Hats off to Michael Bloom for his adaptation of Austen's classic, premiering locally at Little Lake Theatre.
Peter Rosati and Danette Pemberton (in front) with Jenny Malarkey in Little Lake's Emma.
The problem with Jane Austen (and it's a problem all writers should have) isn't just that her characters or plots are so interesting. It's that much of the joy she creates comes from the insight and comedy of the novel's unspecified narrator.
I should say that this becomes a problem when her work is adapted for the theater: How do you get Austen's voice onstage? Most adaptations I've seen have so heavily relied on a live narrator that the production felt like someone reading the book, with occasional scenes acted out.
So hats off to Michael Bloom for his adaptation of Austen's Emma, premiering locally at Little Lake Theatre. The narration is sparse, almost nonexistent, but the few bits Bloom keeps position the emotional context of the story front and center.
Director Jena Oberg is preoccupied with defining the many places against which the story plays out, which occasions a few too many elaborate set changes. But she does a great job with her actors, keeping them honest, bright and on task.


text:
David Madueño i Sentís (Sabadell, 22 de juny de 1976) és poeta, docent i crític literari. Llicenciat en Filologia Catalana a la Universitat Autònoma de Barcelona (UAB).

Compagina l'escriptura amb la docència com a professor de llengua catalana i literatura i amb la crítica literària, que ha exercit en diversos mitjans al llarg dels darrers anys: El Punt Avui, Caràcters, Poetari, Núvol, Llavor digital i Quadern de les idees, les arts i les lletres.

Obra

Poesia
 2008. Desvetlla de mots és feina obscura. Sabadell: Edició de l'autor.
 2012. Poesia per a carnívors. Sabadell: Edició de l'autor.
 2015. Els murs incerts. Barcelona: Témenos edicions. Pròleg de Ricard Mirabete.
 2017. Osques. Barcelona: Témenos edicions. Pròleg de Manuel Costa Fernàndez. Fotografies de Zita Vehil.
 2020. Cua de foc. Sabadell: Papers de Versàlia. Fotografies de Zita Vehil.

Crítica literària
 2007. "Les adaptacions cinematogràfiques de la literatura catalana". Dins AAVV, Catàleg MIDA. Pàg. 18-26 (alemany), pàg. 99-107 (anglès), pàg. 181-189 (català). Barcelona: Institut Ramon Llull.
 2008. "L'escriptura invisible". Dins AAVV, La catosfera literària 08. Primera antologia de blogs en català. Pàg. 128-129. Valls: Cossetània edicions.
 2016. "De l'esclat a l'àtom. Pròleg". Dins Marcel Ayats, El sentit de la vida. Pàg. 5-9. Badalona: Pont del Petroli.
 2017. "L'instint del caçador furtiu. Pròleg". Dins Josep-Ramon Bach, L'instint. Obra poètica 1962-1993. Pàg. 9-37. València: Edicions 3i4.

Premis
 2008. Salvador Estrem i Fa de poesia de Falset.
 2008. Certamen Literari Foment Martinenc de Barcelona.
 2011. Poemes de Mar. Premis "La Mar de Lletres" de Calafell.
 2013. Millor blog sobre literatura en llengua catalana per llunÀtic. Premi Vila de Martorell.
 2022. 8è Concurs de Microrelats iSabadell. Millor microrelat en català.

Referències

Enllaços externs
 Pàgina de l'autor a la web Versos.cat
 llunÀtic. Blog de crítica de poesia i narrativa.

Escriptors sabadellencs
Escriptors catalans del sud contemporanis
Assagistes del Vallès Occidental
Crítics literaris catalans del sud contemporanis
Professors de català

redpajama_set_name: RedPajamaWikipedia


text:
New Report Reveals That Missouri Public De...
New Report Reveals That Missouri Public Defenders May Need Relief from Workload
reports that a new report funded by the American Bar Association provides an in-depth look of the current state of a public defender's work load at the Missouri State Public Defender Office, an office in which public lawyers have become so overtaxed that they have won the right — by the state's supreme court — to refuse taking on new cases. The hope is that the data will garner support for the key changes that the office plans to implement in the coming years.
The study, conducted last year by the accounting firm RubinBrown, and described by legal experts as the most comprehensive and credible of its kind, tracked how 375 public lawyers in Missouri spent their time, broken down by five-minute increments. Independently, a panel of both private and public lawyers also conducted an analysis of average time that public defenders in Missouri need to properly do their jobs.
The analyses revealed that in cases of "serious felonies, defenders spent an average of only nine hours preparing their cases, compared with the 47 hours they needed. For misdemeanors, they spent only two hours while 12 were called for." Director of the State Public Defender System, Cathy R. Kelly, told the
, "We found we are worse off than we thought we were."
reports that many legal experts believe that the "the daily triage required of public lawyers is unconstitutional and forces them to violate their ethical obligations to clients."
Missouri's defender office has requested a funding increase of about $25 million over four years to hire more than 200 new layers and scores of additional investigators and clerks and an additional $4 million cover about 4,000 juvenile cases annually. It's doubtful, however, that those requests will be met fully. According to the
, Missouri Governor Jay Nixon has approved only a small portion of the increased funding requests so far.
Ms. Kelly is hopeful that legislators will back up their appreciation for the report's new data with new funding during the spring's budget process.
Read the full

redpajama_set_name: RedPajamaCommonCrawl


EEPROM adapter should be work together with original OBDSTAR X100 pro auto key programmer to carry out EEPROM programming for some models. How to use the EEPROM adapter?
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3) After activate the authorization, browse to OBDSTAR official website http://www.obdstar.com/ to register and update.
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What's good of Quickly chip code reader comparing with CN900 auto key programmer?
4) Strong compatibility. CN900 4D decoder and CN900 ID46 decoder colder box can be applied to Quickly 4C/4D/46/48 key chip copy machine as well.
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For detail SRS model, please check obdexpress.co.uk webpage.


There are those people who may miss the personal touch of a friendly face at the end of their supermarket shopping trip, but for me, the self-service checkout is a joy. I can scan what I bought quickly, pay quickly, and get back into the open air. Granted, there are those times when an item is too light to have been weighed by the machine, the occasional instance of, "unknown item in the bagging area," and the rare treat of having the machine being opened up because it jammed itself when trying to deliver the change from the note you entered, but other than that, it is fine.
I am not anti-social – at least, I don't think so – but I want shopping to go by quickly, without having to enter into a conversation with store staff about what I wanted, or what they think I should do – I go in, I get what I wanted, and get out. You do not want any experience that involves your unloading your choices onto a conveyor belt, so a cashier can see the results of your decision-making skills. I also prefer have the cashier being redeployed into a better job elsewhere in the shop, instead of watching them scan a particularly tricky barcode on a bag of bananas, a frustration that did not need to happen.
Superficially, self-service checkouts look like a cost-saving measure, putting one member of staff in charge of a number of machines. However, what starts as a test, or trial, only continues because the rest of us find it too useful. Shops in the UK were generally run as a personal service to all, picking the items for their customers before charging for them, until Sainsbury's converted their Croydon branch to a self-service supermarket model in 1950, pioneered by the US store Piggly Wiggly in 1916. From then on, you take your basket, and you take what you need from the shelves yourself. It reduced queuing, and freed up staff to help with merchandising, changing the shopping experience.
From then, the more the customer can be persuaded to do themselves, the more that shopping has changed. Argos, started in 1972 as an online store without the website, have stores that are mainly storage space, where customers give staff the catalogue numbers for the items they want. Argos were primed and ready for the growth of "click and collect" shopping online, because they could now get their customers to make their usual transactions with them at home, a system that other shops have had to introduce, or build warehouses that are able to handle home delivery – Argos, on the other hand, have vans waiting out the back of their larger stores, sending out stock the same way they went in.
Will this mean the end of the shop as we know it? Will we only shop online after a certain point? There once was a time when people had to queue at their bank, within its opening hours, to take cash out of their current accounts. Now, you use an ATM to take out your cash, and make deposits in some cases – ATM makers like NCR and Wincor Nixdorf also produce self-serve checkout machines –while the majority of banking is done online or over the phone. I last walked into the local branch of my bank two years ago, and I'm not sure it will be there for the next time.


