil chemokines Kc and Mip-2, as well as Tnfα, immunoresponsive gene 1 (Irg1) and intelectin1 (Itln1) (Table 2). In addition, there were significant increases of BALF chemokines KC and MIP-1α in SNP-mice (100 and 400 μg SNP) as compared to SAL-mice (Figure 8). These findings corresponded to the increase in BALF neutrophils in 100 and 400 μg SNP-mice (Figure 2B).
Enhanced activation of tracheobronchial lymph node cells from SNP/OVA -mice
The results of the present study indicate that airway exposure to engineered SNP during sensitization of mice to OVA enhances pathologic aspects of allergic airway disease upon secondary OVA challenge. This effect was more profound with increasing SNP doses and was characterized by enhancement of OVA-induced eosinophilic and lymphocytic inflammation, Th2 and Th17 cytokine expression, elevation of serum OVA-specific IgE and IgG1 levels, as well as an enhanced manifestation of mucous cell metaplasia in pulmonary airways. The fact that SNP exposure during allergen sensitization enhanced the pulmonary allergic response to the secondary OVA challenge indicates that SNP exhibits adjuvant-like characteristics in the development of allergic airway disease. Adjuvants are defined as compounds that are not immunogenic themselves, but increase and/or modulate the intrinsic immunogenicity of an antigen. Adjuvants are used in vaccines to induce potent and persistent immune responses, with the additional benefits that less antigen and/or fewer injections are needed [25]. In this study, however, SNP promoted the immunologic response towards the allergen (OVA) and thereby potentiated the adverse allergic responses in the pulmonary airways.
Our results suggest that workplace exposures to engineered NP could have similar adverse health consequences as those reported for UFP in outdoor air pollution associated with high traffic roadways. UFP collected from the Los Angeles Air Basin have been shown to act as adjuvants to enhance the development and severity of allergic airway disease, using a similar OVA-induced murine model of asthma [6]. In addition, epidemiologic studies have reported an increased incidence of asthma in children living in close proximity to highway traffic [26, 27], where ambient UFP concentrations are high [28]. By comparison, relatively few studies have investigated the exacerbation of allergic airway disease by other types of NP exposures. Exacerbation in allergic airway disease, with increased Th2 responses, have been reported in OVA-sensitized and -challenged mice when co-exposed with 50 μg multi-walled carbon nanotubes (MWCNT) [29] or different sized carbon black NP [30]. Similar findings were reported by Hussain et al. in mice sensitized with toluene diisocyanate (TDI) via skin and exposed to TiO2 NP and gold NP via instillation (0.8 mg/kg body weight) [31]. Unlike our study design, mice in these studies were co-exposed to NPs during both the sensitization and challenge phases of the allergen administration. In our study we found that inhaled SNP during sensitization phase alone can act as adjuvants to markedly increase the magnitude of the host's secondary immune response upon subsequent allergen challenge. A similar effect was also found previously for nano-sized crystalline silica particles [32]. However, a recent study by Ban and colleagues [33] addressed the same question in a similar OVA mouse model with iron oxide NP, and found that iron NP exposure during sensitization with OVA results in attenuation of OVA mediated allergic airway disease. This suggests that the adjuvant effect of SNP in the development of allergic airway disease, we observed in our study, is likely to be particle specific. Further research, however, is needed to understand the immune modulatory effects of different NP and the impact of NP material, size and surface coating.
In our study, SNP exposure alone, without the antigen, caused a dose-dependent pro-inflammatory response in non-allergic animals as indicated by a modest increase in BALF neutrophils and elevations of neutrophil-related chemokines and innate immune response genes, namely Kc, Mip-1α, Mip-2, Itln1 and Irg1. These effects were evident at SNP exposure doses of 100 and 400 μg and demonstrate that these engineered SNP at high dose cause a minimal, yet sustained, innate immune responses up to 16 days post-instillation. By comparison, many rodent SNP toxicity studies describe airway neutrophilic inflammation that is accompanied by overt toxicity and tissue injury. For example, persistent pulmonary toxicity [13, 34], including neutrophilic inflammation, apoptosis, tissue injury [13, 35], the induction of pro-inflammatory BALF cytokines such as IL1β, IL6 and TNFα [34] as well as cardiovascular effects [36] have been reported. These studies used smaller sized SNP (14 nm) at similar or higher exposure doses (100 μg/mouse [13]; 3 mg/mouse [35]). Compared to these in vivo studies, we used larger SNP (90 nm), which appear to be less toxic than smaller SNP reported by others [9]. The SNP used in ours study were further modified with a PEG shell which prevents them from agglomeration and gives them the ability to penetrate rapidly through airway mucus barriers [37]. PEG-coatings have been reported to decrease systemic NP interactions and overall toxicity [38–40], which might explain the less severe acute inflammatory response we observed compared to those reported by others.
