In the last three decades, I have seen Mumbai developing quickly especially with so many people migrating to the city to change their destiny and lifestyle. Mumbai has been the City of Opportunities for many people across the country and even across the world and that has worked in the favor and also against the city in many ways. For all my childhood days, I have seen Mumbaikars shopping in the small lanes and shops but with the level of lifestyle going up new trends came in that swept shopaholics off their feet. I remember when the first mall in Mumbai opened up in September 1999 near Haji Ali known as Crossroads at that time and now known as SoBo Central it attracted millions of people to it. Crossroads was jam packed for weeks with shopaholics, casual window shoppers, college students and even curious middle class people who wanted to analyze to concept of malls. Well, a lot of time has already passed by and malls today have become a part and parcel of our life. Today, malls are packed with all sorts of people, you will find families shopping around, college students wandering and dating around while many limited budget consumers feast their eyes with all the glitter and glamour that they get to see around. In my previous blog post I already covered the street shopping destinations in Mumbai so this time let's take a quick look at the top 3 shopping malls in Mumbai that have managed to offer consumers with the right kind of shopping experience that offers them value for money and great brands and products that enrich their lifestyle.
Formerly known as Phoenix Mills brags to be one of the biggest shopping malls not only in Mumbai, but also across the country. With more than 500 stores that combines premium range brands and economy brands it offers a great shopping experience to the consumers. High Street Phoenix also has a five star hotel, a multiplex and commercial space and residential tower that cover 3,300,000 square feet. High Street Phoenix is developed on a land where Phoenix Mills stood earlier and you can see the tall chimneys that indicates the presence of textile mills that provided employment to large chunk of workers in Mumbai in the early 1900s.
High Street Phoenix also sports South Asia's largest 20 lane bowling concourse. The best thing about High Street Phoenix is that budget shoppers can also enjoy shopping here as they have options like LifeStyle, Big Bazaar and FabIndia. For those who are ready to spend extravagantly there is so much to shop here with top brands like Burberry, Chanel, Dolce Vita, Emporio Armani, Jimmy Choo and Canali available here. Enjoy your meal at the five star Shangri-La Hotels and Resorts. However, it lacks food court like other Mumbai malls and has fully fledged restaurants instead. To reach High Street Phoenix you can either get down at Mahalaxmi or Lower Parel on the Western Line if you are using suburban train.
Located right between Vikhroli and Ghatkopar R-City Mall is also one of the best and top shopping malls in Mumbai that offers the best shopping experience to the consumers. Since the mall is located on the Central Line of the city R-City Mall is usually quite popular with Mumbaikars that live around that locality, but you will still find people from all walks of life and different parts of the city. The best part about the mall is that it targets all kinds of shoppers and therefore consumers will find different brands here that cater to different classes across the city.
While there are certain international brands that cater the financially strong consumers there are budget brands as well that cater to the upper middle class consumers. With international architecture the mall attracts many people who want to spend some good time especially on the weekends. It offers central food court that offers a better choice to consumers who want to have a bite from different food stalls at the same time. To get here you can get down at Ghatkopar on the Central Line and take a rickshaw towards LBS Road.
InOrbit Mall certainly has managed to impress many shoppers around Mumbai and you will find InOrbit Mall in Malad as well which was opened four years before the Vashi mall was developed. Although, there are so many malls already around in Vashi, InOrbit has managed to attract large chunk of shoppers especially on weekends.
Similarly, InOrbit attracts many shoppers and youngsters at the Malad location as well.The mall offers an international shopping experience to the consumers with many international brands selling their products here along with economy brands side by side.
InOrbit Mall has always come up with the concept of One Stop Destination for shoppers all over Mumbai and they have done it through various promotions and events that happen here. The mall also anchors with Shoppers Stop and HyperCity at Vashi, Malad and Hyderabad. To reach InOrbit Mall at Vashi you can get down at Vashi on the Harbor Line and you will find the mall just opposite the railway station.
I will definitely save this blog and refer my friends since we are always looking for good places to roam around and shop in the weekend.High Street Phoenix, Lower Parel is also good place to hang out with friends and shop.


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Seven Bucks Productions Announces Partnership with Media Comglomerate AMI and their Mr. Olympia, LLC
Together Create Dynamic Programming to Showcase the Fitness and Bodybuilding Industry
New York, NY – Seven Bucks Productions, co-founded by Dwayne Johnson and Dany Garcia, today announced a long-term production and promotional partnership with Mr. Olympia, LLC, a division of American Media Inc. (AMI). The partnership is backed by a mission to highlight the passion and grandeur of the Mr. Olympia Weekend, the premiere event in the sport of bodybuilding and one of the largest fitness expos in the country. Seven Bucks Productions will work with Mr. Olympia, LLC to develop, produce and promote groundbreaking programs and products designed to entertain and captivate audiences along the road to the 2016 Mr. Olympia Weekend, taking place in Las Vegas from September 15-18.
The partnership marks a first for the production company that produces film and TV projects including HBO's Ballers, the documentary film Rock and a Hard Place, and the upcoming Baywatch movie. Both Ms. Garcia and Mr. Johnson have passionate ties to the fitness industry and look forward to utilizing this new collaboration with Mr. Olympia, LLC to help grow awareness for the industry and the sport of bodybuilding's marquee event, the 2016 Mr. Olympia. Ms. Garcia, in addition to being a successful businesswoman and producer is also one of the country's top female professional bodybuilders. Mr. Johnson was recently named "Man of the Century" by Muscle & Fitness and shares his passion for fitness and exercise daily with his more than 104 million followers on his combined social platforms.
"We have a great respect for the determination, drive and work ethic that is necessary to succeed in this sport and we are excited to share the bodybuilding community with the world," said Johnson.
"This is a passion project for not only myself but the entire Seven Bucks Production family. We are absolutely thrilled to have the opportunity to highlight an event that that has shaped the world of bodybuilding and touches every aspect of the fitness community. We are committed to working with AMI to bring the greatest level of showmanship and entertainment to the athletes, audience and viewers," said Garcia.
"Seven Bucks Productions, Dwayne Johnson and Dany Garcia are a committed, dedicated and creative force in the entertainment industry," said AMI Chairman and CEO David J. Pecker.
"The Mr. Olympia weekend has continued to grow in size, attendance and excitement leading up to last year's record-setting event featuring the largest competitive purse in the sport. Combining the passion, creativity and incredible knowledge of the sport that Dwayne and Dany possess with the momentum of the event ensures that the 2016 Mr. Olympia will be the most exciting in its 50-year history."
The partnership is the first between Seven Bucks Productions (HBO's highly rated comedy series Ballers, Spike TV's Rock the Troops and Paramount's Baywatch) and American Media Inc.
About Seven Bucks Productions
Co-founded by Dwayne Johnson and producing partner Dany Garcia, Seven Bucks Productions is a multi-platform production company pioneering original content for television, film, emerging technologies, and digital networks. Crossing all entertainment verticals, Seven Bucks Productions creates innovative content rooted in authenticity, strong storytelling, and passion.
Seven Bucks Productions has an ever-expanding slate including tent-pole movies such as Paramount's Baywatch, New Line Cinema's Central Intelligence and Shazam, Universal's The Janson Directive and Dreamworks' Alpha Squad Seven. The company also produces original television programming including TNT's The Hero and Wake Up Call, HBO's Ballers, and Spike TV's Rock the Troops (November 2016).
About American Media, Inc.
American Media, Inc. (AMI) owns and operates the leading print and digital celebrity and active lifestyle media brands in the United States. AMI's titles include National Enquirer, Star, OK!, Globe, National Examiner, Soap Opera Digest, Men's Fitness, Muscle & Fitness, Flex and Muscle & Fitness Hers. AMI also manages 10 different digital sites including RadarOnline.com, OKmagazine.com, MensFitness.com and MuscleandFitness.com. AMI's magazines have a combined total circulation of 2.1+ million and reach more than 37 million men and women each month. AMI's digital properties reach an average of 50 million unique visitors and over 350 million page views monthly.

redpajama_set_name: RedPajamaCommonCrawl


WSHA hosted a webinar from 12-1 pm Feb. 23 to provide basic information and answer questions on Link4Health: the state's effort to advance electronic exchange of clinical records and transform health care. This new program, sponsored by the Health Care Authority, is intended to provide real-time information exchange among providers caring for Medicaid patients. The state has partnered with OneHealthPort, the state-designated health information exchange, on this initiative.
Link4Health should support risk-based contracting approaches and more integrated provision of care. The Clinical Data Repository program, the first initiative under Link4Health, allows for easier exchange of clinical information. As of Feb. 1, 2017, the state will require provider organizations that contract with Medicaid-managed care plans and have a certified EHR to participate in the program. The webcast speakers were Melodie Olsen, state health IT manager at the Washington State Health Care Authority; and Rick Rubin, CEO of OneHealthPort.


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Mens Custom Dress Shirts
The first shirt, after the animal skin, was worn as early as the 18th dynasty of ancient Egypt. They were made of a rectangular piece of linen, folded and sewn up the sides, with openings left for the arms and a hole cut at the fold for the head. There are also shirts preserved from ancient Egypt that have long, tight sleeves sewn into the armholes.