text:
Q: ¿Se puede realmente cambiar el directorio de descarga predeterminado para una sesión de Chrome ya abierta usando Selenium en Python? Estoy tratando de descargar unos archivos zippeados de esta página a una ruta específica en  para una sesión de Chrome ya abierta usando el siguiente código:
import time
import numpy as np
from selenium import webdriver
from selenium.webdriver.chrome.options import Options
from selenium.webdriver.chrome.service import Service
from selenium.webdriver.common.by import By

opt = Options() #the variable that will store the selenium options
opt.add_experimental_option("debuggerAddress", "localhost:9222") #this allows bulk-dozer to take control of your Chrome Browser in DevTools mode.
opt.add_experimental_option("prefs", {"download.default_directory": r"C:\Users\ResetStoreX\Downloads\Binance futures data\ADAUSDT-Mark_Prices_Klines_1h_Timeframe"}) #set the path to save the desired zipped data
s = Service(r'C:\Users\ResetStoreX\AppData\Local\Programs\Python\Python39\Scripts\chromedriver.exe') #Use the chrome driver located at the corresponding path
driver = webdriver.Chrome(service=s, options=opt) #execute the chromedriver.exe with the previous conditions

#Why using MarkPrices: https://support.btse.com/en/support/solutions/articles/43000557589-index-price-and-mark-price#:~:text=Index%20Price%20is%20an%20important,of%20cryptocurrencies%20on%20major%20exchanges.&text=Mark%20Price%20is%20the%20price,be%20fair%20and%20manipulation%20resistant.

if driver.current_url == 'https://data.binance.vision/?prefix=data/futures/um/daily/markPriceKlines/ADAUSDT/1h/' :
    number = 2 #initialize an int variable to 2 because the desired web elements in this page starts from 2
    counter = 0
    the_dictionary_links = {}
    while number <= np.size(driver.find_elements(By.XPATH, '//*[@id="listing"]/tr')): #iterate over the tbody array
        data_file_name = driver.find_element(By.XPATH, f'//*[@id="listing"]/tr[{number}]/td[1]/a').text
        if data_file_name.endswith('CHECKSUM') == False:
            the_dictionary_links[data_file_name] = driver.find_element(By.XPATH, f'//*[@id="listing"]/tr[{number}]/td[1]/a').get_attribute('href')
            print(f'Saving {data_file_name} and its link for later use')
            counter += 1
        number += 1
    print(counter)
    i = 0
    o = 0
    for i,o in the_dictionary_links.items():
        driver.get(o)
        print(f'Downloading {i}')
        time.sleep(1.8)

Y desafortunadamente no funciona, este código arroja el siguiente error:

InvalidArgumentException: invalid argument: cannot parse capability:
goog:chromeOptions from invalid argument: unrecognized chrome option:
prefs

Entonces, me gustaría saber ¿qué pudo haber salido mal? Hice el programa de arriba teniendo en cuenta esta otra solución pero este último parece que sólo funciona en una nueva sesión de Chrome, y necesito que el directorio de descargas predeterminado sea modificable para una sesión ya abierta. ¿Alguna idea?

redpajama_set_name: RedPajamaStackExchange


There are times when one needs a quick, fun and easy game to spend a few minutes between chores, or while waiting for something. It doesn't have to be a masterpiece, it doesn't have to be amazing, it just have to be fun. Forbidden Island does that really well.
To give you an idea of how easy it is go get to understand it, we learned it and played it while we were waiting for a friend to read another game's rules. Oh… and we played twice!
So what's all about? Forbidden Island is a cooperative game, where the mission of the game is to rescue four different treasures before all the islands where they reside get flooded and disappear under the waters. You can take the role of a few characters, each one with different abilities to help undertake the task, and each round you can take actions like move or recover an island from flooding, and then draw cards for treasure and flooding.
To make things more difficult and tense, some cards will require you to shuffle and return to the main pile the locations that have been flooded already. They can also increase the number of locations that will get flooded every round, increasing the difficulty quite a bit!
Once all the treasures have been found, something similar to an orb, a chalice, a Sumerian winged lion and some sort of fire, players have to go back to the heliport island, if it hasn't been flooded and then lost, of course.
The mechanics are a watered down version of Pandemics (no pun intended), they're quick, easy, but they allow for enough strategy to make for fun times. Don't get me wrong, you're unlikely to want to play this game every day, or even more than a couple of times at a time, but as a time filler and family game, you simply can't go wrong with it.
Nice review Paco. I've still not picked this up because I've been promised review copies (and never received them, (not offers from Gamewright I should point out)) but I think I'll just have to cave and give it a go. Pandemic has definitely grown on me and I think Forbidden Island will have more appeal to my younger brother.


text:
Death crash driver who killed young Cambridge pharmacist 'distracted by Snapchat'
Dishant Patel, 25, was killed in the crash on the B1050 in April 2016
Tara Cox
The parents of a much-loved pharmacist from Cambridge who died in a crash have spoken of their grief as the driver responsible has been jailed.
Dishant Patel, 25 from Longstanton, was travelling in a car as a rear seated passenger along the B1050 towards Longstanton from the A14 at Bar Hill on Saturday, April 23, 2016, when the accident happened.
He was taken to Addenbrooke's Hospital, Cambridge, and died in theatre in the early hours of the morning on April 24.
On Friday (March 23) Jignesh Patel, 27 from Wellingborough was found guilty of causing death by dangerous driving.
Dishant Patel from Longstanton was killed in the fatal crash in April 2016
Today (Monday, March 26) Patel was sentenced to three years in prison. He has also been disqualified from driving for three and a half years.
The trial heard that the vehicle Dishant was travelling in entered a right-hand bend and lost control, causing it to leave the road and roll into a nearby field. Jignesh Patel suffered minor injuries in the crash.
During the investigation it emerged that a front seated passenger in the car, driven by Jignesh Patel, had been recording the journey on his mobile phone and using the Snapchat social media app.
The terrible toll of hit-and-run drivers in Cambridgeshire revealed
Giving his evidence, Patel claimed that the back torch light from the front passenger's phone had interfered with his vision on the dark unlit road when the collision occurred.
Dishant, who lived with his parents, was a registered pharmacist and worked for worked for Lloyds Pharmacy in Cambridge at the Arbury branch.
He had been offered a post at Lloyds branch in Addenbrooke's Hospital in the city, but had not been able to take the position up before his accident.
Jignesh Patel was handed three years in prison at Cambridge Crown Court
"Nothing will ever bring Dishant back"
Speaking after the conclusion of the case Dishant's father, Shailesh Patel, said: "Dishant's death absolutely devastated the family. We still have not come to terms with the fact that he is no longer with us.
"He was a pivotal member of the family, who was ambitious and dedicated to his new career. It's so upsetting that we will not see him flourish in his professional life, which he would no doubt have done.
"Despite the verdict, the hard fact is nothing will ever bring Dishant back and that is just heartbreaking.
A new death by dangerous cycling law could be introduced
"As a family we really want to thank the team from Cambridgeshire police and the Crown Prosecution Service who were involved in the case and were completely dedicated to their job.
"They worked incredibly hard throughout and kept us informed and supported."
Before delivering his sentence, Judge Gareth Hawkesworth read a victim impact statement prepared by Dishant's family.
Death crash driver was friends with young pharmacist
He also considered substantial mitigating factors, noting that Dishant had been a very close friend of Jignesh Patel's.
He also said that the front seat passenger had clearly contributed to Jignesh Patel losing control of the vehicle.
Following the crash, Dishant's parents, Sangita and Shailesh Patel, instructed fatal accident specialist solicitors at Irwin Mitchell to investigate the crash and to support them through the proceedings.
This drug driver's 'cavalier and reckless' actions led to a fatal crash three days before Christmas
Neil Whiteley, the serious injury specialist at Irwin Mitchell representing Dishant's parents, said: "Dishant's death was extremely tragic and has been felt by a lot of people as he was very well regarded.
"As this trial has shown, the consequences of dangerous driving can be fatal and devastating. We will continue to work with Sangita and Shailesh to ensure they receive the support they need.
Download the CambridgeshireLive App for iPhone and Android
If you're looking for a way to keep up to date with the latest news from across Cambridgeshire, the CambridgeshireLive app has you covered.
Available for free on both iOS and Android devices, the clean and tidy interface is the easiest way to follow our news updates.
The team at CambridgeshireLive are committed to bringing our readers the latest breaking news, in-depth features, and exclusive content from all across the county.
Once installed, you can even select the area in which you live to receive dedicated notifications whenever we post a story that is relevant to you.
If you have an iOS device, you can download it from the App Store online by clicking here.
If you have an Android device, you can find the app on the Google Play Store here.
"Although there was clear evidence that the driver had been speeding at times leading up to the crash, sadly this incident goes to demonstrate the risks around mobiles and videos distracting the driver.
"I would also urge people to take note of the #dontstreamanddrive campaign which seeks to raise awareness of these dangers, currently being promoted online."