Adjuvant effects described in the current study had a mixed Th2/Th17 cytokine response, similar to findings by Li et al. who used ambient UFP in a similar OVA model [6]. Mechanisms by which SNP may induce an adjuvant Th2/Th17 cytokine response can only be speculated, since this study was not designed to investigate the underlying mechanisms of adjuvancy in detail. SNP are known to induce oxidative stress [9], which plays an important role in the pathogenesis of asthma [7, 41]. This connection has been shown by Li et al. for ambient UFP where co-administration with the anti-oxidant N-acetylcysteine in OVA challenged mice diminished the adjuvant allergic airway response of UFP [7]. In another study, oxidative stress, caused by diesel enriched PM, was also shown to skew the immune response from a Th1 to a Th2 cytokine profile [42]. It has been suggested that oxidative stress-induced activation of transcription factor NF-E2-related factor 2 (NRF2), during the sensitization phase with an allergen, can down regulate the production of Th1 cytokines IL12 and IFNγ [43] and thereby leads to a Th1/Th2 imbalance. Furthermore results from in vitro studies on amorphous, colloidal SNP in a size range of 14 to 80 nm show a size- and dose-dependent cytotoxicity of SNP with induction of oxidative stress and/or glutathione (GSH) depletion [44, 45]. At the time point after challenge, however, we did not detect any signs of oxidative stress. Further investigations are therefore needed to assess oxidative stress and Th1/Th2 cytokine balance during the sensitization phase of our protocol in order to address these potential mechanisms of SNP-associated adjuvancy.
Recently IL17 has been associated with more severe forms of asthma, especially those cases complicated by persistent airway neutrophils [46]. Th17 responses in allergic airway disease are promoted by IL6, tumor growth factor β (TGFβ), IL23, SAA3 as well as IL1β [47, 48]. In our study we detected significant increases in allergic SNP/OVA-mice of IL17A, IL6 and IL1β (cytokine and gene expression) as well as Saa3 (gene expression). SAA3 has been shown to activate the NLRP3 inflammasome and promote an allergic Th17 response in mice in combination with other mediators [47]. The NLRP3 inflammasome is a protein complex required for splicing pro-IL1β into its active form [49], and was recently found to have an immuno-stimulatory function for aluminum adjuvants in vaccination [50]. Though we did not measure inflammasome activation in the present study, SNP treatment caused a dose- dependent increase in BALF IL1β, and a recent study has shown that SNP can activate the inflammasome [51]. It is therefore possible that the SNP-mediated adjuvant effects in allergic models are related to inflammasome activation and/or oxidative stress. However, further studies are needed to elucidate their roles in the adjuvant effects of SNP in more detail.
Besides Th2 and Th17 cytokine responses, we also detected an increase in TNFα and Th1 cytokine IFNγ in the SNP/OVA-mice which was not detectable in SNP- or OVA-mice. Therefore it may be possible that a Th1 component is present as well in the adjuvant response of SNP/OVA-mice. It has been shown previously, that Th1 cells do not attenuate Th2 cell–induced airway hyperreactivity in OVA-immunized BALB/c mice, but rather cause severe airway inflammation [52]. We therefore suggest that the increase in BALF cytokines TNFα and IFNγ are augmenting rather than attenuating the allergic response in our model.
Besides stimulation of Th2 response, NP have also been shown to influence maturation, antigen presentation and co-stimulation of DC [53], and the analysis of TBLN cell populations in our study confirmed these findings. In response to DNA and RNA viruses, pDC secrete large amounts of IFN-α and IFN-β that play important roles in activating other cells in the immune system. For example, IFN-α and IFN-β produced by pDC have been shown to increase CD69 expression and IFN-γ production from CD4+ T cells [54, 55], and also activate CD8+ T cells upon influenza challenge [56]. Up-regulation of CD69 on pDC in response to influenza infection has been found to cause down-regulation of sphingosine-1-phosphate (S1P) on pDC, resulting in elongated transit time of pDC and their accumulation in LN [57]. An OVA-induced CD69 up-regulation on pDC might therefore lead to retention of pDC in TBLN where they could interact with lymphocytes and stimulate an immune response. The CD69 expression on pDC was further increased by co-exposure with SNP. Although AM have been suggested to prevent development of airway hyperresponsiveness upon OVA challenge [58], AM are also known to produce proinflammatory cytokines that enhance Th2 cytokine production by pulmonary CD4+ T lymphocytes [59]. In our study, the activation of AM was further increased in the presence of SNP and an increase in BALF Th2 cytokines was measured. In addition, OVA-induced maturation of CD11c+ cells, including APC, such as DC and macrophages, was also exaggerated by SNP. Koike et al. further confirmed that effects on APC parallel those on allergic pathology in vivo in their overall trend [60]. These mechanisms might partly explain the SNP-mediated immune enhancement; however, further research is still required to understand interaction of NPs with the immune system, which could include additional surface markers to further refine effects of NPs on various myeloid subpopulations.