Toward the end of the Middle Ages, when clothing became rather closely fitted, the shirt gradually increased in importance. During the 14th century, shirts worn by the Normans developed a neckband and cuffs. By the end of the 15th century, shirts were made in a variety of fabrics, such as wool, linen, and sometimes silk, for royalty.
Shirts began to be embroidered, laced, and frilled in the 16th century, and men's outer garments-the doublet, a precursor to the jacket-had a low neckline so that the shirt showed across the chest. By the end of that century, the shirt frill had developed into the ruff, which was a mark of the aristocracy.
Photo of rare undergarments, the first shirts, surviving from the late 16th and early 17th century periods.
The man's shirt (left) dating from the time of Queen Elizabeth I, around 1580-90.
The woman's shift is a little later, dating from the reign of James I, in about 1610.
Both are made of linen decorated with delicate embroidery in black thread. The needlework on the man's shirt includes naturalistic motifs of birds, insects and flowers - while that on the woman's shift has a geometrical pattern.
A law was passed in England that forbade persons without social rank to wear elaborately decorated shirts. At the beginning of the 17th century, the doublet had become so short that the ruffled shirt was visible between it and the breeches. A new style of men's dress was initiated in 1666, when Charles II of England adopted the long waistcoat, which covered up most of the shirt.
"Ruff and ready". The collar was derived from the "ruff", a detachable pleated collar of linen, supported by a wire frame. It's that frilly circular neck covering from the late 16th and 17th centuries. Think of Shakespeare or Queen Elizabeth I, and you'll remember what I'm talking about.
The roots of the ruff were in East where Indians wore collars stiffened with rice water. In 1540 the Queen of Navarre (in the Pyrenees of northern Spain and southwest France, an independent kingdom until it was incorporated into the French crown lands in 1589) began to widen the frill to hide her ugly throat. Catherine de Medici of Italy adopted it and the style spread to England after Mary Tudor married Phillip II of Spain.
The popularity of the "ruff" spread to gentlemen's shirt cuffs which were decorated with bands of lace until around 1690 when the frills disappeared leaving just the long sleeve.
The 1800's saw the last burst of glamour in men's shirts until the 1960's with chin high collars and rows of pleated ruffles on the chest. In 1840 there is some relief as collars turn down their stiff points to form wings, but shirt bodies sport stripes and floral patterns and studs are more popular than buttons.
As the 18th century unfolded, so did the cravat, becoming longer and lacier at the ends. France's Republican "incroyables" ("unbelievables") were known for their 15-foot cravats wrapped around the neck and tied in a bow. They were worn so high a man could not move his head. But they did show their colors, usually red, and allegiance to the French Revolution.
The first half of the 19th century in England was the golden age of cravat. The neck cloth was so elaborate and voluminous that George Bryan "Beau" Brummell (1778-1840) and his valet sometimes spent a whole morning getting it to sit properly.
Classes were given on how to tie the Ascot, and other knots as decreed by dandy, "Beau" Brummell.
Brummell was born in London, and educated at Eton College and Oriel College, University of Oxford. At the age of 17, Brummell became an intimate companion of the Prince of Wales, later George IV, (1762-1830), king of Great Britain and Ireland (1820-30), and was for many years regarded by court society as a wit and an authority on all matters of dress and etiquette.
While his fortune lasted, Brummell kept an elegant establishment in London, but he finally lost his royal friends, gambled recklessly, and in 1816 fled from his creditors to France. He obtained an appointment as British consul at Caen from 1830 to 1832, but eventually was forced into debtor's prison and died in Caen in a mental institution.
Brummell popularized a new English style of menswear in the early 1800s. He wore suits made of dark wool rather than of silk, and he promoted the idea that simplicity in dress could be elegant. He also stressed the importance of personal cleanliness as a fashionable virtue, insisting that a plain but clean white shirt was preferable to a fancy one that was dirty. Brummell set the mode in 1806 for the ruffled shirt for both day and eveningwear. Brummell was also responsible for introducing the fashion of wearing only black and white for eveningwear.
Men's clothing became more somber in the Victorian age especially during the morning of the death of Prince Albert, Queen Victoria's husband. High neck cloths were abandoned for collars and ties more or less the same as those worn in the 20th century.
Shirts then were tunic style, which pulled directly over the head. In 1871 the first "coat style" shirt was registered in England. It opened with buttons all the way down the front and was not pulled over the head. The first modern shirt collars appeared in the late 1800's and were high, starched, and white collars that attached to the "new style" shirts. The wing collar is a direct descendant of these stand-up collars.
Detachable Shirt Collars:
Housewife, Mrs. Hannah Lord Montague (1794 -1878), of 139 Third Street, Troy, New York "invented" the detachable collar in 1827. She got tired of washing her husbands entire shirt when only the collar was dirty.
Her husband, Orlando Montague, showed-off his wife's invention to the guys around town, and soon all the wives of Troy saw the time saving value of this new invention. Soon after, merchants got interested in marketing the collar, and begin manufactured collars in mass quantities for sale to the outside world.
Troy, New York, became "Collar City" to the rest of America, and went from a sleepy little upstate town to the center of American shirt and collar manufacturing. Woodsheds, garrets and store fronts became collar making places as teachers, farmers, carpenters, butchers and ironmongers were drawn to this new trade by its apparent simplicity: a table, a pair of scissors, a bolt of cloth and a spool of cotton was all that was necessary.
By 1897, twenty-five manufacturers in Troy were producing a total of eight million dozen collars and cuffs a year. Linen collars were offered in a breathtaking variety of styles and had become the status symbol of the growing office-worker class (i.e. "white collar" workers). Mail order catalogs like Sears-Roebuck, Montgomery-Ward, and Bloomingdale's sent detachable collars to every part of America, along with the often-colorful collarless shirts with which they were worn. By the Turn-of-the-Century, nearly every man in America had his neck in the vise-like grip of an iron-hard starched linen detachable collar.
If you were rich your collars were in cotton or linen and heavily starched, hence the name "stiff collar". Collars were stored in a round leather collar box, usually with a little lidded box inside for studs. The detachable collar had to be attached with a collar stud in the front and back. Those stiff collars had to go to commercial laundries, which was quite expensive. Poorer people wore collars made from lacquered cardboard, which were discarded once they were dirty.
Beginning in the 1920's collars were even made in that new material Bakelite, (just wipe clean). Of course really poor people never wore collars; even with their Sunday best they wore collarless shirts. The term white and blue-collar workers would have been more accurate as collar and no collar workers.
Also in the 1920s collars (still separate) were available in the same fabric as the shirt, not just white! Shirts could be purchased with two additional collars. Collars in matching fabric were never as heavily starched as white ones.
Disposable collars reached the height of popularity in the 1860's, but by 1928 the detached collar was gone, replaced by the semi-stiff attached collar.
The Oxford button-down collar shirt:
Polo players originally fastened their shirt collars down to keep it from flapping in their faces. There is no historical evidence (but some great folklore which may or may not be true) that the polo players used buttons. It seems more authentic that pins of some sort were used.
In any case, as the story goes,John Brooks, grandson of Brooks Brothers' founder Henry Sands Brooks was watching a polo match in England and noticed the button-down collars that the players wore (or pinned!). He decided to adapt the button-down collar for shirts. Now a business classic, it was introduced to the USA by Brooks Brothers in 1896. This was a bold move since most men's shirt collars were stiff and detachable.
THE BROOKS CLAN
On April 7, 1818 Henry Sands Brooks ,age 45, opened H. & D. H. Brooks & Co. on the Northeast corner of Catharine and Cherry Streets in New York City. After his death in 1833, control of the company was assumed by his eldest son, Henry, Jr.
In 1850, Henry's sons Daniel, John, and Elisha inherited the family business, and Brooks Brothers was born.
Button-down collars were popular in the late Forties after WWII and synonymous with the Ivy League Look of the 1950's.
Legal trivia - Brooks owns the name "Polo collar" for its button-down, which is why Ralph Lauren can't use the name Polo on his button-down collar shirts.
Soft attached collars were made popular by the English Duke of Windsor (later briefly King Edward VIII) who preferred the attached softer collar and the stiff collar became obsolete. Also collars and cuffs that matched the shirt fabric became fashionable.
Pinned or eyelet collars were popular during the late 1920's and early 1930's, the pin pierced the fabric under the tie pulling the wings of the collar together raising the tie. Collar bars came later which slipped or slid on the edges of the collar. The tab collar in the late l920's, which used tabs to fasten under the tie, followed this style. (It was popular again in the 30's 60's and 90's).
Next were straight collars. The collar points were lengthened or shortened to create fashion changes.
English Spread, Cutaway, or Windsor collar was said to accommodate the Duke of Windsor's (later briefly King Edward VIII) thick tie knot (also know as the full Windsor knot). This collar has a wider spread than the semi-cutaway or spread collar.
The club or Eaton, a rounded collar was popular in the 1930's.
In the 1960's technology caught up with the fabric industry with a wealth of permanently pressed synthetics.
The shirt was a major focus of the Peacock Revolution in the 1960's with shirts appearing in a variety of stripes, patterns, and flamboyant colors previously not worn, close-to-the-body shapes, and collars wide enough to accommodate the widest ties ever.
In the early 1970's knit dress shirts came on the market.
Keep your shirt on:
To remain calm or patient: The plane doesn't land for another hour, so keep your shirt on.