The termination of an employee is rarely an easy thing, but some of the dislocation and emotional upheaval can be avoided if preparations are made beforehand. A civilized termination also makes it less likely that any legal claim will be made by the former employee.
The procedure for employee termination should be designed, as far as possible, to protect the dignity of the employee being terminated and to provide for the safety of coworkers, visitors, and property. During the termination, it's a good idea to have the employee's supervisor or another employee present.
Have you treated the employee the same way you treated others in the same circumstances? Treating employees differently for the same behavior is the most common cause of discrimination and unlawful termination claims.
Have you documented verbal and written warnings?
Has someone else, preferably someone in the human resources department (if there is one), reviewed the employee file to verify the propriety of dismissal?
Is there any company property that should be given back, such as a cell phone, laptop, tools, books, etc.
Are all keys, cardkeys, and similar access devices being surrendered?
The owner should prepare a list like this before the need to terminate an employee comes up, so that it is done without being under pressure.
If the employee asks for a copy of the letter, give it to him after it's been signed. If the employee refuses to sign it (which will happen sooner or later), have the witness write near the bottom "Employee declined to sign" and put his (the witness's) initials or signature.
Any company, no matter how well run, no matter how big or small, has to fire an employee eventually. It is frequently not pleasant but the unpleasantness can be minimized if the right steps are taken. In fact, many of those steps must be taken long before the employee is terminated, but that's a topic for a different day.


Yao Han introduced me to this movie, at the time when we first met and were in love. I was 27, she was 19. I don't know how many times I have watched, listened to, and dreamed of, this movie. Each time it touches me deeper and deeper, makes me wonder why two people would love then go apart, marry yet think back, miss the other person but continue to live a life without him/her. why?
It's a wonderful movie, a wonderful story. The guitar at Chen Li's funeral always gets me, every, single, time. It's beautiful, it's sad, it is a memory one can only dare to look at when the memory is about to disappear forever, still pains, still hurts, still live, still real.
I don't know. Sadness, a boundless ocean of sadness, surrounds me when the music plays. I was talking to Noah's mom last night that I don't get overwhelmed in emotion, thus I hardly found myself cry.
But this one, gets me, every, single, time. If one day I fall into a coma, play the music, the piece when Chen Li wrote a letter to Xiao Rou, confessing her thoughts for the last time, I will answer, for it reaches deep inside, that I, will tear, will sigh.


text:
Available for Review
Link to Publisher's Website
dc Talk's Jesus Freak
(33 1/3)
Will Stockton, D. Gilson
New York, NY:
, November
9781501331664.
For other formats: Link to Publisher's Website.
If, as Rob Sheffield suggests, "Love is a mixtape," then for a generation of Christians growing up in the nineties, that mixtape was dominated by the group dc Talk. By declaring a fierce loyalty and love for all things Christian, Jesus Freak then emerges as the song and album that dominates that mix. In dc Talk's Jesus Freak—their contribution to Bloomsbury's 33⅓ Series—Will Stockton and D. Gilson explore this 1995, genre-crossing, hit-making album by the Christian pop/rock/rap trio. As queer scholars with evangelical roots, Stockton and Gilson offer aunique understanding of how the band—with this album in particular—served to shape the theological and cultural understandings of young Christians. Simultaneously, the album provides a subversive subtext which may inform the identity and sexual orientation trajectories of many who enjoyed the music, a trajectory certainly never intended by the message of Jesus Freak.
In fact, both Stockton and Gilson assert "Jesus Freak presented Christ as the solution to despair, loneliness, isolation, and apathy—problems that ostensibly afflicted the new generation of youth consumers … both song and album invited listeners to join a sacred, but also countercultural, community of Jesus Freaks" (15). Comprised of Toby McKeehan, Michael Tait, and Kevin Max, dc Talk formed when the three were students at Jerry Falwell's conservative Liberty University, and with the release of the 1995 album and its title track, "self-consciously worked to replicate and sanctify the sonic patterns and lyrical terrain of grunge and alternative music..." (26), while the central message of the album and title song "Jesus Freak" seeks to provide clarity where "historical distance, theological differences, and political complexities all vanish in the album's creation of a universal Jesus Freak identity, an ahistorical community of nonconformist in which modern believers mingle with ancient ones" (36). The message becomes increasingly abstruse as Stockton and Gilson examine the lyrical content and received meanings from multiple perspectives. For example, the lyrics of the album's second track "Colored People" offers insight into the often-problematic nature that its intended verses communicate. The performance and lyrical content of "Colored People," a song which pays homage to the neoliberal zeitgeist of the mid-1990's that embraced "racial difference when economically or politically beneficial, while casting such differences as divisive when they are not" (64), demonstrates the further difficulties of the band's attempts to at best impersonate, or at worse appropriate, the musical and cultural styles of people of color. As Gilson observes, "the lyric discourages the audience from perceiving any significant difference between Tait, McKeehan, and Max. It encourages the audience to perceive all three men as both white and black, to consider whiteness as itself a color, and therefore to recognize, even if only tacitly, McKeehan's claim to black artistic and cultural forms" (81). What some readers may find the most challenging about Stockton and Gilson's critique is their claim that dc Talk's work served to inform certain sexual angsts and identity anxieties, stating that dc Talk "were men teenage Christian boys wanted to emulate and girls wanted to marry, and at times whom D. and I wanted to both emulate and marry" (44). Stockton and Gilson make it clear that their examination of dc Talk—as both artists and authors of Jesus Freak—isn't a mere dismantling of an artifact of their Christian youth, but rather, they argue that "perhaps in queering Jesus Freak, we have shattered nostalgia for our Christian youths into some useful force of shame" (130). What all readers will find helpful is the understanding that the past, including all cultural artifacts, are mitigated. We interpret, understand, reflect, keep, and dispense with the items of our history based upon the lens through which we see them, and the utility with which we still find them helpful or deem them hurtful. Music is one such artifact, that even when possessing a sense of romantic nostalgia, can serve to inform or infuriate. As Stockton and Gilson conclude, "in our look backwards, that stubborn queer desire colors what we see. It minimizes the angsty memory of salvation anxiety, but it also encourages us to probe the queer faultiness of the very theology that sustained us as young Christian men" (130). Nostalgia often romanticizes our past experiences and influences. With dc Talk's Jesus Freak, Stockton and Gilson demonstrate that nostalgic reflections may also reveal complicated conclusions, resulting in unexpected answers to the query, "what will people think when they hear that I'm a Jesus Freak?"
About the Reviewer(s):
Scot Loyd is Professor of Communication Studies at Oklahoma Baptist University.
Date of Review:
About the Author(s)/Editor(s)/Translator(s):
Will Stockton is Associate Professor of English at Clemson University and author of several books including Members of His Body: Shakespeare, Paul, and a Theology of Nonmonogamy (2017), and Playing Dirty: Sexuality and Waste in Early Modern Comedy (2011).
D. Gilson is Assistant Professor of English at Texas Tech University. His books include I Will Say This Exactly One Time (2015) and Brit Lit (2013).
dc Talk, rap music, Jesus Freek
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PYEONGCHANG: The Pyeongchang Winter Olympics will open in sub-zero temperatures on Friday, with North and South Korea marching together at a gala ceremony to launch what could be the coldest Games on record.
North Korean leader Kim Jong Un has dispatched a delegation of more that 500, including his increasingly influential sister Kim Yo Jong. She is the first ever member of the ruling Kim dynasty to visit the south.
Twenty-two North Korean athletes are taking part, but only pairs skaters Ryom Tae Ok and Kim Ju Sik have a real shot at a medal.
For the South Koreans, major home hopes rest of the slender shoulder of yet another teenage girl. In short track speedskating, 19-year-old Choi Min-jeong is among the favourites for a gold-medal slam of all four races.