In our treatment protocol, mice were IN instilled with a wide range of SNP doses (0, 10, 100 or 400 μg SNP per instillation). The applied doses of SNP used in our study are comparable to those used in similar studies performing intratracheal (IT) or IN administration [13, 29, 30, 32, 33]. IN has been shown to be an easily applicable and efficient method to administer particle suspensions in murine models [61]. Nevertheless, particle inhalation represents a more realistic model for NP workplace exposure. Intrapulmonary NP delivery and distribution might differ between inhalation and instillation exposure which may result in different dose responses. The potential workplace exposure levels for SNP, however, are currently not known and it is therefore difficult to estimate a realistic dosing regimen. It has been reported that airborne, crystalline silica particles may reach as high as 0.28 mg/m3 in some workplace conditions such as cement mason/concrete finisher [62].
In summary, the results of our study indicate that engineered SNP can act as adjuvants to enhance the development of allergic airway disease in mice. This finding further suggests that individuals exposed to SNP might be more prone to develop allergic airway diseases, establishing a new aspect of NP toxicity that has particular relevance to occupational NP exposure. More research, however, is needed to clarify the potential risks of NP exposure in the development of allergic airway diseases in humans. Nevertheless, the murine allergic OVA model we used in our study, which involved IN instillation of allergen and SNPs during sensitization followed by IN challenge with allergen only, may be used to test the adjuvant potential of other NPs in allergic airway disease.
Material and methods
Particle preparation and characterization
Plain SNP (LUDOX® TM-40 colloidal silica, 40 wt% suspension in H2O), aminopropyltriethoxysilane (APTES), propargyl chloroformate and sodium azide were purchased from Sigma-Aldrich. Anhydrous dimethylformamide (DMF) was freshly distilled from powered BaO. Cu(PPh3)Br and 1-azido-2-(2-(2-(2-methoxyethoxy)ethoxy)ethoxyethane (PEG-N3) were prepared as previously described in detail [63, 64]. PEG-modified SNP were synthesized from commercially available LTM40 SNP in three steps (shown in Additional file 1: Figure S1). To avoid aggregation, the modified SNP were purified by several steps of washing the NP with solvent, followed by centrifugation. Resulting particles were used directly to the following step and aggressive drying such as drying under vacuum was avoided [65]. First, APTES was condensed on plain SNP. The resulting amine-modified SNP (aSNP) were washed twice with reagent grade DMF, and then three times with anhydrous DMF to remove ethanol and water. aSNP were reacted with propargyl chloroformate to afford the alkyne-modified particles (aaSNP) which were purified by centrifugation. The resulted particles were clicked with PEG-N3 catalyzed by 10% of Cu(PPh3)Br in DMF. The resulted SNP were purified by centrifugation to receive the PEG-coated SNP. Note that the SNP suspension was not tested for endotoxin content. It has been shown previously that LPS exposure during sensitization with OVA might suppress the development of a Th2 cytokine response [66], which was not the case in our study.
Dynamic Light Scattering (DLS) was performed with a Malvern NanoZS ZetaSizer to measure the particle hydrodynamic size and intensity average diameters. The particle samples for DLS analyses were sonicated prior to DLS measuring. Particle size, surface grafting amount at different step of synthesis are shown in Table 1. The final SNP used for the animal exposure study had a hydrodynamic diameter of 90 nm. Zeta potential of the particles was not measured, however, the size distribution measurements of the particles (Additional file 1: Figure S4) only shows a single peak at 90 nm and therefore suggesting single SNP dispersion. A more detailed description of particle synthesis and characterization can be found in the supplementary information (Additional file 1).
Experimental protocol
Female BALB/c mice (~20 grams body weight; 6–8 weeks old) were obtained from Charles River (Portage, MI). Mice were maintained at the Michigan State University (MSU) animal housing facilities at room temperature of 21°C–24°C and relative humidity of 45–70%, with a 12 h light/dark cycle starting at 7:30 A.M. All animal procedures and experimental protocols were approved by the MSU Institutional Animal Care and Use Committee; MSU is an AAALAC accredited institution.