Lose your shirt:
To lose everything one has or owns.
The Shirt off (one's) back:
The maximum one is able to give or lose:
The only thing those swindlers didn't take was the shirt off my back.
"With the kind courtsey of Andy Gilchrist" - Ask Andy About Clothes
A Brief History of Clothes in Time
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Dinesh D'Souza Just Used the 'N-Word' During a Speech at Liberty University
Conservative provocateur Dinesh D'Souza has just used the n-word on stage while giving a speech at Liberty University. It was captured on video which is embedded here. Please note that the word is not bleeped.
D'Souza uses the word during a rant against President Lyndon Johnson, who he accused of using the racial epithet. "[Johnson] is a guy who would use the word 'n*****'," says D'Souza.
Ever since garnering a presidential pardon from President Donald Trump for his role in campaign finance violation, D'Souza has made it his goal to defend the Republican Party against allegations of racism, while pinning responsibility for slavery and Jim Crow on the Democratic Party. His most recent attempt was a "documentary" Death of a Nation.
Nation, Social Justice
Clemency Has Been Granted for Julius Jones
This comes on the heels of several high profile resignations in the wake of racist language. Megyn Kelly was dismissed from NBC less than a week after her comments about blackface, and Papa John's founder John Schnatter resigned after his own use of the n-word was made public
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Differential regulation of neurotrophin expression in human bronchial smooth muscle cells
Cecilia Kemi1,
Johan Grunewald1,
Anders Eklund1 &
Caroline Olgart Höglund1,2
Human bronchial smooth muscle cells (HBSMC) may regulate airway inflammation by secreting cytokines, chemokines and growth factors. The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to be elevated during airway inflammation and evoke airway hyperresponsiveness. We studied if HBSMC may be a source of NGF, BDNF and NT-3, and if so, how inflammatory cytokines may influence their production.
Basal and cytokine (IL-1β, IFN-γ, IL-4)-stimulated neurotrophin expression in HBSMC cultured in vitro was quantified. The mRNA expression was quantified by real-time RT-PCR and the protein secretion into the cell culture medium by ELISA.
We observed a constitutive NGF, BDNF and NT-3 expression. IL-1β stimulated a transient increase of NGF, while the increase of BDNF had a later onset and was more sustained. COX-inhibitors (indomethacin and NS-398) markedly decreased IL-1β-stimulated secretion of BDNF, but not IL-1β-stimulated NGF secretion. IFN-γ increased NGF expression, down-regulated BDNF expression and synergistically enhanced IL-1β-stimulated NGF expression. In contrast, IL-4 had no effect on basal NGF and BDNF expression, but decreased IL-1β-stimulated NGF expression. NT-3 was not altered by the tested cytokines.
Taken together, our data indicate that, in addition to the contractile capacity, HBSMC can express NGF, BDNF and NT-3. The expression of these neurotrophins may be differently regulated by inflammatory cytokines, suggesting a dynamic interplay that might have a potential role in airway inflammation.
Allergic asthma is characterised by an inflammatory airway obstruction induced by specific allergen [1]. In addition, structural cells of the allergic airways are often hyperresponsive to non-specific stimuli, which synergises with the inflammatory response to aggravate disease [2]. While the pathogenesis of allergen-induced inflammatory airway obstruction is relatively well understood [1], we know much less about the regulation of airway hyperresponsiveness. According to current models, it involves proliferation and phenotypic changes of structural cells (e.g. smooth muscle cells, fibroblasts) and nerve cells in the airways, which contribute to enhanced airway resistance [2, 3].
Neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were initially discovered as factors that regulate development, differentiation and survival of neurones (for review see [4]). However, neurotrophins have recently been implicated also in inflammatory responses. For example, NGF enhances survival, activation and mediator release from multiple cell types of the immune system, such as mast cells, lymphocytes and eosinophils (for review see [5, 6]). In agreement with a pathogenic role in allergic asthma, elevated levels of NGF, BDNF and NT-3 have been detected in blood and locally in the airways in patients with asthma [7–10], a response that can be further augmented after allergen challenge [9, 11]. In animal models, NGF and BDNF may contribute to the development of bronchial hyperresponsivness (BHR) [12–14]. A direct action on neurones may be one part of this response since NGF increases the number of neurones and the neuropeptide content in the airways [15, 16]. However, NGF has also been suggested to participate in tissue remodelling processes and fibrosis in the airways [17, 18], suggesting important roles for neurotrophins in many pathogenic processes that characterise pulmonary inflammation and allergic disease.
Different members of the neurotrophin family often show distinct functional effects, a phenomenon that has been mostly studied in the nervous system [19–21], but has also been observed in epithelial cells [22]. NGF and BDNF may also play distinct roles in airway inflammation. For example, antibody blockage of NGF affected early inflammatory responses in murine asthma while neutralisation of BDNF reduced only chronic airway obstruction and BHR but not inflammation [23, 24]. In addition, BDNF has been shown to enhance pollen-specific IgE production while NGF and NT-3 were without effect [25]. It is unknown whether similar functional differences between the neurotrophins are operating in human airway inflammation.
The source and regulation of neurotrophin expression in the airways is not fully understood. A local NGF production by structural cells, including airway smooth muscle cells, epithelial cells, fibroblasts and infiltrating inflammatory cells has been suggested in vivo and in vitro [10, 11, 26–28]. Compared to NGF, less is known about the cellular sources of BDNF and NT-3 in human airways, but the presence of BDNF and NT-3 in human bronchial smooth muscle (HBSMC) and epithelium has been implied using immunohistochemistry on bronchial biopsies from non-asthmatic subjects [28]. In the present study, we used primary HBSMC to study the expression patterns of three members of the neurotrophin family, NGF, BDNF and NT-3, after stimulation with inflammatory cytokines.
Our data show that HBSMC constitutively expressed all neurotrophins analysed. NGF and BDNF, but not NT-3, were induced by IL-1β, but with markedly different kinetics. While NGF was induced more rapidly and peaked at 6 hours after the start of stimulation, BDNF was maximally induced only after 24 hours. A recent study has identified a critical role for cyclooxygenase (COX) for BDNF production [29]. This led us to explore the possibility that an intermediary mediator, such as a COX-2-derived mediator, would be involved in the IL-1β-dependent BDNF secretion. Interestingly, the induction of BDNF was shown to involve a COX-2-dependent pathway. Furthermore, the addition of Th1 and Th2 cytokines affected synthesis of NGF but affected BDNF marginally. Our data suggest that HBSMC display a differential and complex transcriptional regulation of three different members of the neurotrophin family, which may provide a framework for differential functional effects of neurotrophins in airway inflammation and asthma, controlled by HBSMC and regulated by inflammatory cytokines.
Culture of human bronchial smooth muscle cells (HBSMC)
HBSMC in primary culture from a healthy donor (Promocell, Heidelberg, Germany) were grown in monolayer in DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS (Invitrogen, Rockville, MD, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 μg/mL amphotericin B (Fungizone®, all Sigma-Aldrich) and 0.12 IU/mL insulin (Lilly, St Cloud, France) in 25 cm2 flasks (Becton Dickinson Falcon, Franklin Lakes, NJ, USA). Cells at passage 9 were used in all experiments. Cells were positive for smooth muscle specific α-actin and in light microscopy the cells displayed the reported characteristics of viable smooth muscle cells in culture [30].
At 80% confluence cells (corresponding to 800 000 cells/25 cm2 flask) were growth arrested for 24 h in a low-FBS (0.3%) insulin-free DMEM. Time-dependent pattern of neurotrophin expression was studied with and without cytokines, IL-1β 10 U/mL (Boehringer Ingelheim, Mannheim, Germany), IL-4 and IFN-γ 10 ng/mL (both R&D Systems, Oxon, United Kingdom), for 0.5, 1, 2.5, 6, 24 and 48 h in fresh low-FBS medium. Dose-dependent effects of IL-1β, IL-4 and IFN-γ were studied by stimulating cells in fresh low-FBS medium for 6, 24 and 48 h with or without increasing cytokine concentrations; IL-1β 0.1–30 U/mL, IL-4 and IFN-γ 0.1–30 ng/mL. The effects of COX-inhibition on IL-1β-dependent neurotrophin secretion were studied by using the unselective COX-inhibitor indomethacin (10 μM) and the COX-2 inhibitor NS-398 (10 μM). The chosen dose of indomethacin and NS-398 has been shown to effectively inhibit IL-1β-stimulated COX activity [31]. The inhibitors were added 1 h prior to the addition of IL-1β (10 U/mL), and NGF and BDNF were measured in the cell culture supernatants following 48 h of IL-1β stimulation.
In preliminary studies IL-1β gave a maximal increase in NGF mRNA-expression at 6 h, whereas IFN-γ gave a significant increase at 48 h. Therefore, to evaluate effects of IFN-γ on IL-1β-stimulated NGF mRNA-expression, HBSMC were treated with IFN-γ (0.1–30 ng/mL) for 42 h, where after IL-1β (10 U/mL) was added and the stimulation continued for an additional 6 h. The effects of IFN-γ on IL-1β-stimulated BDNF and NT-3 mRNA expression were studied at both 6 and 48 h of stimulation with a combination of IL-1β (10 U/ml) and IFN-γ (0.1–30 ng/ml). The effects of IL-4 (0.1–30 ng/mL) on IL-1β (10 U/mL)-stimulated NGF mRNA-expression were studied following 6 h of combined stimulation. The effects of IL-4 (0.1–30 ng/mL) on IL-1β (10 U/mL)-stimulated BDNF and NT-3 mRNA-expression were studied following both 6 and 48 h of combined stimulation.