text:
Home Technology Technology Focus
Market Verticals
CES 2009: Thin and Trim is 'In'
In spite of economic slowdown, the Consumer Electronics Show—held at Las Vegas from 8th to 11th January—attracted a large number of new exhibitors and products. Here is a brief report from EFY's editor who has been visiting CES regularly since 2007 -- Ramesh Chopra
FEBRUARY 2009: All screens and devices seem to be on diet to get slim and trim. In particular, the mobile, the PC and the TV screens—the most important ones in our daily lives. And they are all getting well-connected to each other and the 'cloud.' The cloud here refers, of course, to the Internet. This is one of the clear messages I got at this year's Consumer Electronics Show at Las Vegas in the USA.
You have already seen much slimmer and lighter PCs introduced last year, starting with Mac's. Preference for thinner TVs is also well-known, although they cost hell of a lot at present for anyone to be able to afford one. I also notice preference for thin, sleek mobile phones when I look around-especially in international forums such as the CES.
Is it due to the global economic downturn that the manufacturers have started saving on the material cost? Of course, not! The reason is to make the devices look better as these become our lifestyle statements. The slim is the 'in' thing nowadays. This must have taken a while to register in the US where big was always considered to be better.
In place of Bill Gates, who has been giving keynote address almost regularly at CES, Steve Balmer, Microsoft's CEO, took the stage this year for the first time to show the shape of things to come from their end. As he disclosed, beta version of 'Windows 7' has just been launched worldwide. It can be downloaded from the Internet for checking, according to Steve. He showcased its salient features to the audience.
Janet Galore, program manager of Microsoft, demonstrates their new ultra-thin, flexible monitor
"There is no turning back from the connected world," said Steve. No matter what happens with the economy, or how long the recession lasts, the companies that continue to pursue advancement in technology will do much better than those who lag behind, he feels. Microsoft had invested $8 billion in R&D last year alone for this purpose.
"There is so much opportunity ahead of us," said Steve. First and foremost is the convergence of the three screens that people use every day—the PC, the phone and the TV. Microsoft's original aim was to put a PC on every desk. Today, a billion people already have PC. But that means there are five billion people who have never owned a PC. So we have a big task ahead of us to make computing more affordable.
There is also the challenge and opportunity for creating anytime, anywhere computing through the convergence of the PC, phone and TV, and their connectivity through the Internet. Soon smart phones will make up 50 per cent of the mobile phone market. Also, we shall soon see TV becoming more sophisticated and more connected, and the boundary between the PC and TV will dissolve.
In the next couple of years, according to Steve, the computer will be able to 'hear' and 'see,' besides recognising gestures.
Intel's chairman Dr Craig Barret chose to speak at length on 'Small Things Challenge' (instead of processors) in his keynote address. This project is being run together with Kiva.org to raise money for Save the Children's programme titled 'Rewrite the Future.' The plan is to urge the people world-wide through 'smallthingschallenge.com' website to help in development and education of children in emerging countries.
Barret unveiled the third generation of low-cost Classmate PC tablet for use in schools in developing countries. Also, a video was shown to demonstrate how low-cost solutions were being found in India to take education to villages in buses fitted with computers, and to provide healthcare facilities in remote areas using telemedicine techniques.
(From L to R) Ed Granger-Happ, CIO, Save the Children and chairman of NetHope, explains the features of the Intel-powered classmate PC to Carolyn Miles, COO of Save the Children, and Intel chairman Craig Barrett, at the International Consumer Electronics Show (CES) in Las Vegas. Intel is teaming up with Save the Children and Kiva.org on a year-long global campaign called the 'Small Things Challenge' to raise awareness and money to educate kids and help their parents in developing nations
Barret, however, believes that we shall continue to be surrounded by screens, including the large TV, medium-size computer and small-size hand-held displays, for a long time. Anyone who thinks that there won't be these three predominant screens or devices in the near future is crazy, according to him.
Sony's chief executive Sir Howard Stringer showed a thin, flexible, coloured OLED screen which could be bent. He also showed the audience a couple of short futuristic digital 3D movie clips with the help of sunglasses which had polarised lenses made by RealD.
Since the technology for screening 3D movies is complicated, it is not likely to be available for showing at home theatres. So 3D movies are once again expected to drive the viewers to the theatres for this new kind of realistic experience. Dream-Works Animation's chief executive Jeffery Katzenberg disclosed that their company had already retooled their studio to make 3D movies, and from this year they would be making 3D movies only.
Stringer pulled out the prototype of a Vaio 'Lifestyle PC' from his pocket which he said was as thin as a pack of playing cards and weighed just 640 gm. It has a 20cm (8-inch) display but full capabilities of a standard notebook, unlike the smaller netbooks being offered nowadays, he claimed. Priced much higher at $900 than other netbooks, which are priced at around $400 only, it is a fan-less device which would be available in such bold colours as garnet red and emerald green. It should be in market by February end.
3D HDTV
One of the novelties noticed at CES was the advent of 3D television, which could be viewed without the need of using special eyeglasses. Samsung as well as Panasonic were seen demonstrating their respective 3D TV sets. Of course, the effect was much better when the movie was viewed directly from the front—at least in the Samsung TV personally checked by me.
If 3D TV catches on, it may not be necessary once again to go to cinema halls to watch 3D movies, as expected by Sony's Howard Stringer. But there is no news on pricing and commercial availability of 3D TV as yet.
Miniaturised
Woo Paik, president and chief technology officer of LG Electronics, introduced their fully functional wristwatch cell phone with 3G video telephony (VT) service and GSM quad-band network capabilities. Though it is a follow-up of the prototype he introduced at CES2008, it includes such upgrades as a touch-screen interface, 3G capabilities and video calling. Its voice recognition features, for use with or without a Bluetooth headset, make call placing and looking up contacts easy. This good-looking, wearable phone will be available in Europe soon this year.
World's first projector phone by Logic Wireless
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Have you heard about CompTIAs new stackable certifications? If you've earned more than a couple of CompTIA certifications, you may already have one of them.
As an example, if you an A+ and Network+ certification, you now also have the CompTIA IT Operations Specialist stackable certification.
If you have the CompTIA A+, Network+, and Security+ certifications, you you now also have the CompTIA Secure Infrastructure Specialist stackable certification.
CompTIA IT Operations Specialist (A+ / Network+).
CompTIA Systems Support Specialist (A+ / Linux+).
CompTIA Cloud Admin Professional (Network+ / Cloud+).
CompTIA Network Infrastructure Professional (Network+ / Server+).
CompTIA Linux Network Professional (Network+ / Linux+).
CompTIA Secure Infrastructure Specialist (A+ / Network+ / Security+).
CompTIA Secure Cloud Professional (Security+ / Cloud+).
CompTIA Security Analytics Professional (Security+ / CSA+).
CompTIA Security Analytics Expert (Security+ / CSA+ / CASP+).
Type in a search term (such as path) in the Google Custom Search box.
Check out the blogs I've written on the topic including this one Recommended Certification Path.
With these new stackable certifications, people can choose a path based on their interest: infrastructure or cybersecurity.
Within the context of information technology (IT), infrastructure refers to the hardware and network components used to support an IT network or system. This includes things such as user computers (desktop PCs, laptops, and mobile devices), network devices such as routers and switches, and servers.
CompTIA certifications A+ and Network+ provide a solid foundation for someone starting on an IT infrastructure pathway. Because many servers and other devices use Linux, Linux+ is a logical next step for technicians working in an infrastructure environment. Additionally, technicians can add some specialization with the Cloud+ and/or the Server+ certification.
These stackable certifications help validate your skillsets needed in a broad number of IT infrastructure roles.
Cybersecurity refers to the knowledge, technologies, practices, and procedures used to protect IT resources from threats. This is an extremely wide field with multiple career paths.
The CompTIA A+, Network+, and Security+ certifications provide a solid foundation for someone starting a career in IT security. The next step in this CompTIA path is the CSA+ (or CySA+) certification, for someone wanting to expand their knowledge in cybersecurity. The Cloud+ certification helps someone show some specialization in cloud technologies.
Last, the CASP+ certification (combined with CSA+ and CASP+) helps people demonstrate an Expert level of knowedge in cybersecurity.
You many have noticed that these certifications include the following three words at the end of each: Specialist, Professional, or Expert. These refer to an implied experience level.
Specialist: Early-career IT professionals with 0–2 years of experience.
Professional: Mid-level IT professionals with 2–5 years of experience.
Expert: Established IT professionals with more than 5 years of experience.
You may have already received an email indicating you earned one of these stackable certifications. If you missed, you can login to the CompTIA site and click on the Stackable Certifications tab to see if you've earned one of them already.