On days 1, 3, 6 and 8, BALB/c mice were intranasally sensitized with 0.02% OVA (allergen; Sigma-Aldrich) in saline or saline alone (vehicle control) at a volume of 15 μL per nostril (total intranasal volume of 30 μL) (n = 6 animals/group). SNP were co-administered with intranasal doses of 0, 10, 100, or 400 μg (Figure 1). On days 22 and 23, OVA-sensitized mice were challenged intranasally with 30 μL of a 0.5% OVA in saline solution. All mice were anesthetized with 4% isoflurane prior to each intranasal aspiration. Animals were sacrificed 24 hours after the last intranasal challenge (Day 24).
Necropsy, lavage collection and tissue preparation
Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight). A midline laparotomy was performed and approximately 0.5 mL of blood was drawn from the vena cava and collected in heparinized tubes (BD Microtainer, Franklin Lakes, NJ) for separation of plasma. Animals were exsanguinated via the abdominal aorta. Immediately after death, the trachea was exposed and cannulated and the heart and lungs were excised en bloc. A volume of 0.8 mL sterile saline was instilled through the tracheal cannula and withdrawn to recover bronchoalveolar lavage fluid (BALF). A second intratracheal saline lavage was performed and the collected BALF was combined with the first sample for analysis.
After the BALF was collected, the right lung lobes were ligated, removed and placed in RNAlater (Qiagen, CA). Samples were kept at - 20°C until further processing for RNA isolation. The left lung lobe was intratracheally fixed with neutral-buffered formalin at a constant pressure (30 cm H2O) for 2 h and then stored in a large volume of the same fixative until further tissue processing for light microscopy.
Twenty-four hours later, two sections were excised at the level of the 5th and 11th airway generation along the main axial airway (G5 and G11), to sample proximal and distal bronchiolar airways, respectively [67]. Tissue blocks were then embedded in paraffin and 5- to 6-μm-thick sections were cut from the anterior surface. Lung sections were stained with hematoxylin and eosin (H&E) for routine light microscopic examination and with Alcian Blue (pH 2.5)/Periodic Acid–Schiff (AB/PAS) for identification of intraepithelial neutral and acidic mucosubstances in pulmonary bronchiolar epithelium. To detect eosinophils, slides were immunostained using a polyclonal rabbit antibody directed against murine eosinophil-specific major basic protein (MBP; 1:500; Mayo Clinic, AZ).
In OVA- and OVA/SNP-mice (10 μg SNP), TBLN were removed en bloc from the mice, and homogenized in 1× PBS. Single-cell suspensions were prepared, and 1 × 106 cells from each sample were collected in a 96 well round-bottom plate for flow cytometric analysis.
BALF cytometry
Total number of cells in the collected BALF was estimated using a hemocytometer. All intact cells were counted, but no trypan blue exclusion test of dead cells was performed. Cytological slides were prepared by centrifugation at 400 g for 10 min using a Shandon cytospin 3 (Shandon Scientific, PA) and stained with Diff-Quick (Dade Behring, DE). Differential cell counts for neutrophils, eosinophils, macrophages/monocytes, and lymphocytes were assessed from a total of 200 cells. Remaining BALF was centrifuged at 2400 g for 15 min and the supernatant fraction was collected and stored at -80°C for later biochemical analysis.
Flow cytometric analyses for inflammatory cytokines
BALF supernatants were assayed for the inflammatory cytokines IL-1β, IL-2, IL4, IL-5, IL-6, IL-13, IL-17A, TNFα, IFN-γ, MCP-1, MIP-1α and KC. All cytokine kits were purchased as either Flex Set reagents or as preconfigured cytometric bead array kits (BD Biosciences, San Jose, CA). Cytokine analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). 50 μL of BALF was added to the antibody-coated bead complexes and incubation buffer. Phycoerythrin-conjugated secondary antibodies were added to form sandwich complexes. After acquisition of sample data using the flow cytometer, cytokine concentrations were calculated based on standard curve data using FCAP Array software (BD Biosciences).