After stimulation cell supernatants were collected, centrifuged (+4°C, 400 × g, 10 min) and stored at -70°C until analysis. Cells were collected in 1 mL RLT lysis buffer (Qiagen Inc, Valencia, CA, USA) and kept at -70°C until analysis.
Extraction of total RNA and cDNA synthesis
Total RNA was extracted from the RLT lysis buffer using the RNeasy extraction kit and genomic DNA was removed by DNAse I (all products from Qiagen Inc), according to the manufacturer's protocol. RNA was reverse transcribed in a 20 μl final volume using 10 μl of total RNA, 20 mM random primers, 200 μM of each deoxyribonucleoside triphosphate (dNTP), 40 units RNAsin (all products from Pharmacia Biotech, Uppsala, Sweden) and 200 units SUPERSCRIPT™ II RNase H- Reverse Transcriptase (Invitrogen), according to the protocol and with the buffers supplied by the manufacturer. To enable detection of eventual genomic DNA contamination, non-reversed transcribed total RNA was diluted 1:1 with water whereafter it was analysed the same way as cDNA.
Quantification of neurotrophin cDNA by real-time PCR
The basal and IL-1β-, IL-4- and IFN-γ-stimulated NGF, BDNF and NT-3 mRNA expression was quantified following 0.5, 1, 2.5, 6, 24 and 48 h of cell culture using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) utilising the 5' nuclease method (TaqMan) with a two-step polymerase chain reaction (PCR) protocol (95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min). The PCR reaction was set up in a volume of 25 μl, with 2 μl cDNA diluted 1/5 and a final concentration of 1× Buffer A, 5 mM MgCl2, 0.05 U/μl AmpliTaq Gold (all from Applied Biosystems), 200 nM dNTP-mix (Pharmacia Biotech), 300 nM of each primer and 200 nM probe. All primers and probes were purchased from Applied Biosystems, including primers and probe for the housekeeping gene 18S rRNA, which sequences were commercially available. The neurotrophin primer and probe sequences were designed using the PRIMER EXPRESS Software (Applied Biosystems) according to the manufacturer's guidelines and the target primers and probes were for technical reasons placed within a single exon. None-reversed transcribed RNA was analysed in the same way as cDNA as a negative control. Primer and probe sequences are shown in Table 1. The probes were synthesised with the fluorescent reporter dye FAM (6-carboxy-fluorescein) attached to the 5'-end and a quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine) to the 3'-end. Relative quantification of the cDNA levels were performed using the 'Relative Standard Curve method', described in detail in User Bullentin #2 (Perklin Elmer Applied Biosystems, 1997), with amplification of neurotrophins and the housekeeping gene 18S in separate tubes. Briefly, standard curves for each neurotrophin and 18S were created using five serial dilutions (1:1, 1:2, 1:10, 1:20 and 1:100) of cDNA from the human foetal fibroblast cell line HFL-1 (American Type Culture Collection, Rockville, MD, USA). By plotting the values for the threshold cycle (CT = the first cycle in which the amount of amplicon exceeds the threshold) against the different dilutions a standard curve was obtained. All samples were run in duplicates and the mean values were used in further analysis. The relative amount of neurotrophin mRNA in each sample was then calculated as the ratio between the neurotrophin mRNA and the housekeeping gene 18S rRNA before samples were compared.
Table 1 PCR primers and TaqMan probes
Detection of neurotrophin expression by conventional PCR
Constitutive mRNA expression of neurotrophins by HBSMC was analysed following 0.5, 1, 2.5, 6, 24 and 48 h of cell culture by conventional PCR on a GeneAmp PCR System 9600 (Applied Biosystems). PCR was performed in a total volume of 20 μl using 2 μl of 1/5 diluted cDNA and a final concentration of 200 nM dNTP-mix (Pharmacia Biotech), 300 nM of each primer (Tabel 1), 1 × TaqGold-buffer II, 1.9 mM MgCl2 and 0.05 U/μl AmpliTaq Gold (all Applied Biosystems). The thermal cycling conditions were: 94°C for 12 min, followed by 30 cycles for 18S and 40 cycles for the neurotrophins of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, before ending with 72°C for 9 min. The products were analysed on a 1.5% agarose (Invitrogen) gel complemented with 0.025‰ ethidium bromide (Pharmacia Biotech), where a 1 kb DNA-ladder (Invitrogen) was run in parallel to the samples. The gels were pictured using a Kodak Digital Science 1D™ analysing system (Kodak Scientific Imaging Systems, New Haven, CT, USA).
Quantification of neurotrophin protein by enzyme-linked immunosorbent assay (ELISA)
To quantify the NGF, BDNF and NT-3 protein levels in cell culture supernatants, commercially available ELISA kits were used, according to the manufacturer's instructions (Promega, Madison, WI, USA). All measurements were performed in duplicate. Constitutive protein secretion was analysed following 2.5, 6, 24, and 48 h of cell culture for NGF and BDNF, and 2.5, 6, 24, 48 and 72 h of cell culture for NT-3. IL-1β stimulated protein expression was analysed at 2.5, 6, 24 and 48 h for all three neurotrophins. Protein expression following IL-1β stimulation and COX-inhibition was analysed following 48 h of stimulation. Prior to analysis of NT-3 the supernatant was concentrated using the Amicon Ultra-15 Centrifugal Filter Units (Millipore, Carrigtwohill, Ireland), with a molecular weight cut off of 5,000 Da, according to the protocol obtained from the manufacturer. The detection range for NGF and BDNF was 2.0–250 pg/mL, and for NT-3 2.4–150 pg/mL.
NGF, BDNF and NT-3 mRNA expression was formulated as the ratio of neurotrophin cDNA to 18S rRNA and expressed as neurotrophin cDNA/18S rRNA. Effects of cytokines on neurotrophins mRNA expression and protein secretion were expressed as change in mRNA (%) and protein release (pg/mL) from baseline level of time-related control. Results are presented as mean ± SEM of 3–10 independent experiments performed in duplicate. Raw data were compared using one-way analysis of variance (ANOVA) followed by Tukey's post test. Differences were considered significant at p≤0.05. All analyses were performed using GraphPad InStat 3.01 (Graph Pad Software, San Diego, CA, USA).
Neurotrophin mRNA and protein expression in HBSMC
In order to study the expression of NGF, BDNF and NT-3 by unstimulated cultured HBSMC, we analysed cellular mRNA as well as secreted protein in the culture supernatants, at six different time points (0.5, 1, 2.5, 6, 24 and 48 h) after the start of the cultures. HBSMC constitutively expressed all three neurotrophins at the mRNA level. In Figure 1A a representative expression is pictured following 2.5 h of incubation. Furthermore, NGF and BDNF proteins accumulated in the culture supernatants over time. Hence, NGF and BDNF reached detectable levels following 2.5 h of cell culture and increased further over the next 48 h (Figure 1B). In contrast to NGF and BDNF, NT-3 protein was made in very low amounts, reaching detectable levels (3.1 ± 0.7 pg/mL) only after 72 h of culture (not shown).
Constitutive expression of neurotrophins by non-treated HBSMC evaluated by RT-PCR and ELISA. (A) Expression of NGF, BDNF, NT-3 mRNA and 18S rRNA. Reversed transcribed (RT+) and non-reversed transcribed (RT-) mRNA or rRNA are displayed. (B) NGF (white bars) and BDNF (black bars) protein secretion determined by ELISA. Data presented as mean ± SEM of 3–4 independent experiments performed in duplicate. In (B) ***: p < 0.001 for NGF, and #: p < 0.05 and ##: p < 0.01 for BDNF, versus 2.5 h
Effects of IL-1β on neurotrophin mRNA and protein expression
We next investigated the ability of the proinflammatory cytokine IL-1β to induce neurotrophin expression in HBSMC. At 2.5 h after addition of IL-1β (10 U/mL), NGF mRNA expression was significantly increased compared to control cultures. A maximal increase was reached after 6 h of stimulation, after which mRNA levels dropped to near basal levels (Figure 2A). A corresponding increase in NGF protein secretion was detected, reaching a maximal increase after 24–48 h of IL-1β stimulation (Figure 2B). Interestingly, the induction of BDNF mRNA expression by IL-1β (10 U/mL) showed a completely different kinetics. At 6 h, no increase was seen and only at 24 h after stimulation (which was our next observation point) we observed a significant upregulation of BDNF mRNA (Figure 2C). A corresponding elevation of BDNF protein secretion was evident at 24 h, a secretion that was further enhanced at 48 h (Figure 2D). The increases in NGF and BDNF mRNA expression by IL-1β- were both dose-dependent (0.1–30 U/mL) (Figure 3). NT-3 mRNA expression was unaltered at any tested dose of IL-1β as evaluated at 2.5, 6, 24 h (not shown) and 48 h (Figure 3).