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text:
Frontiers in Immunology
Vaccines and Molecular Therapeutics
Lee M. Wetzler
School of Medicine, Boston University, United States
John S. Gunn
The Research Institute at Nationwide Children's Hospital, United States
María Marcela Barrio
Fundación Cáncer, Argentina
Coalition for Epidemic Preparedness Innovations (CEPI), Norway
Open Supplemental Data
Front. Immunol., 10 January 2017 | https://doi.org/10.3389/fimmu.2016.00677
Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines
Sudeep Kumar, Raju Sunagar, Giang Pham, Brian J. Franz, Sarah J. Rosa, Karsten R. O. Hazlett and Edmund J. Gosselin*
Center for Immunology and Microbial Diseases, Albany Medical College, Albany, NY, USA
Francisella tularensis (Ft) is a category A biothreat agent for which there is no Food and Drug Administration-approved vaccine. Ft can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. Furthermore, Ft expresses distinct sets of antigens (Ags) when inside of macrophages (its in vivo host) as compared to those grown in vitro with Mueller Hinton Broth (MHB). However, in contrast to MHB-grown Ft, Ft grown in Brain-Heart Infusion (BHI) more closely mimics the antigenic profile of macrophage-grown Ft. Thus, we anticipated that when used as a vaccine, BHI-grown Ft would provide better protection compared to MHB-grown Ft, primarily due to its greater antigenic similarity to Ft circulating inside the host (macrophages) during natural infection. Our investigation, however, revealed that inactivated Ft (iFt) grown in MHB (iFt-MHB) exhibited superior protective activity when used as a vaccine, as compared to iFt grown in BHI (iFt-BHI). The superior protection afforded by iFt-MHB compared to that of iFt-BHI was associated with significantly lower bacterial burden and inflammation in the lungs and spleens of vaccinated mice. Moreover, iFt-MHB also induced increased levels of Ft-specific IgG. Further evaluation of early immunological cues also revealed that iFt-MHB exhibits increased engagement of Ag-presenting cells including increased iFt binding to dendritic cells, increased expression of costimulatory markers, and increased secretion of pro-inflammatory cytokines. Importantly, these studies directly demonstrate that Ft growth conditions strongly impact Ft vaccine efficacy and that the growth medium used to produce whole cell vaccines to Ft must be a key consideration in the development of a tularemia vaccine.
Whole cell-based vaccines are preferred in the absence of well-defined protective immunogens. In addition, whole cell-based vaccines provide a broad range of potential protective antigens (Ags) and immune stimulatory molecules, which can include proteins, polysaccharides, and pathogen-associated molecular pattern molecules (PAMPs). In the absence of an identified protective Ag, directing the immune response toward an array of Ags that the host is likely to encounter during a natural infection maximizes the potential for a protective immune response to be generated upon vaccination. In this regard, the composition of the antigenic repertoire expressed by the constituent whole cell vaccine becomes very important, while PAMPs can provide molecular components required for the activation of Ag-presenting cells (APCs). Furthermore, activation of APCs is a key requirement for the generation of an optimal adaptive immune response against cognate Ags contained within such vaccines.
Importantly, the in vitro culture medium utilized to generate whole cell-based vaccines can significantly impact the expression of antigenic determinants and PAMPs on the infectious agent. Several microbes have been shown to differentially express Ags depending on the growth medium utilized, including Escherichia coli, Salmonella enterica (1), Listeria monocytogenes (2), Helicobacter pylori (3), and Mycobacterium bovis (BCG) (4, 5). Direct evidence supporting an impact of growth medium on the efficacy of whole cell-based vaccines comes from a study involving BCG where its differential cultivation in Sauton versus Middlebrook medium significantly altered its protective efficacy (6). However, the mechanism responsible for this apparent difference has not been well established.
In regard to Francisella tularensis (Ft), it has been found that its antigenic repertoire can change depending on the growth medium employed for in vitro cultivation. Specifically, we previously demonstrated that the protein profile expressed by Brain-Heart Infusion (BHI)-grown Ft closely resembles that of in vivo circulating Ft, while Ft grown in Mueller Hinton Broth (MHB) exhibits a distinct protein profile (7, 8). Interestingly, other investigations have revealed that BHI- and MHB-grown Ft also differ in their ability to interact with complement and antibodies (9). In addition, BHI-grown Ft exhibits a diminished ability to elicit pro-inflammatory cytokines by various APCs compared with inactivated Ft (iFt)-MHB (10, 11). These features of BHI- versus MHB-grown Ft provide unique and necessary tools to investigate the dichotomy between the antigenic repertoire versus APC-activating components of whole cell-based Ft vaccines.
In this study, we examined the differences in protective efficacy of iFt vaccine prepared by growth in MHB or BHI and further evaluated if the difference in protective efficacy observed reflects changes in not only the immune response but also APC function. While we initially hypothesized that BHI-grown iFt, which more closely mimics the antigenic repertoire of in vivo macrophage-grown Ft, would provide the superior vaccine immunogen, we found that iFt-MHB generates superior protection compared to iFt-BHI. Other indicators of protection, such as bacterial burden, tissue damage, and production of pro-inflammatory cytokines, also correlated with the improved protective efficacy of iFt-MHB versus iFt-BHI. Furthermore, we found that iFt-MHB exhibited significantly higher binding to APCs, higher induction of dendritic cell (DC) maturation, and increased Ag presentation to Ft-specific T cells. Overall, this investigation suggests that APC activation components are important for protective efficacy of inactivated whole cell-based vaccines. Moreover, growth conditions employed to prepare these vaccines can significantly influence their immunological and protective efficacy.
Six- to 8-week-old male and female C57Bl/6 mice were obtained from Taconic laboratories (Hudson, NY, USA). Mice were housed in the animal resource facility at Albany Medical Center. Food and water were provided ad libitum during the entirety of experiments. Animal studies were reviewed and approved by Institutional Animal Care and Use Committee of Albany Medical Center according to NIH guidelines.
Preparation of iFt
Francisella tularensis LVS was grown in MHB (Becton, Dickinson, Franklin Lakes, NJ, USA) or BHI (Becton, Dickinson) medium at 37°C to a density of ~0.5–1 × 109 colony-forming units (CFU)/ml. To inactivate Ft LVS, 1 × 1010 CFU/ml of live bacteria were incubated in 1 ml of sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) containing 2% paraformaldehyde (Sigma-Aldrich) for 2 h at room temperature with gentle shaking. Subsequently, fixed bacteria (iFt) were washed with sterile PBS three times. Inactivation was verified by culturing a 100 µl of sample (1 × 109 iFt organisms) on chocolate agar plates (Becton, Dickinson) for 7 days. Protein contents were quantified using a bicinchoninic acid assay (Sigma-Aldrich) as described (12). The iFt preparations were resuspended in PBS and stored in 1 ml aliquots at −20°C until use. Ft LVS for challenge was grown similarly and stored in liquid nitrogen until use.
Immunization and Challenge
Groups of six to eight mice with equal numbers of males and females were immunized on day 0 and 21 via the intranasal (i.n.) route. Each mouse was anesthetized by intraperitoneal injection of 20% ketamine plus 5% xylazine and subsequently administered 20 µl of PBS (vehicle), iFt-MHB, or iFt-BHI at a concentration of 37.5 ng, 75 ng, 150 ng, or 300 ng/20 μl. Immunized mice were then challenged on day 35 i.n. with 2× LD50 of Ft in 40 µl of PBS (single nostril) and subsequently monitored for survival for 21 days.
Quantification of Bacterial Burden
Following immunization and challenge, mice were euthanized at various time intervals, and lung and spleen were collected aseptically in PBS-containing protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA) and subjected to mechanical homogenization using Mini Bead Beater-8 (BioSpec Products, Bartlesville, OK, USA) using 1-mm zirconia/silica beads. Tissue homogenates were then diluted 10-fold in sterile saline, and 10 µl of each dilution was spotted onto chocolate agar plates in duplicate and incubated at 37°C for 48 to 72 h. The number of colonies on the plates were counted and expressed as CFU per milliliter for the respective tissue. Tissue homogenates were then centrifuged at 14,000 g for 20 min, and the clarified homogenates were stored at −20°C for subsequent cytokine analysis.