Flow cytometric analyses for surface markers
Cells from TBLN were incubated with purified rat anti-mouse CD16/CD32 (Fc block; BD Pharmingen, San Diego, CA) in FACS buffer (1× HBSS, 1% bovine serum albumin (BSA), 0.1% sodium azide, pH 7.6). Cells were then stained for surface markers using following antibodies from Biolegend (San Diego, CA): phycoerythrin (PE)/cy7-conjugated anti-mouse CD4 (clone RM4-5), Pacific Blue-conjugated anti-mouse CD11b (clone M1/70), allophyeocyanin (APC)/cy7-conjugated anti-mouse CD11c (clone N418), fluorescein isothiocyanate (FITC)-conjugated anti-mouse MHC II (I-A/I-E, clone M5/114.15.2), APC-conjugated anti mouse Gr-1 (granulocyte differentiation antigen 1, clone RB6-8C5), and PE-conjugated anti-mouse CD69 (clone H1.2 F3). Cells were fixed with Cytofix (BD Pharmingen) and analyzed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). A graph, explaining the applied gating strategy, is presented in the supplementary information (Additional file 1: Figure S6). Samples were concatenated (n = 6) and further analyzed using Kaluza 1.1 software (Beckman Coulter, Miami, FL). The fluorescence intensity values for CD69 or MHC II expression by different populations were presented in histograms. To determine the statistically significant changes between OVA and SNP/OVA groups, an unpaired t-test was performed on non-concatenated data. The percent values of CD69 expression by CD4+, CD11c+, Gr-1+, AM, and pDC populations, respectively, or the percent values of MHC II expression by CD11c+ population from individual animal were transformed and analyzed using GraphPad Prism v4.0 (Graphpad Software, San Diego, CA).
ELISA assay OVA- IgE/IgG1
OVA-specific IgE and IgG1 expression in serum was analyzed with an ELISA kit (BD PharMingen, CA) as described previously [6]. 96 well plates (Costar, NY) were coated overnight with 50 μg/mL OVA. After washing with phosphate buffered saline (PBS) and blocking with 10% fetal bovine serum (FBS)/PBS, samples and standards were loaded and incubated overnight. Following additional washing steps, detection antibody (biotin-conjugated rat anti-mouse IgG1 or IgE Antibody (BD PharMingen, CA) was applied and incubated for 4 h. Finally, streptavidin-horseradish peroxidase mixture was added for 30 min, followed by 20 min incubation with reaction substrate. Optical density (OD) was read at 405 nm wave length with a Spectra Max Gemini plate reader (Molecular Devices, CA). The OVA-IgG1 standard was a monoclonal anti-chicken egg albumin (Sigma-Aldrich, MO). Since no standard was available for IgE, OVA-specific IgE was determined only by OD. All samples were loaded and measured on the same 96-well plate, therefore a standard was not essential for comparing relative increases in IgE concentrations.
Real-time PCR of pulmonary tissues
Total RNA was isolated from right lung lobes using RNeasy Mini Kit (Qiagen, CA) according to the manufacturer's instructions. Briefly, tissues were homogenized in lysis buffer (Buffer RLT) containing 2-mercaptoethanol with a 5 mm rotor-stator homogenizer (PRO Scientific, CT). During RNA purification, DNase digestion was performed on-column using Qiagen RNase-Free DNase Set. Purified RNA was quantified using a GeneQuant Pro spectrophotometer (BioCrom, England). CDNA was generated from 2 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit. The reaction mixture was incubated at 25°C for 10 min and then 37°C for 2 h. PCR array analysis was performed by pooling aliquots of cDNAs from samples in each experimental group. Quantitative gene expression analysis was performed using Taq Man Gene Expression Assay reagents on the ABI PRISM 7900 HT Sequence Detection System. The PCR cycling parameters were 48°C for 2 min, 95°C for 10 min, and 50 cycles of 95°C for 15 s followed by 60°C for 1 min. Relative gene expression levels were reported as fold-change using the ΔΔCt method where FC = 2-ΔΔCt. The mRNA expression of each gene was normalized by subtracting the geometric mean of the Cts from four endogenous controls (Actb, Arbp, Gapd, Gusb). Selected genes that had expression levels at least 2-fold greater in experimental groups relative to the control group were confirmed by relative quantitative real-time RT-PCR using individual animal cDNAs as described above. Statistical differences between ΔCt values of different groups were determined with two-way ANOVA (SigmaStat, Ashburn, VA; P ≤ 0.05).
Airway morphometry
Each study group consisted of 6 mice and all data were reported as group means ± standard error of the mean (SEM). Grubbs outlier test was performed and recognized outliers were removed from the analysis. Differences among groups were analyzed by a two-way ANOVA followed by a pair-wise comparison (Student-Newman-Keuls). When normality or variance equality failed, a Kruskal-Wallis ANOVA on ranks was performed. All analyses were conducted using SigmaStat software (SigmaStat; Jandel Scientific, San Rafael, CA). Significance was assigned to p values less than or equal to 0.05.