Time-course effects of IL-1β (10 U/mL) on NGF and BDNF mRNA and protein expression in HBSMC evaluated with real-time RT-PCR and ELISA, respectively. (A) NGF mRNA; (B) NGF protein; (C) BDNF mRNA and (D) BDNF protein. The IL-1β-dependent effects on neurotrophin mRNA expression and secreted protein are expressed as change in neurotrophin mRNA (%) and protein release (pg/mL) from baseline level of time-related control. Unstimulated control is set to 0 (% or pg/mL, respectively). Data are presented as mean ± SEM of 4–10 independent experiments performed in duplicate. In (A) and (C): **: p < 0.01 versus 0.5 h, ***: p < 0.001 versus 0.5 h. In (B) and (D): ***: p < 0.001 versus 2.5 h.
Dose-dependent effects of IL-1β on neurotrophin mRNA expression in HBSMC evaluated by real-time RT-PCR. NGF mRNA expression evaluated at 6 h (solid squares); BDNF mRNA expression evaluated at 48 h (open triangle); NT-3 mRNA evaluated at 48 h (closed triangles). The IL-1β-dependent effects on neurotrophin mRNA expression are expressed as change in neurotrophin mRNA (%) from baseline level of time-related control. Unstimulated control is set to 0 %. Data are expressed as mean ± SEM of 3 independent experiments performed in duplicate. *: p < 0.05 for NGF and #: p < 0.05 for BDNF versus unstimulated control.
Effects of COX-inhibitors on IL-1β-stimulated NGF and BDNF protein secretion
We hypothesised that the slower induction kinetics of BDNF compared to NGF after IL-1β stimulation reflected an involvement of COX in the induction of BDNF but not of NGF. To test this, we evaluated the effects of the COX-inhibitors indomethacin and NS-398 (both 10 μM) on the secretion of these two neurotrophins. BDNF secretion after IL-1β (10 U/mL) stimulation was significantly inhibited by both indomethacin and NS-398 (Figure 4). None of the COX inhibitors altered basal NGF or BDNF secretion (not shown) and they were inefficient in preventing IL-1β-stimulated NGF secretion (Figure 4). These data suggest that the production of BDNF, but not of NGF, in HBSMC involves COX-2 and the synthesis of prostaglandins.
Effects of the COX-inhibitors indomethacin and NS-398 (both 10 μM) on IL-1β (10 U/mL)-stimulated NGF (A) and BDNF (B) secretion by HBSMC, evaluated by ELISA. Data are presented as mean ± SEM of 6 independent experiments. ***: p < 0.001 versus control, and #: p < 0.05, ##: p < 0.01 versus IL-1β (10 U/mL) alone.
Effects of IFN-γ and IL-4 on neurotrophin mRNA expression
Allergic asthma is characterised by a disturbed balance between Th1 and Th2 cytokines towards Th2 dominance [1]. To test the hypothesis that Th1 and Th2 cytokines displayed different effects on neurotrophin gene expression by HBSMC, we treated HBSMC with either IFN-γ or IL-4 and measured the relative mRNA content of NGF and BDNF at various time points after addition of the cytokine. IFN-γ enhanced NGF mRNA expression in a time- (Figure 5A) and dose (0.1–30 ng/mL, not shown)-dependent manner, displaying a maximally enhanced NGF mRNA level at 48 h after the start of stimulation (Figure 5A). In contrast to NGF, BDNF mRNA expression was not induced. If anything, we observed a small decrease in relative BDNF mRNA content during the first 6 h of IFN-γ stimulation, after which it returned to baseline levels at 48 h after the start of the culture (Figure 5B). NT-3 expression was unaltered in response to IFN-γ under the tested conditions (not shown). In contrast to IFN-γ, IL-4 (0.1–30 U/mL) did not have any affect on basal NGF, BDNF or NT-3 expression at any of the tested intervals (0.5, 1, 2.5, 6, 24, 48 h) (not shown).
Time-course effects of IFN-γ (10 ng/mL) on NGF (A) and BDNF (B) mRNA expression in HBSMC evaluated by real-time RT-PCR. The IFN-γ-dependent effects on neurotrophin mRNA expression are expressed as change in neurotrophin mRNA (%) from baseline level of time-related control. Unstimulated control for each time-point is set to 0 %. Data are presented as mean ± SEM of 6 independent experiments performed in duplicate. *: p < 0.05, **: p < 0.01, ***: p > 0.001 versus 0.5 h.
Effects of different combinations of IL-1β, IFN-γ, IL-4 on neurotrophin mRNA expression
During an inflammatory response in vivo, T cell-derived cytokines such as IFN-γ and IL-4 are likely to act in concert with proinflammatory cytokines, such as IL-1β. An interesting question was therefore whether IFN-γ or IL-4 would work in synergy with IL-1β. Since the kinetics of NGF induction in HBSMC was different between IFN-γ (peak at 48 h) and IL-1β (peak at 6 h) we cultured HBSMC with IFN-γ for 42 h and added IL-1β during the last 6 h of culture to maximise the effect of each cytokine. Under such culture conditions, we observed a potent and dose-dependent synergistic behaviour on NGF secretion by IFN-γ (0.1–30 ng/mL) and IL-1β (10 U/mL) (Figure 6A). We also tested IL-4 (0.1–30 ng/mL) in this protocol. To our surprise, we noted that IL-4 reduced the stimulatory effects of IL-1β (10 U/mL) on NGF mRNA expression, reaching a maximal reduction with an IL-4 dose of 10 ng/mL as evaluated at 6 h of combined stimulation (Figure 6B), despite the fact that IL-4 did not affect NGF secretion by itself.
Dose-dependent effects of IFN-γ and IL-4 on IL-1β (10 U/mL)-stimulated NGF expression in HBSMC evaluated by real-time RT-PCR. Solid triangles illustrate effects of IFN-γ (A) and IL-4 (B) alone and solid squares illustrate IL-1β in combination with IFN-γ (A) and IL-4 (B). The IFN-γ- or IL-4-dependent effects on IL-1β-stimulated NGF mRNA expression are expressed as change in NGF mRNA (%) from baseline level of time-related control. Unstimulated control is set to 0 %. Data are presented as mean ± SEM of 3–4 independent experiments performed in duplicate. *: p < 0.05, **: p < 0.01, ***: p < 0.001 versus IL-1β (10 U/mL) alone.
In contrast, the IL-1β (10 U/mL)-stimulated expression of BDNF mRNA was not altered by IFN-γ or IL-4 at any tested dose (0.1–30 ng/mL) or time (6 and 48 h, data not shown). In addition, NT-3 mRNA expression was not altered by combining IL-1β (10 U/mL) with either INF-γ or IL-4 at any tested dose (0.1–30 ng/mL) or time (6 and 48 h, data not shown).
IL-1β is a pro-inflammatory cytokine of known importance in asthma pathogenesis [32]. The effect of IL-1β on NGF expression by structural cells in the airways, including HBSMC, has already been studied in some detail [27]. However, our study is the first to use HBSMC to systematically investigate how IL-1β-induced expression of NGF relates to IL-1β-induced expression of BDNF and NT-3, two other members of the neurotrophin family. This is an important question to address since these three neurotrophins have been shown to display different functional activities, including in asthmatic disease [19–22, 24, 25]. We found several differences in the regulation of these three neurotrophins. First, mRNA synthesis of NGF and BDNF, but not of NT-3, was stimulated in a dose-dependent fashion by IL-1β. Since nonstimulated HBSMC expressed mRNA from NT-3, the lack of induction of NT-3 cannot be explained by lack of basal gene transcription. Rather, differences in critical IL-1β response elements in the NT-3 gene may be crucial. Alternatively, NT-3 expression may require a different set of intermediate factors compared to NGF and BDNF that may be lacking in our HBSMC. It is interesting to note that NT-3 expression, but not NGF or BDNF expression, is transcriptionally downregulated in chronic obstructive pulmonary disease, suggesting that cells from the airways may be poor at producing NT-3 compared to other neurotrophins [33].
A second difference we observed was that the two neurotrophins that were induced by IL-1β (NGF and BDNF) showed markedly different kinetics of induction. While NGF mRNA induction was transient and high already at 6 h after addition of IL-1β, BDNF showed a more sustained expression pattern with a slower onset and a maximal induction after 24 h. With regard to the kinetics of NGF induction, our data is in line with that of Freund and co-workers, who similarly demonstrated a rapid and transient increase of NGF by IL-1β in airway smooth muscle cells, although Freund and co-workers reported a maximal upregulation at 2.5 h of IL-1β stimulation [27]. It may be speculated that the difference in time-course effects by IL-1β on NGF expression is dependent on the origin of the smooth muscle cells. Hence, the question of whether cells from different locations in the bronchial tree have different synthetic properties needs further investigation. In both our, and in the study by Freund and co-workers, NGF protein induction was detected with a significant delay. The dramatic differences in mRNA and protein induction between NGF and BDNF shown in our study demonstrate remarkable heterogeneity in the regulation of neurotrophin expression in HBSMC. One possible explanation for this difference could be the involvement of additional intermediary mediators in BDNF induction. Our finding that secretion of BDNF, but not NGF, was inhibited by the unselective COX inhibitor indomethacin and the COX-2 inhibitor NS-398 imply the prostaglandins as such intermediary mediators. A regulatory role of COX-2 in BDNF secretion adds to recent data showing that prostaglandins can stimulate the expression of BDNF in mouse astrocytes [34] and that COX inhibitors are able to counteract BDNF-mediated effects of spatial learning in a mouse in vivo model [29]. Interestingly, Toyomoto and co-workers found that also NGF expression was stimulated by prostaglandins [34]. More work is needed to investigate whether our observation of a differential role of COX inhibitors on NGF and BDNF secretion in HBSMC will also be observed in vivo.