Lactate Dehydrogenase (LDH) Assay
Serum LDH levels were quantified following the manufacturer's instructions (Sigma-Aldrich). Briefly, mice were immunized and challenged as described for quantification of bacterial burden. Sera were collected on days 1, 3, 5, and 7 postinfection. Prechallenge sera were collected 11 days after booster immunization. Serum samples were diluted 1:100 with LDH assay buffer. Fifty microliters of diluted serum samples, standard, and blanks was added to separate wells in a 96-well microtiter plate. Fifty microliters of LDH substrate was subsequently added to each well. LDH converts NAD to NADH, which can be read calorimetrically at 450 nm. The amount of NADH generated between Tinitial (absorbance at 450 nm immediately after the LDH substrate) and Tfinal (absorbance at 450 nm immediately after the LDH substrate) by the LDH activity is first calculated (B). The LDH activity of the sample is determined by the formula LDH activity = (B × sample dilution factor)/(reaction time × V), where V is the sample volume added to well. LDH activity is reported as milliunit per milliliter.
Antibody (Ab) Titer Determination
Anti-Ft-Ab production in response to immunization with iFt-MHB versus iFt- BHI was measured by ELISA. Briefly, ELISA plates (Corning, Corning, NY, USA) were coated with 50 µl of Ft LVS (5 × 107 CFU/ml) in carbonate buffer [4.3 g/l sodium bicarbonate (Sigma-Aldrich) and 5.3 g/l sodium carbonate (Sigma-Aldrich) at pH 9.4] for 16 h at 4°C. The plates were then washed with washing buffer [PBS (Sigma-Aldrich) containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich)] and blocked for 2 h with 200 µl of PBS containing 5% BSA. Serial threefold dilutions of sera (starting with 1/10) were added to the plates (100 µl/well) and incubated for 2 h at 4°C. After three washes with washing buffer, alkaline phosphatase-conjugated anti-mouse Ab specific for IgG (Sigma-Aldrich) was added and incubated for 1 h at 4°C. Plates were then washed three times with washing buffer, and then 100 µl of BCIP/NBT alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were subsequently incubated for 2 h, and absorbance at 405 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis
Samples derived from 10 µg (~1 × 108 cells) of Ft were mixed with Laemmli sample buffer (BioRad, Hercules, CA, USA) and boiled for 10 min prior to resolution through 4–12% gradient SDS-PAGE precast gels (Invitrogen, Carlsbad, CA, USA). The running buffer was NuPAGE MES SDS buffer (Invitrogen). Gels were run at 90–160 V. Resolved gels were stained with either Coomassie blue (Bio-Rad) or transferred to nitrocellulose membranes. Coomassie-stained gels were scanned into Adobe Photoshop. Membranes were then blocked for 30 min with PBS, 0.05% Tween 20, 5% non-fat dry milk. Primary Abs were applied for overnight incubation at dilutions ranging from 1:500 to 1:60,000 in PBS-Tween. Following five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with biotinylated goat anti-mouse IgG (H + L, Southern Biotech, Birmingham, AL, USA). Following an additional five washes with PBS-Tween, the membranes were incubated for 1 h with agitation with streptavidin-conjugated horseradish peroxidase (Southern Biotech). Development of the chemiluminescent substrate (SuperSignal West Pico, Pierce, Rockford, IL, USA) was visualized using an Alpha Innotech imaging system in movie mode. Densitometric analysis of developed blots was performed as previously described (7, 9).
Ag-Presenting Cells
Dendritic cells were generated from bone marrow cells using mouse recombinant FLT-3 (rFLT-3) (R& D systems, Minneapolis, MN, USA) following a previously described method (13). Briefly, bone marrow cells were obtained from 6- to 8-week-old female C57Bl/6 mice and were incubated for 6 days in bone marrow-derived DC (BMDC) generation medium [Roswell Park Memorial Institute (RPMI) 1640 medium] supplemented with 10% fetal bovine serum (FBS), 50 ng/ml rFLT-3, 1 mM sodium pyruvate (Cellgro, Tewksbury, MA, USA), 2 mM l-glutamine (Cellgro), 1× non-essential amino acids (Cellgro), 0.05 mM 2-Mercaptoethanol (Bio-Rad), and 1× penicillin streptomycin (Cellgro). Fifty percent of the medium was replaced with fresh BMDC generation medium on day 3 during 6 days of incubation. Peritoneal exudate cells (PECs) were isolated from naïve mice. PECs were cultured with RPMI 1640 supplemented with 10% FBS, and non-adherent cells were removed by aspiration of medium after 24 h. Adherent cells, which comprise >90% macrophages, were used as APCs.
iFt Binding Assay
Binding of iFt was visualized by flow cytometry using GFP-expressing iFt, as previously described (13). GFP-expressing Ft LVS organisms were provided by Dr. Mats Forsman (Swedish Defense Research Agency, Stockholm) (14). Briefly, 1 × 106 BMDCs were incubated with 5, 10, 15, or 20 µg of GFP-expressing iFt-BHI or iFt-MHB in PBS. After 2 h incubation at 4°C, BMDCs were washed twice with PBS and stained with rat anti-mouse CD11c-Alexa 700 Ab and subsequently analyzed by flow cytometry (LSRII, BD, Franklin Lakes, NJ, USA). The percent of GFP-positive CD11c DCs (indicative of iFt bound to DCs) were analyzed in respective samples.
In Vitro Analysis of Costimulatory Molecule Expression and Cytokine Secretion
Expression of costimulatory molecules on BMDCs was analyzed by flow cytometry as previously described (13). Briefly, 2 × 105 BMDCs were incubated for 24 h with PBS or 10 µg of iFt- MHB or iFt- BHI in an equivalent volume of PBS. Cells were then harvested and stained with Abs to the following surface markers: rat anti-mouse CD40-PE, rat anti-mouse CD80-APC, and rat anti-mouse CD86-Alexa 700. Cells were subsequently analyzed via flow cytometry.
Cytokine secretion by BMDCs was assayed following previously described methods (13). Briefly, 2 × 105 BMDCs were incubated with PBS or with 10 µg iFt- MHB or iFt-BHI for 24 h at 37°C. Culture supernatants were then collected and analyzed for cytokines using the Bio-Plex assay system as described below.
Cytokine Analysis
Cytokines were measured using Bio-Plex assay kits (Bio-Rad) following the manufacturer's instructions. Briefly, 50 µl of a 1× mixture of magnetic beads coupled with capture Abs for capturing desired cytokines were added to separate wells of flat bottom 96-well microtiter plate. Beads were washed (2×) with Bio-Plex wash buffer. Unless stated otherwise, all incubations were done at room temperature with plates sealed with a plastic sealer and covered with aluminum foil with gentle shaking. All washing was done with Bio-Plex washing buffer. Fifty microliters of diluted standards, blank, or samples was added to individual wells. Plates were incubated for 30 min and washed 3× with Bio-Plex washing buffer. Then 25 µl of 1× mixture of biotinylated detection Abs were added to each well and incubated for 30 min. Plates were washed again, and 50 µl of phycoerythrin-coated streptavidin was added to each well and incubated for 10 min. Plates were then rewashed, and 125 µl of assay buffer was added to each well. Plates were read via Luminex reader (Bio-Rad), and the quantity of each cytokine was deduced using a standard curve included in the assay.
Ag Presentation Assays
Antigen presentation assays were done as previously described (13). Briefly, 2 × 105 BMDCs or PECs were incubated overnight at 37°C with PBS or 5 µg of iFt-MHB or iFt-BHI. A total of 1 × 105 Ft-specific T cells (FT25 6D10 hybridoma cells) (13) in 100 µl medium (RPMI 1640 supplemented with 10% FBS) were then added to each well. The plate was then incubated at 37°C for 24 h in CO2 incubator, and supernatants were collected. Supernatants were then assayed for interleukin (IL)-5 (secreted by these cells in response to Ag) using the Bio-Plex assay as described earlier.
The log-rank (Mantel-Cox) test was used for survival curves. One-way analysis of variance or the unpaired Student's t-test was used for the remaining figures. Data analysis was performed using Graph-Pad Prizm 5 (Graph-Pad Software, San Diego, CA, USA).
iFt Exhibits Protein Profiles Similar to Its Live Counterpart
Before using iFt as immunogen, we first used SDS-PAGE and western blot analysis to examine the impact of our fixation procedure on the protein composition of Ft LVS. The overall protein profile of live and fixed Ft LVS was largely unchanged, although the fixed samples did show some expected evidence of cross-linked Ags (Figure 1A). Consistent with previous reports (7, 9), the BHI-derived samples contained elevated levels of ~23 and ~55 kDa proteins [Figure 1A (asterisks)], which were apparent in both the live and fixed bacteria. Western blot analysis indicated that the live and fixed BHI-derived samples contained levels of IglB and IglC that were elevated relative to their MHB-grown counterparts (Figure 1B). Densitometric analysis revealed that these increases were on the order of threefold to fivefold (Table S1 in Supplementary Material), as reported previously (7, 9). As our fixed iFt preparations appeared to be faithful representations of their live counterparts, we next examined the vaccine efficacy of these immunogens.