- aaSNP:
Alkyne-modified silica nanoparticles
- aSNP:
Amine-modified silica nanoparticles
Alcian Blue/Periodic Acid–Schiff
- Actb:
Alveolar macrophages
- Amuc:
Area of mucus
Antigen presenting cell
- Arbp:
Acidic ribosomal phosphoprotein P0
Bronchoalveolar lavage fluid
Chemokine (C-C motif) ligand
Chemokine (C-X-C motif) ligand
Dendritic cells
Fetal bovine serum
- FoxP3:
Forkhead protein P3
Histological sections at the 5th and 11th airway generation of the main axial airway
- Gapdh:
Glyceraldehyde-3-phosphate dehydrogenase
- Clca3:
Chloride channel calcium activated 3 (Gob5)
- Gusb:
- IgE:
Immunoglobulin isotype E
- IgG1:
Immunoglobulin isotype G1
- IFNγ:
Interferon γ
Inflammatory protein (CXCL10)
(CXCL1) keratinocyte chemoattractant
Length of basal lamina
LUDOX® TM-40 colloidal silica 40 wt% suspension in H2O
Major basic protein
(CCL2) monocyte chemoattractant protein 1
(CCL8) monocyte chemoattractant protein 2
- MIP-1α:
(CXCL3) macrophage inflammatory protein 1 alpha
Matrix metalloproteinase 12
Mucin 5 AC
Optical density
Phosphate buffered saline
Polyethylene glycol
Particulate matter
- Saa3:
Serum amyloid A3
Silica nanoparticles
Tracheobronchial lymph nodes
Tissue inhibitor of metalloproteinase 1
- TNFα:
Tumor necrosis factor α
- T-reg:
Regulatory T cells
Ultrafine particles
- VDmuc:
Volume density of mucus.
We would like to thank Amy Porter and Kathy Joseph from the Michigan State University Histopathology Laboratory for their excellent technical support. This manuscript is in memory of our friend and colleague Dr. Gregory Baker who passed away unexpectedly while this paper was being written. Dr. Baker was responsible for the generation and characterization of the nanoparticles used in this study.
This research was funded by NIH/NIEHS 5RC2ES018756-02.
Authors' Affiliations
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An investigation of the writing strategies three Chinese post-graduate students report using while writing academic papers in English
Mu, Congjun (2006) An investigation of the writing strategies three Chinese post-graduate students report using while writing academic papers in English. PhD thesis, Queensland University of Technology.
Congjun Mu Thesis
Administrators only |
Due to a lack of effective writing strategies and inhibition of English language proficiency, university students in China are found to produce little and shallow content in their English academic writing. Similar problems are also embodied in the academic writing of Chinese overseas students who struggle to survive in the target academic community. The purpose of this study was to investigate the writing processes of second language (L2) writers, specifically examining the writing strategies of three Chinese post-graduate students in an Australian higher education institution. The study was prompted by the paucity of research in the writing strategies used by Chinese students in English academic writing in an authentic context. Although it was too small in scale to generalise in the field of L2 writing, the study will stimulate research in L2 writing theory and practice. Based on a review of theories related to L2 writing and research in Chinese and English writing strategies, the writing strategies used by three Chinese post-graduate students while writing academic papers in English were investigated. Their understandings of English and Chinese writing processes, the issue of transfer of Chinese writing into English writing and cultural influence of native language on L2 writing were explored as well.
Qualitative hermeneutic multi-case study methods were employed to provide a richer description of the writing strategies used by the three students to develop a deeper understanding of the L2 writing process. Data were provided by three Chinese post-graduate student writers in Public Health who were observed undertaking different tasks. Ally, a Masters student, was observed completing one of the assignments for a course. Susan and Roger, both doctoral students, were observed working on a second stage proposal and a journal paper respectively. Data collected from semi-structured interviews, questionnaires, retrospective post-writing discussions and papers were categorised and analysed using topical structure analysis and cohesion analysis.
The findings suggest that writing in a second language is a complicated idiosyncratic developmental process influenced by cognitive development, social/educational experience, the writer's first language (L1) and second language (L2) proficiency and cultural factors as well. These proficient writers were found to utilise a broad range of writing strategies while writing academic papers in English. This study in some degree supports Silva's (1993) finding that the L2 writing process is strategically, rhetorically, and linguistically different from the L1 writing process. Most of the metacognitve, cognitive, communicative and social/affective strategies except rhetorical strategies (operationally defined in this study as organisation of text or paragraphs) were found to transfer across languages positively. These student writers were noticed to have difficulties in acculturating into the target academic discourse community because of their background of reader-responsibility which is regarded as a crucial feature in Eastern rhetoric and is distinguished from writer-responsibility in English rhetoric (Hinds, 1987, 1990).