We also found that the lymphocyte-derived cytokines IFN-γ and IL-4 affected neurotrophin expression differently. For example, the Th1 cytokine IFN-γ induced NGF expression but not BDNF expression, and IFN-γ synergised with IL-1β to enhance NGF gene transcription, implying an important role for IFN-γ in the airway inflammation. In addition, we found IL-4, a typical Th2 cytokine, to downregulate IL-1β stimulated NGF expression. In relation to the asthma, these results are surprising, but may reflect a more complex involvement of Th1/Th2 cytokines in the asthmatic airway inflammation, as also suggested from recent studies in humans [35, 36] as well as from animal models of asthma [37]. More work is needed to clarify the role and pathogenic importance of Th1/Th2 cytokines and neurotrophin release in asthma pathogenesis.
Proliferation and survival of mast cells, eosinophils and lymphocytes in the airways may be of importance in establishment and maintenance of a chronic inflammation. In asthmatic subjects, NGF, BDNF, NT-3 and NT-4 significantly enhanced airway eosinophil survival [38]. Mast cells located in human asthmatic bronchus have been demonstrated to express the neurotrophin receptor TrkA [11] and NGF may enhance mast cell proliferation and survival [6]. Since mast cells have been shown to be in close contact with smooth muscle cells in the asthmatic bronchus [39], smooth muscle cell-derived NGF has the potential to influence airway mast cells. B and T lymphocytes, including CD4+ T cells, express the neurotrophin receptors TrkA and TrkB, and NGF has been shown to enhance proliferation of B and T cells and survival of B cells [6]. Thus, there is evidence for a broad regulatory role for neurotrophins in the airways. In addition to cells of the immune system, neurones may also be targets for the neurotrophins in the airways. Hence, NGF has been shown to increase the number of neurones and the neuropeptide content in the airways [15, 16], and both NGF and BDNF may evoke airway hyperreactivity in the airways [12–14]. In this respect, it is interesting to note that while both NGF and BDNF have been shown to evoke airway hyperreactivity in animal studies [12–14], they may have distinct roles in allergen-dependent airway obstruction [24]. Hence, NGF may play a role in the early airway response (EAR) in asthma, since anti-NGF attenuated the early allergen-induced bronchoconstriction [13, 40, 41], and NGF causes histamine release from mast cells [42] and basophils [43]. Anti-NGF-treated mice also reduced the eosinophil recruitment to the airways and decreased IL-4 and IL-5 production [40], and IL-1β-induced airway hyperactivity in isolated human bronchus [44]. BDNF, on the other hand, seems not to influence the EAR but rather to abrogate chronic airway obstruction [23]. Thus, NGF and BDNF may influence different phases of the allergic response. Interestingly, there are also indications of a differential in vivo production of the neurotrophins in allergic airway inflammation. Hence, following allergen challenge, NGF levels in bronchoalveolar lavage were increased fourfold at 18–24 h, whereas the increase in BDNF peaked after 1 week [24]. An interesting possibility is that that the differential functional effects and secretion observed between NGF and BDNF in vivo might somehow reflect a differential release patterns, such as the one described in our study.
In the present study, HBSMC were obtained from a healthy donor. An important next step will be to obtain airway smooth muscle cells from asthmatic donors, and ask whether such cells are similar to HBSMC from normal individuals or in some way biased towards production of a different set of neurotrophins [45]. Also, an important next step will be to test whether inflammatory and asthma-associated mediators might have different effects on asthmatic as compared to non-asthmatic bronchial smooth muscle [45, 46].
In conclusion, this study shows that HBSMC are a source of NGF, BDNF and NT-3 in the airways, that IL-1β, IFN-γ and IL-4 may alter this production differently, and that the IL-1β-dependent BDNF, but not IL-1β-dependent NGF, secretion is COX-2-dependent. Taken together, we propose a paracrine interaction between smooth muscle cells, neurones, inflammatory cells and structural cells in the vicinity, in which neurotrophins derived from bronchial smooth muscle may promote airway hyperreactivity, inflammation and tissue remodelling.
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Pang L, Knox AJ: Effect of interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells. Br J Pharmacol 1997, 121:579–587.
Barnes PJ, Chung KF, Page CP: Inflammatory mediators of asthma: an update. Pharmacol Rev 1998, 50:515–596.
Groneberg DA, Peiser C, Eynott PR, Welker P, Erbes R, Witt C, Chung KF, Fischer A: Transcriptional down-regulation of neurotrophin-3 in chronic obstructive pulmonary disease. Biol Chem 2005, 386:53–59.
Toyomoto M, Ohta M, Okumura K, Yano H, Matsumoto K, Inoue S, Hayashi K, Ikeda K: Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures. FEBS Lett 2004, 562:211–215.
Corrigan CJ, Kay AB: CD4 T-lymphocyte activation in acute severe asthma. Relationship to disease severity and atopic status. Am Rev Respir Dis 1990, 141:970–977.
ten Hacken NH, Oosterhoff Y, Kauffman HF, Guevarra L, Satoh T, Tollerud DJ, Postma DS: Elevated serum interferon-gamma in atopic asthma correlates with increased airways responsiveness and circadian peak expiratory flow variation. Eur Respir J 1998, 11:312–316.
Dahl ME, Dabbagh K, Liggitt D, Kim S, Lewis DB: Viral-induced T helper type 1 responses enhance allergic disease by effects on lung dendritic cells. Nat Immunol 2004, 5:337–343.
Nassenstein C, Braun A, Erpenbeck VJ, Lommatzsch M, Schmidt S, Krug N, Luttmann W, Renz H, Virchow JCJ: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 are survival and activation factors for eosinophils in patients with allergic bronchial asthma. J Exp Med 2003, 198:455–467.
Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID: Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002, 346:1699–1705.
Path G, Braun A, Meents N, Kerzel S, Quarcoo D, Raap U, Hoyle GW, Nockher WA, Renz H: Augmentation of allergic early-phase reaction by nerve growth factor. Am J Respir Crit Care Med 2002, 166:818–826.
De Vries A, Engels F, Henricks PA, Leusink-Muis T, Fischer A, Nijkamp FP: Antibodies directed against nerve growth factor inhibit the acute bronchoconstriction due to allergen challenge in guinea-pigs. Clin Exp Allergy 2002, 32:325–328.
Bruni A, Bigon E, Boarato E, Mietto L, Leon A, Toffano G: Interaction between nerve growth factor and lysophosphatidylserine on rat peritoneal mast cells. FEBS Lett 1982, 138:190–192.
Bischoff SC, Dahinden CA: Effect of nerve growth factor on the release of inflammatory mediators by mature human basophils. Blood 1992, 79:2662–2669.
Frossard N, Naline E, Olgart Hoglund C, Georges O, Advenier C: Nerve growth factor is released by IL-1beta and induces hyperresponsiveness of the human isolated bronchus. Eur Respir J 2005, 26:15–20.
Chambers LS, Black JL, Ge Q, Carlin SM, Au WW, Poniris M, Thompson J, Johnson PR, Burgess JK: PAR-2 activation, PGE2, and COX-2 in human asthmatic and nonasthmatic airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2003, 285:L619–27.
Burgess JK, Ge Q, Boustany S, Black JL, Johnson PR: Increased sensitivity of asthmatic airway smooth muscle cells to prostaglandin E2 might be mediated by increased numbers of E-prostanoid receptors. J Allergy Clin Immunol 2004, 113:876–881.
The present work was funded by the Swedish Research Council, Swedish Heart and Lung Foundation, Swedish Society of Medicine, Swedish Asthma and Allergy Association, Magnus Bergvall's Foundation, Tore Nilson's Foundation, Torsten and Ragnar Söderberg's Foundations, Åke Wiberg's Foundation, and Karolinska Institutet. We thank Dr P. Hoglund for valuable discussions.
Department of Medicine, Division of Respiratory Medicine, Lung Research Laboratory, Karolinska Institutet and Karolinska University Hospital Solna, 171 76, Stockholm, Sweden
Cecilia Kemi
, Johan Grunewald
, Anders Eklund
& Caroline Olgart Höglund
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77, Stockholm, Sweden
Caroline Olgart Höglund
Search for Cecilia Kemi in:
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Correspondence to Caroline Olgart Höglund.
The author(s) declare that they have no competing interests.
CK carried out the majority of the experiments, did the statistical analysis and participated in writing the manuscript. JG participated in the design of the study and writing the manuscript. AE participated in the design of the study. COH conceived of the study and its design, performed its co-ordination, did parts of the experiments and statistical analysis and participated in writing the manuscript. All authors have read and approved the final manuscript.
Kemi, C., Grunewald, J., Eklund, A. et al. Differential regulation of neurotrophin expression in human bronchial smooth muscle cells. Respir Res 7, 18 (2006). https://doi.org/10.1186/1465-9921-7-18
Airway Smooth Muscle Cell
Nerve Growth Factor Expression
Nerve Growth Factor mRNA
BDNF mRNA Expression


text:
Building an Inclusive, Human Culture
Brittany Decker tells people she gets to talk on the phone for a living – and enjoys it. As one of our Talent Acquisition Specialists, she's the first person a potential Continental employee meets. Here's what an engineer of our culture has to say about it.