Figure 1. Fixed Francisella tularensis (Ft) LVS used herein for immunization studies retain the protein composition of their unfixed counter parts. Ten micrograms of live and fixed Ft LVS previously grown in Mueller Hinton Broth and Brain-Heart Infusion (BHI) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie blue (A) or transferred for western blot analysis (B). Asterisks in A mark bands, which are visibly more abundant in the western blot of BHI-grown Ft (B). The membrane was probed with a cocktail of monoclonal antibodies specific for IglB, IglC, and a FopA-specific polyclonal mouse serum. IglB and IglC have previously been shown to be more abundant in BHI-grown Ft (7, 9). FopA is used here as a loading control. The ~18-kDa band visible under IglC is an unidentified, endogenously biotinylated Ft protein (likely AccB) detected by the streptavidin–horseradish peroxidase conjugate used during development of the western blots.
iFt-MHB Generates Superior Protection against Ft LVS-Induced Mortality versus iFt-BHI
First, we evaluated whether the differential expression of antigenic repertoire and PAMPs affected by cultivation in MHB versus BHI has any impact on the protective efficacy of iFt. We used iFt generated in MHB or BHI to immunize mice. Following a lethal challenge with Ft LVS, we observed that iFt- MHB-immunized mice were better protected than iFt-BHI-immunized mice (Figure 2). Of the four doses used, the 150- and 300-ng doses exhibited statistically significant differences in the protection afforded by iFt-MHB versus iFt-BHI (Figures 2C,D). Importantly, in Figures 2A–D mice were challenged with MHB grown Ft LVS. Thus, to evaluate any potential bias of growth medium used to culture the challenge organism (Ft LVS) on the outcome of survival, we similarly immunized mice with PBS or 150 ng of iFt-MHB or iFt-BHI, but challenged with BHI-grown Ft LVS Once again, we observed that iFt-MHB provided better protection compared to iFt-BHI (Figure 2E).
Figure 2. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) provides superior protection compared to iFt-Brain-Heart Infusion (BHI) against lethal Ft LVS challenge. Mice were immunized twice on days 0 and 21 with phosphate-buffered saline or iFt-MHB or iFt-BHI at a dose of 37.5 ng (A), 75 ng (B), 150 ng (C,E), or 300 ng (D). (A–D) Mice were infected 2 weeks after booster immunization with 2× LD50 of Ft LVS grown in MHB. (E) Mice were infected with 2× LD50 of Ft LVS grown in BHI. This figure represents Kaplan–Meier survival curves of three independent experiments with a total of 20 mice per group (A–D) or 1 experiment with 12 mice per group (E). *p < 0.05.
iFt-MHB-Immunized Mice Are Better Protected against Bacterial Replication and Tissue Damage following Ft LVS Infection, as Compared to That of iFt-BHI
Following the above observation of differential protection afforded by iFt-MHB versus iFt-BHI, we investigated the ability of the two iFt immunogens to protect against bacterial replication and tissue damage exerted by Ft LVS infection. Following immunization and Ft LVS challenge, we found significantly lower bacterial burdens in the lungs (Days 5 and 7) (Figure 3A) and spleen (Day 7) (Figure 3B) of mice immunized with iFt-MHB as compared to that of iFt-BHI.
Figure 3. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) better reduces bacterial burden following Ft LVS infection as compared to that of iFt-BHI: mice were immunized as in Figure 2 with phosphate-buffered saline (white bars), iFt-MHB (gray bars), or iFt-BHI (black bars) and challenged with Ft LVS grown in MHB. Subsequently, Ft LVS colony-forming units were enumerated in the lung (A) and spleen (B) homogenates at indicated days postinfection. Each bar represents the mean ± SE (error bar) of two independent experiments with a total of eight mice per group. ***p < 0.001.
Francisella tularensis LVS infection inflicts systemic tissue damage in infected mice, and serum LDH levels are commonly used as a biomarker of tissue damage (15). Thus, we evaluated serum LDH levels at early and late stages of Ft LVS infection in mice immunized with iFt-MHB versus iFt-BHI. At days 5 and 7 after Ft LVS infection, significantly lower levels of LDH activity were found in iFt-MHB-immunized mice versus that of iFt-BHI-immunized mice (Figure 4).
Figure 4. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) induces superior resistance to Ft LVS-induced systemic tissue damage compared to that of iFt-Brain-Heart Infusion (BHI): mice were immunized on days 0 and 21 with phosphate-buffered saline (white bars), iFt-MHB (gray bars), or iFt-BHI (black bars) and challenged with Ft LVS grown in MHB. Lactate dehydrogenase activity was quantified in serum drawn on indicated days postchallenge. Prechallenge sera were drawn 11 days after boost. Each bar represents mean ± SE (error bar) of two independent experiments with a total of eight mice per group. *p < 0.05.
Differential Protection against Ft LVS-Induced Inflammation by iFt-MHB versus iFt-BHI
Systemic inflammation and/or sepsis are hallmarks of the tularemia in advanced stages of infection. We previously observed that mice protected by immunization exhibit lower levels of pro-inflammatory cytokines in the lungs and spleens following Ft infection, compared to those of unprotected mice (16, 17). In this case, analogous to our previous studies, we observed that mice immunized with iFt-MHB, which are better protected, also exhibited lower levels of IL-6 (Figure 5A), interferon (IFN)-γ (Figure 5C), and monocyte chemoattractant protein (MCP)-1 (Figure 5D) in the lungs in the latter stage of disease (day 7 postinfection). MCP-1 levels were also lower at day 3 postinfection in the lungs of iFt-MHB immunized mice compared to that of iFt-BHI immunized mice. MCP-1 is a chemoattractant for inflammatory monocytes, which can induce to tissue damage. Therefore, regulation of MCP-1 levels is important for protection against systemic tissue damage and subsequent mortality inflicted by Ft LVS infection. Interestingly, higher levels of IL-17 were also found at day 3 postinfection in iFt-MHB immunized mice compared to that of iFt-BHI immunized (Figure 5B). IL-17 has been found to be protective against mucosal infections with bacteria including Ft (18, 19). Thus, higher IL-17 levels at early stages of infection correlate with superior protection afforded by iFt-MHB compared to iFt-BHI. Similarly, in the spleens, significantly lower levels of tumor necrosis factor (TNF)-α (at day 7 postinfection) (Figure 5F), IFN-γ (day 5 postinfection) (Figure 5G), and MCP-1 (day 7 postinfection) (Figure 5H) were observed in mice immunized with iFt-MHB compared to that of iFt-BHI. However, level of IL6 (Figure 5E) in the spleens were comparable among mice immunized with iFt-MHB and iFt-BHI.
Figure 5. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) is superior to iFt-Brain-Heart Infusion (BHI) in controlling Ft-induced inflammation in the lung. Mice were immunized with phosphate-buffered saline (white bars) or 150 ng of iFt-MHB (gary bars) or iFt-BHI (black bars) and challenged with Ft LVS Indicated cytokines were quantified in homogenates of lung (A–D) and spleen (E–H) at the indicated days postinfection. Values represent mean ± SE (error bar) of two independent experiments with a total of eight mice per group. *p < 0.05.
Increased Ab Responses Are Induced by iFt-MHB versus iFt-BHI
Next we investigated whether iFt generated in MHB versus BHI medium also has a differential impact on the humoral immune response against Ft. We quantified Ft-specific serum-IgG by ELISA as a measure of humoral immune response to Ft. Live Ft LVS grown in BHI and MHB medium were coated onto ELISA plates and reacted with sera obtained from iFt-MHB versus iFt-BHI-immunized mice. IgG induced by iFt-MHB compared to iFt-BHI reacted more strongly to Ft surface Ags expressed on both MHB- or BHI-grown Ft LVS, as evidenced by their relative titers (Figures 6A,B). This is consistent with superior protection afforded by iFt-MHB compared to that of iFt-BHI. The choice of Ags employed [Ft-MHB (Figure 6A) versus Ft-BHI (Figure 6B)] to detect Ab titers did not alter the relative pattern of Ab production observed in iFt-MHB or iFt-BHI-immunized mice.