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ID Code: | 16426 |
Item Type: | QUT Thesis (PhD) |
Supervisor: | Carrington, Suzanne, Crawford, Jane, & McKay, Penelope |
Keywords: | second language writing, Chinese students, rhetorical strategies, metacognitive strategies, cognitive strategies, communicative strategies, social/affective strategies, multi-case study, hermeneutic approach |
Divisions: | Current > Schools > School of Cultural & Professional Learning Current > QUT Faculties and Divisions > Faculty of Education |
Department: | Faculty of Education |
Institution: | Queensland University of Technology |
Copyright Owner: | Copyright Congjun Mu |
Deposited On: | 03 Dec 2008 04:03 |
Last Modified: | 25 Mar 2013 08:17 |
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The author is a Forbes contributor. The opinions expressed are those of the writer.
Looking today at week-over-week shares outstanding changes among the universe of ETFs covered at ETF Channel, one standout is the Proshares Ultra QQQ (AMEX: QLD) where we have detected an approximate $27.7 million dollar outflow -- that's a 5.1% decrease week over week (from 8,775,000 to 8,325,000). Among the largest underlying components of QLD, in trading today Altera Corp. (NASD: ALTR) is off about 0.4%, Analog Devices, Inc. (NASD: ADI) is down about 0.2%, and Autodesk Inc. (NASD: ADSK) is lower by about 1.1%. For a complete list of holdings, visit the QLD Holdings page »
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Company news: Amgen and DeCODE Genetics; Porter Novelli
Amgen is buying deCODE Genetics, an Icelandic firm, for $415 million in cash. DeCODE Genetics' work has focused on identifying genetic risk factors for diseases ranging from cardiovascular disease to cancer. "This capability will enhance our efforts to identify and validate human disease targets," said Robert A. Bradway, president and CEO of Amgen. "This fits perfectly with our objective to pursue rapid development of relevant molecules that reach the right disease targets while avoiding investments in programs based on less well-validated targets."Porter Novelli named Paul George director of health. George, who joins from Omnicom healthcare communications agency TogoRun, where he was SVP and partner, starts Jan. 1. In that most recent role, George led global integrated marketing communications programs for clients including BMS and Gilead. He also served as the lead for TogoHealth in an Omnicom initiative to identify and pitch new healthcare business across agencies. Porter Novelli New York senior partner and managing director Karen van Bergen said of George: "As more and more clients look to holding companies to create multi-agency marketing partnerships, Paul's experience will be invaluable as we build the cross-border, cross-discipline solutions that clients need to grow their businesses."


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Welcome to the Diva Holiday Gift Guide! Every day in November I will be highlighting some of the hottest new products with fantastic giveaways from Party Plan Consultants around the USA! Help support small businesses this Holiday Season by shopping from Party Plan Consultants and make a difference in someone's life! You are going to be shopping this season for your loved ones, why not help some one directly by buying through an Independent Consultant in Party Plan? Happy Shopping!
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As someone who is impressed with attention to detail and presentation, I was also very impressed with the "extra touches" that Origami Owl gives each piece. My necklace arrived in a cute little "take out" box, with a woven "fortune cookie" type jewelry box. It was certainly a fun way to get into my new necklace!
My new Origami Owl Necklace included a seashell charm (I live on the beach), a wine glass (my personal blog), a crystal cross, a heart with "husband" for my hunny, a ruby and diamond stone for my Girls birth stones, and then pink and black dangle charms for Party Plan Divas! Like I said, it was PERFECT for ME! (The other necklace in the picture… that's the giveaway!!!) You can check out many more Living Lockets that others have created on the Origami Owl Facebook Page.
Inspired by the desire to create jewelry that would be beautiful and meaningful yet whimsical + fun, young entrepreneur Isabella Weems launched Origami Owl. With the goal of saving enough money to buy her first car, she introduced the "Living Locket", a customizable necklace that one can create with meaningful pieces that tell a story of their lives.
From humble beginnings selling to friends + at boutiques to a burgeoning retail business just months later, Origami Owl's success has continued to catapult + customers are still clamoring for more! To meet the demand, Origami Owl is launching its home party business opportunity nationwide in 2012. Our trend-setting customizable jewelry brings a new level of fun into today's home party concept.
Starting in January 2012, Origami Owl will begin opening up exclusive opportunities for Founding Designers who want to begin designing and selling Origami Owl Jewelry. Origami Owl Parties will allow guests to come together for a unique and memorable jewelry shopping experience, and create a meaningful keepsake that they can take home the night of the Party. Each party is an amazingly fun, hands-on event where you help guests create one-of-a-kind jewelry pieces that reflect who they are + what they love. With a generous Hostess Program and Compensation Plan, Origami Owl is sure to grow quickly! For more information on ordering, hosting, or on the new Opportunity, Contact Christine Strong at firstname.lastname@example.com.