What Brought Me to Continental.
I was recruited by Continental's head of Talent Acquisition through an external recruiting service, which is funny because I used to recruit for Continental at a previous company. I learned about the culture and benefits and I made a point to say I want to work for them some day. It's almost like I put it in the universe. It'll be two years with the company this September. I've been in the automotive recruiting industry for 10 years.
A big part of Talent Acquisition's responsibility is to help hire the right candidate, but also to ensure we are building a diverse and inclusive team. We're always working to improve our diversity recruiting and eliminate unconscious biases, and as someone who is a part of the LGBTQ community, it's something I'm especially passionate about. When we're talking about diversity and inclusion, it's more than ethnicity or sexual orientation, it's an ever-evolving thing. It's how employees feel coming to work every day.
Even though this is the biggest company I've ever worked for, I don't feel like a number. Everyone is professional, yet open and human. My team and I were sitting at the lunch table one time and our CEO came and sat with us. We were talking about dogs and boats. It's the small things like this that contribute to a culture of inclusion.
But an Inclusive Culture Is not Built Overnight.
One of the ways we're promoting diversity and inclusion is through Continental Young Professionals – an idea a colleague of mine and I had and started a year ago. Now, we already have around 250 members. We get a lot of the "Well, I'm not young. I don't fit into that category."
But we have a saying: If you feel young, you look young, you want to be young, or you're young at heart, we want you to join. We can't have a full, diverse resource group – or company – if we don't also include people who are further along in their careers.
We have a few things we really like to focus on. One is career development. Another is community outreach. There's a networking aspect, too. Our kickoff was a bowling event. We had 60 available slots and they filled up within 48 hours. We also team up closely with the Continental Women's Network to host events.
As Someone Who Is out at Work, I Feel Supported by Continental.
I mentioned earlier that our culture is very open and human, and I feel that every day – and it goes beyond my immediate colleagues. Last June, our CHRO raised rainbow flags alongside our Continental flags at our global headquarters and is now a Pride month tradition. For the automotive industry, this was a progressive statement and meant a lot to me that our executive leadership wanted to be a part of it.
Being a part of the LGBTQ community is just one aspect of who I am, my colleagues respect me for my hard work and knowledge. I have always been accepted amongst my team and throughout the organization.
I am not an engineer, well maybe one at heart. One day during a meeting, I was talking to some of my engineering colleagues about the Ford F-150, because I drive one. They were telling me about some of the technology in my truck. They spoke with me like I was one of the team, even though I only know enough to be dangerous to explain the role to a new candidate. It's so cool to learn from the people who are creating the technology.
If You Are Looking to Help Change the World…
Thomas T
Employer Branding Specialist based in the Silicon Valley
people@conti.de
5 Tips For How to Be a Successful Field Sales Rep – Virtually
Father and Son – United in Safety
June 19, 2020 Elmar Fuchs (left) and his son, Lucas (right), work at each end of the engineering spectrum. Together, they're making the world safer for all road users.
Virginia Plant Shifts Gears, to Make COVID-19 Face Shields
May 18, 2020 The plastic windows that you see on a Jeep Wrangler with a soft top roof?
Jon Stone: "That Was a Sign to Me: Keep Working on This"
April 29, 2020 In the wake of tragedy, it's Jon Stone's mission to make wrong-way driving fatalities a thing of the past.
How These Friends Help Support Women at Work
December 13, 2019 Carolyn and Janet founded the Women's Network in the Detroit area and now sponsor 28 global networks.
What it's like to be a military veteran at Continental
November 05, 2019 Joe Wright had the same fears other veterans shave when they transition from the armed forces to civilian life.
An executive chef finds his bliss in… coal mine conveyor belts?
September 23, 2019 Mike Fishbeck has a strong sense of adventure and a willingness to learn.


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Triton Submarines Announces Sale of First Triton 7500/3 Model to Rev Ocean
Sebastian, Florida, 19 September 2019 –Triton Submarines LLC is proud to announce the sale of the first Triton 7500/3 series submersible to Project REV Ocean – a not for profit foundation established and funded by Norwegian businessman and visionary Kjell Inge Røkke. The Triton 7500/3 will be the deepest diving acrylic pressure hull equipped submersible ever conceived and iscapable of diving to 7,500 feet or 2,286 meters with three people aboard.
The revolutionary Triton 7500/3 features a transparent acrylic sphere as the pressure hull, which provides occupants with an immersive and completely unobstructed view of their surroundings while keeping them safe and comfortable at one atmosphere regardless of the diving depth. The Triton 7500/3 acrylic sphere is made using proprietary technology pioneered by Triton's exclusive acrylic manufacturing partners, which results in a finished material that is stronger, clearer and superior to anything previously made. In addition to offering unprecedented levels of passenger comfort, Triton submersibles are simple to operate, easy to maintain, safe and reliable.
"We are thrilled Project Rev Ocean chose to add a Triton 7500/3 submersible to the vast arsenal of subsea tools and equipment on their entirely unique and groundbreaking vessel. The capacity to visit such extreme depths and see things not possible any other way will dramatically enhance the experience of being aboard and create more ocean advocates. Project Rev Ocean is a once in a lifetime opportunity and we are honored to be a part of Mr. Røkke's ambitious and exciting vision," said Triton President Patrick Lahey.
Said CEO of REV Ocean Nina Jensen, "Having the Triton 7500/3 onboard our research vessel truly elevates REV Ocean to a level of unparalleled opportunity. Putting three people in a transparent acrylic sphere submersible with the capacity to reach such incredible depths will certainly inspire, educate, and motivate the global community to support our core mission of ensuring we have one healthy ocean."
REV Ocean is the world's largest and most advanced research and expedition vessel. The ship is 182.9 meters in length overall and has the capacity to carry up to 55 scientists and 35 crew members. Equipment onboard includes scientific trawls, sonar systems, laboratories, auditorium and classrooms, a moonpool, AUV, Triton 7500/3 submersible, and an ROV with a 6,000 meter diving depth and advanced communication equipment.
REV Ocean, will be equipped for conducting missions that cover the entire marine ecosystem. The vessel will be used by scientists and innovators for 'solutions'-oriented research that examine issues such as the impact CO2 emissions have on the ocean, plastic pollution and unsustainable fishing practices and highlights the possibility for positive, sustainable and much needed change.
Added REV Ocean Science Director Alex Rogers, "I have dived with Triton subs on numerous expeditions and they provide unique platforms for visiting the ocean and viewing seascapes in a way that is not possible with other deep diving equipment. The new Triton 7500/3 will enable REV Ocean scientists to visit vulnerable marine ecosystems as well as reach the most productive zones of seamounts and other features such as deep-sea canyons and hydrothermal vents."
About Triton Submarines, LLC
Triton Submarines of Sebastian, Florida, is the most experienced civil submarine producer in the world today – and the only contemporary manufacturer of acrylic and titanium pressure-hull-equipped personal submarines to deliver multiple classed and certified vessels capable of diving to 3,300 feet (1,000 meters) or more. Triton Submarines' senior staff have over 350 years of combined experience with more than 80 different submersibles, and their operations team members have together logged over 25,000 dives. Triton clients also enjoy superlative after-sales service and technical support from a company dedicated to their total satisfaction.
Triton Submarines, through its recently delivered 36000/2 model and contribution to the Five Deeps Expedition, engaged in record-breaking dives to the deepest point of the world's five oceans with the first submersible ever certified for Full Ocean Depth operation.
For further information and imagery
Media Enquiries, high-resolution imagery and additional technical details can be found in the press pack available from Kelly Downey of Shamin Abas Ultra-Luxury Brand Marketing & Business Development (+1 212 734-5500) or +(1 517 231-7775).
kelly@shaminabas.com
Download Press Release Here
Caroline Alex-Ramsay2019-09-19T20:51:31+01:00
Home|Triton Submarines Announces Sale of First Triton 7500/3 Model to Rev Ocean


text:
MM Lee finds climate change a 'woeful' topic
Governments still lack the will to stop environmental degradation, he says
By Lynn Lee
Today Online 26 Sep 07
Melting ice cap chills MM
Nazry Bahrawi
HE WILL be sad if the polar bears vanish from the North Pole, but sadder still should the ice cap melt at the South Pole.
These dire consequences of global warming featured prominently in Minister Mentor Lee Kuan Yew's inaugural Singapore Maritime Lecture at the Fullerton Hotel yesterday.
"When the polar bear disappears from the Arctic, I will be sad when I see them at the zoo. But I will be sadder when the Antarctic ice cap melts � because the water level will rise so much," he said.
The calamity confronting humanity has no easy solutions nor are governments doing enough to tackle this problem, said Mr Lee.
"I am not optimistic about the will of governments to attack this cause," he told some 300 top maritime executives, in response to a question about how Singapore can contribute to climate change.
"I do not see any leaders saying let us eat less, eat more vegetables, eat less meat."
Giving examples of how the global environment has changed over the years, Mr Lee recounted seeing clear blue skies when he visited Beijing in 1999 to attend China's 50th anniversary celebrations only to learn later that this was so because the factories had stopped operations for two weeks.