I am very excited to be able to offer one of my readers their very own Origami Owl "Diva" Living Locket! As seen in the image above, the "Diva" has crystal letters spelling "DIVA" with pink stones and a pearl dangle. It is beautiful!
Sign up to receive the Origami Owl eNewsletter via the sign up form, then come back here and leave me a comment letting me know you did so!
Make sure you leave 1 comment for each entry to get credit!! Contest ends December 12, 2011.


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About This Gigapan
Toggle- Taken by
- Fernando da Rosa
- Explore score
- 63
- Size
- 0.30 Gigapixels
- Views
- 25737
- Date added
- Feb 03, 2008
- Date taken
- Feb 02, 2008
- Gear
Nikon D7000
- Categories
- Galleries
- Competitions
- Tags
- beach, fedaro, montevideo, iemanja, uruguay
- Description
Iemanja is an orisha, originally of the Yoruba religion, who has become prominent in many Afro-
American religions. For the believers she is the ocean, the essence of motherhood, and a
protector of children.
Africa: Yemoja, Ymoja, Iemanja Nana Borocum, Iemanja Bomi, Iemanja Boci
Brazil and Uruguay: Yemanjá, Iemanjá, JanainaThis deity is revered by thousands of people, believers and non-believers in the Umbandismo. On
2 February, thousands of people visit the beaches of the country, some motivated by faith, and
many for the simple power of curiosity.
Believers carry small boats with gifts, which throw the sea as an offering to Iemanja.
Gigapan Comments (2)
Toggle Minimize gigapan_comment-
rnuffer: the beach is usually clean, but the celebration is to bring offerings Iemanja (jewelry, food, fruits, drinks) to give them to the Virgin of Iemanja, and left on the beach. All that is cleaned by the city the next day.
I am suprised by the amount of trash on the beach. Seams to be a large quantity of watermelons


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Turntable Arm
From VEX Wiki
The VEX Turntable Arm was created as a robot to demonstrate the capabilities and uses of the Turntable Bearing Kit released in April 2011 at the VEX Robotics Competition World Championship. The arm was based off of industrial arm robots which often use low-friction, high-load bearings in applications with lots of side load.
VEX Turntable Arm Specifications
Ideas for Future Revisions


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Invaluable cannot guarantee the accuracy of translations through Google Translate and disclaims any responsibility for inaccurate translations.
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Lot 2: ALBY, HENRI. La Vie de S. Henry Premier Empereur de ce Nom. MANUSCRIPT IN FRENCH, written in
BOOKS & AUTOGRAPHS from the Collection of Mr. & Mrs. Stephen Byer and the Byer Museum of the Arts
by Swann Auction Galleries
May 3, 1990
New York, NY, USA
No Image Available
Looking for the realized and estimated price?
Description: neat cursive hand in black ink, recto and verso, ON VELLUM. FULL-PAGE PEN DRAWING OF S. HENRY facing title. 23 leaves. 121 x 71 mm, early 19th century black straight-grain morocco gilt; 1/4 morocco pull-off case. Mortimer L. Schiff morocco book-label. Np, later 17th century?


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Did you hear? Microsoft is pulling the plug on Xbox Live support for original Xbox games, like, tomorrow, so we thought it'd be a good idea to say goodbye in style. Specifically, in the style of playing some Halo 2! So tonight, Wednesday, the 14th of April, we're asking readers to log on and frag with us!
I'll be online at 7PM ET, ready to rumble; merely send me an Xbox Live message (Gamertag: KnifefightYaDad), and I'll do my best to get you into the game, as space permits. Tell your friends, tell your mom and, uh, tell your mom's friends! This is the last time we'll get to play together before the servers are shut down tomorrow.
Note: You may want to download all of the extra maps before joining us online, if you don't have them already. It'll save time and hopefully get us gaming together as soon as possible.
Finally, we're hoping to turn X3F Game Nights into a regular thing, so if you keep showing up, we'll keep hosting -- all you have to do is ask!
What: Multiplayer Halo 2 send-off
Where: Your house (or wherever your Xbox/Xbox 360 is set up)
When: Wednesday, April 14 at 7pm ET until ... dark!
Why: Because Xbox Live won't support original Xbox games after April 15
How: Jump into Halo 2, send KnifefightYaDad a message and wait for the invite