"I do not see them stopping for two years. So we all face a problem," said Mr Lee.
Australia is taking a step in the right direction, he said, by setting aside about $200,000 to conserve Borneo's rainforests.
But closer to the hearts of the audience was the impact global warming would have on the maritime industry. He said: "We are witnessing the opening moves by various countries over the North-west and Northern Passage, as a result of the melting of polar ice caps over the Arctic. In the next few decades, shipping lanes between the Pacific and the Atlantic may switch to these Arctic routes during the summer season."
Mr Lee said he is open to suggestions on how to make the maritime industry greener, saying: "If you can feed us as to how the maritime industry can improve itself without becoming too uncompetitive ... we will push it."
During the nearly hour-long session, Mr Lee had also shared his views on international relations and terrorism.
CLIMATE change is a topic Minister Mentor Lee Kuan Yew finds 'woeful' because he is not optimistic about the will of governments to stop environmental degradation.
Already, rising global temperatures have caused ice in the Canadian Arctic to melt, he said. Ice caps in the Antarctic are also under threat.
'When the polar bears disappear from the Arctic, I'll be sad, but I will be sadder when the Antarctic ice cap melts,' Mr Lee told his audience of maritime industry bigwigs during an hour-long dialogue at the inaugural Singapore Maritime Lecture.
Ice in the Arctic is already in the sea, so water levels will not rise much when it melts, he explained. In the Antarctic, on the other hand, ice rests on land. When it melts, water levels will rise significantly, he said.
'So when I sit back and think about all these things, I get rather pensive,' Mr Lee said at the Fullerton Hotel. 'But at the same time, one mustn't give up hope,' he said.
The possibility of rising sea levels is high on Singapore's radar. The Republic has already engaged the Dutch to teach it how to build dykes.
Mr Lee also responded to a question on what Singapore could do to promote a cleaner world for the shipping industry, particularly in Asia. He said he had not fully studied ways to make shipping more green, but he knew that the type of fuel used could make a difference, though it could come at a higher cost.
His verdict: More ideas are needed on how to balance environmental protection with staying competitive.
'So if you can feed us with how the maritime industry can improve itself without becoming too uncompetitive and cutting the pollution, we will push it,' he said.
He pointed out that when it came to climate change, it was hard to get rising powers like China and India to agree to stem the process. He said their answer would probably be: Other countries have polluted the world before us, and if we stop now, it will affect industrialisation.
What can work, said Mr Lee, is for climatologists to predict what will happen in 20 or 30 years if nothing is done to stop environmental degradation. They can, for instance, stress that if glaciers in the Himalayas and the Tibetan plateau melt away, floods will ensue, he said.
But past the rainy season, rivers will dry up and scores of people who live along their banks will have to find a new way of life. Countries will then have to accept this truth:
'Never mind who gave what in the past. We're now at this junction. If you continue like this and I continue like this, we're both dead.'
Mr Lee's comments struck Mr Felix Scheder-Bieschin Jr, managing director of shipping firm Macs Maritime Carrier, who said: 'For a man of his wisdom, he was very open about not having the answer to the climate change issue.'
Vulnerable to rising seas, Singapore envisions a giant seawall
By Wayne Arnold International Herald Tribune 29 Aug 07
Global impact of shorter shipping route Straits Times 16 Sep 07
Green future: Why MM is not optimistic By Lynn Lee Straits Times 7 Aug 07
Related articles on Singapore: general environmental issues


I want to get the S9-R Mini Sedan (SILVER).
Maukilo is an online retailer that specializes in heirloom-quality European and American designed toys. Their motto is "Buy Once. Play Forever." so you know that durability and craftsmanship are their top priority. Maukilo is owned by HABA and shares a warehouse space with the venerable German toymaker in Skaneateles, New York, allowing them to offer more than 1,500 HABA items, the widest selection of HABA products in the U.S.
We were offered a chance to review an item from Maukilo's vast catalog and chose the HABA' Arranging Game Corner to Corner. This "game" is a set of small building blocks that can be combined in an endless variety of patterns and 3-D structures.
My husband is a sculptor and woodworker and he was very impressed with the Teutonic precision of the blocks and their durable paint job. There are 62 pieces in the HABA' Arranging Game Corner to Corner and Roo and Jasper had a great time trying to decide what to do with them.
HABA' Arranging Game Corner to Corner is a great gift idea for kids beyond the sticking-everything-in-the-mouth phase. The blocks are pretty small, around LEGO-sized, so I might worry about them being a choking hazard for younger children. Otherwise, they are totally engaging on their own and make a nice companion set to other building blocks.
To enter and win a $25 gift certificate to Maukilo, please visit Maukilo.com and comment on this post with what you'd like to spend it on.
Entries will be accepted through December 8th and I'll pick the winner using Random.org. US readers are eligible.
For an additional entry, post my button on your blog (see code on sidebar), follow my blog, follow me on Twitter, fan me on Facebook, fave me on Technorati or subscribe via email or subscribe via reader. Leave an extra comment letting me know what you did for each additional entry.
Splashing Set is a cute gift!
i would probably get more pieces to accompany our ball track, maybe the sound staircase or the domino rally.
i subscribed to your emails via feedburner.
I would get the: Stacking Toy Zoolino Pull Toy, great for my 2 year old daughter!
The Stacker is made from recycled milk jugs!!! How cool is that. I'd buy that for sure.
I like their Bouncy Rody Horses. They look so fun!
I follow you on Twitter as wyomingmom.
I like you on Facebook. Jaimie K****.
I would get the Dado Cubes.
I would love the Automoblox T9 Pickup Truck (GREEN) for my son!
i would love to spend it on the fun stacker and the ice cream cone!
I like the mini-knit dolls.
I'd get the Twinkle Wink Suction Cup Wire Maze, how fun!


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More flights: Benin to Botswana. No direct flights from Cotonou (COO) to Gaborone (GBE), Botswana found. Prices of flights to Gaborone sourced on Wednesday 1st August 2018 at 11:07.


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Most master's and doctoral students at UNL are required to complete a written comprehensive exam, also known as "comps", to enter candidacy for their degree program. This is usually the last major task you need to complete before you can begin your thesis or dissertation research.
Comprehensive exams can be scheduled at any time. Many students choose to schedule their comprehensive exam in the semester after completing their last course. Comps are the middle step in the road to your degree. It is usually sandwiched between completion of your formal course work and the writing of your dissertation.
What your written exam will look like depends on your academic department. You may be asked to respond to a series of 4-8 questions to test your broad knowledge of your academic area. In some fields, you may be asked to write papers or literature reviews and, in other fields, you may be asked to take an exam on content you should know. In most instances, the questions will be based on the coursework you have completed in your degree plan of study or will be directly related to the anticipated topic(s) of your thesis or dissertation. In some academic departments, the written exam may be followed by an oral defense or exam.
The administration and format of the exam can also vary by academic department. Some programs require students to "sit" for their comps and these can be given on multiple days. In this instance, the student is placed in a conference room or classroom, given the questions, and a time limit to write their answer. Another department might give students the option to receive questions at various times and respond in a "take home" fashion, answering the each question by writing a paper and submitting the paper by a certain date set by the department. The length of the paper may vary by department and the number of questions asked. The amount of time you have to complete your comps will vary by department; so ask your adviser what the time frame will be.
If given a choice, the format can be a matter of personal preference. For example, the writer of this article chose to "sit" for his comps. It involved two Mondays, four questions each day, two-hours allowed per question. While it was fairly intense, and he had to do revisions later, it was more convenient for him personally as a working student. On the other hand, a fellow student and writer's friend, another part-time student, opted for the take-home format, receiving a question every Friday for six weeks, writing the paper over the weekend, and submitting it Monday. A full-time student might choose to do a more drawn out format if they are less pressed for time or depending on the structure of comps in that department.
Regardless of the format, preparation is key to successfully passing your comps. Reviewing lecture notes, assigned readings, and your written papers are good study strategies. Think through some questions that you might get from your committee and outline some written responses to them. If you are likely to produce an article or literature review, you may want to compile and organize your research literature in preparation. You may be required to generate a grant proposal or prepare full articles for publication in a professional journal. Discuss what is expected with your adviser/supervisor. If this is the case, you may want to develop a strategy to manage the research literature or other materials you will be using to write your comps. Talk with fellow students in your department about what their comps were like—they may have specific tips for how best to prepare. As mentioned earlier, some academic departments also require an oral exam, usually in front of a faculty committee. These can be stressful, but if you are well prepared, you should have nothing to worry about.
Anticipate the questions and practice answering them out loud, speaking slowly and deliberately. Nervousness during your exam, can make you rush your speech.
Proper eye contact and body language is important. Be professional and academic.
Rephrase questions to make sure you understand what the questioner is asking. If necessary, ask for clarification. This can help you change your perspective on the question and give you more time to develop your answer.
The oral exam is meant to demonstrate what you know. But sometimes you get a question that's beyond your area of knowledge and you might have to say something like, "I haven't thought about that. But if I were to broaden my research into that, I suspect…." This acknowledges you need to broaden your area of expertise.
So, now you've got a plan to succeed on this important milestone in your graduate education. Good luck on preparing for your comps. It can be a rewarding experience!