Figure 6. Increased Francisella tularensis (Ft)-specific antibody production is induced by inactivated Ft (iFt)-Mueller Hinton Broth (MHB) versus iFt-Brain-Heart Infusion (BHI). Mice were immunized twice at a 3-week interval with 20 µl phosphate-buffered saline (PBS) or 150 ng of iFt-BHI or iFt-MHB via the intranasal route in 20 µl PBS. Sera obtained 2 weeks postboost were analyzed for Ft-MHB-specific (A) and Ft-BHI-specific (B) IgG. Values represent mean ± SE of two independent experiments (n = 16 mice per group). *p < 0.05.
To further characterize these Ab responses, we also used the above sera to probe by western blot lysates of MHB- and BHI-grown Ft LVS along with a BHI-grown mutant Ft (WbtA), which is deficient for O-Ag. As shown in Figure 7, the anti-iFt-MHB sera recognized a more diverse array of Ags than did the anti-iFt-BHI sera. One of the Ags preferentially recognized by the anti-iFt-MHB sera appeared to have a ladder pattern typical of LPS, and consistent with this, this ladder pattern was markedly reduced/absent in the WbtA mutant Ft. In addition, the Ag specificity of the anti-iF-BHI sera appeared to be more focused on a ~43 kDa protein with reduced O-Ag-dependent reactivity. Collectively, the data in Figures 6 and 7, as well as in data not shown, indicate that immunization with iFt-MHB induces higher titers of anti-Ft Ab and that a larger proportion of this Ab is dependent on O-Ag for binding.
Figure 7. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) elicits antibody (Ab) against broader range of antigens compared to iFt-Brain-Heart Infusion (BHI). Ten micrograms of Ft LVS or WbtA LVS grown as indicated (M = MHB, B = BHI) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for western blot analysis. Membranes were incubated overnight with 1:500 dilutions of the indicated sera. Bound Abs were detected through sequential incubations with biotinylated goat anti-mouse and streptavidin-conjugated horseradish peroxidase (HRP). The prominent ~18 kDa band visible in all three blots is an unidentified, endogenously biotinylated Ft protein detected by the SA-HRP conjugate.
iFt-MHB and iFt-BHI Differentially Bind to BMDCs and Differentially Induce Expression of Costimulatory Molecules
Having observed differential protective efficacy between iFt generated in MHB versus BHI, we sought to further identify the immune mechanisms underlying the differences in protective efficacy of iFt-MHB versus iFt-BHI. In our previous studies (7, 9) and Figure 1, we demonstrated that iFt-BHI exhibits an antigenic profile more similar to that of in vivo circulating Ft, as apposed to that of Ft-MHB. This is in contrast to our expectation that by virtue of exposing the host to iFt-BHI, which is more similar antigenically to in vivo replicating Ft, a better protective immune response would be achieved. This led us to further investigate the immune mechanisms responsible for this unexpected observation.
By using a GFP-expressing Ft LVS, we investigated whether iFt generated in MHB versus BHI have different abilities to bind to and activate APCs. In fact, we observed significantly higher binding of iFt-MHB-(GFP) to BMDCs compared to iFt-BHI-(GFP) (Figure 8). Increasing the concentration of the iFt-MHB-(GFP) successively increased the number of BMDCs harboring iFt-MHB. In contrast, iFt-BHI-(GFP) exhibited relatively poor binding to BMDCs at all concentrations used in this assay. Furthermore, costimulatory molecules are essential components for T cell activation. As demonstrated in Figure 9, iFt-MHB induced significantly higher expression of the costimulatory molecules CD80 (Figures 9A,B) and CD86 (Figures 9C,D), as compared to iFt-BHI, although CD40 (Figures 9E,F) expression was stimulated to a similar degree by both immunogens.
Figure 8. Enhanced binding of inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) to antigen-presenting cells (APCs) as compared to iFt-Brain-Heart Infusion (BHI). Bone marrow-derived dendritic cells (BMDCs) were incubated with GFP-expressing iFt-BHI or iFt-MHB. Cells were washed and stained with CD11c antibody before analyzing with flow cytometry for GFP signal associated with BMDCs (A). (B) Percentage of GFP-positive (+) BMDCs as evaluated by flow cytometry. Values represent mean ± SE of two independent experiments. ***p < 0.001.
Figure 9. Induction of higher expression of costimulatory molecules on antigen-presenting cells by inactivated Francisella tularensis (iFt)-Mueller Hinton Broth compared to iFt-Brain-Heart Infusion (BHI). Bone marrow-derived dendritic cells were incubated with iFt-BHI and iFt-MHB for 24 h. Expression of indicated cell surface markers were analyzed via flow cytometry (A,C,E). (B,D,F) The MFI of respective markers. Values represent mean ± SE of two independent experiments. *p < 0.001.
iFt-MHB Mediates Higher Pro-inflammatory Cytokine Production and Increased Ag Presentation to Ft-Specific T Cells
In addition to Ag binding and expression of costimulatory molecules on APCs, secretion of pro-inflammatory cytokines is also critical for DC-mediated orchestration of the adaptive immune response. We observed that iFt-MHB induces higher levels of the pro-inflammatory cytokines [IL-1β (Figure 10A), IL-6 (Figure 10C), and TNF-α (Figure 10D)], as compared to iFt-BHI, while both immunogens induced similar levels of IL-10. Finally, we investigated the presentation of iFt-MHB versus iFt-BHI using an Ft-specific T cell hybridoma (FT25 6D10), which is specific to an epitope corresponding to Ft 50 s-ribosomal protein (20) and secretes IL-5 on engagement with cognate major histocompatibility complex (MHC)-peptide complex (13). Co-cultures of FT25 6D10 and APCs (PECs or BMDCs) exposed to iFt-MHB exhibited higher levels of IL-5 compared to that of iFt-BHI (Figure 11). Together, data presented in Figures 8–10 suggest that iFt-MHB harbors a superior ability to stimulate APC function as compared to iFt-BHI.
Figure 10. Inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) elicits greater pro-inflammatory cytokine production compared to iFt-Brain-Heart Infusion (BHI). Bone marrow-derived dendritic cells were incubated with phosphate-buffered saline, iFt-BHI, or iFt-MHB for 24 h. Supernatants were analyzed for (A) IL1B, (B) IL10, (C) IL6, and (D) TNF-α. Values presented are mean ± SE of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 11. Relative presentation of inactivated Francisella tularensis (iFt)-Brain-Heart Infusion (BHI) and iFt-Mueller Hinton Broth (MHB) by antigen-presenting cells (APCs): APCs [peritoneal exudate cells (A) or bone marrow-derived dendritic cells (B)] were exposed to phosphate-buffered saline or 10 µg of iFt-MHB or iFt-BHI for 24 h. FT25 6D10 cells were then mixed with the APCs and incubated for 24 h. Supernatants were then collected analyzed for interleukin-5. Values represent mean ± SE of two independent experiments. *p < 0.05, **p < 0.01.
In the present investigation, we observed that iFt propagated in MHB versus BHI medium exhibits a differential ability to stimulate an immune response to and protection against Ft challenge. Specifically, iFt-MHB immunogen induced improved protection against lethal Ft LVS challenge as compared to iFt-BHI. Consistent with the superior protective activity of iFt-MHB immunogen, bacterial burden and tissue damage were also reduced. Furthermore, immunization with iFt-MHB leads to reduced inflammation in the lungs and spleens. Importantly, it has been demonstrated that mice protected against Ft LVS challenge via immunization exhibit lower pro-inflammatory cytokines in their lungs and spleen late in infection (16, 17), which we also observed in the case of iFt-MHB immunization. Notably, in contrast to the above, however, we observed elevated levels of IL-17 in lungs of mice immunized with iFt-MHB on day 3 postinfection with Ft LVS IL-17 has been implicated in protection against mucosal infections including Ft LVS (18, 19). Immunization with iFt-MHB also induced higher levels of Ft-specific IgG, which have been shown to play a role in protection against Ft LVS (21). Apart from quantitative differences in IgG titers, Abs produced in response to iFt-MHB and iFt-BHI also differed qualitatively. While the dominant reactivity of anti-iFt-BHI was to a 43 kDa protein, the anti-iFt-MHB response was more broadly distributed among a range of Ags including Ft LPS (O-Ag). Ft LPS has been found to be an important Ag for protection against Ft infection (22). Although, at present, we do not know the significance of the differences in the Ab responses elicited by iFt-MHB versus iFt-BHI, further studies in this regard may reveal their impact on protection.
In regard to the mechanisms(s) by which iFt-MHB is a better immunogen, APCs are the gateway to adaptive immune response. APCs present Ags to T cells in association with MHC, which provides the primary signal for T cell activation. Other signals required for T cell activation include costimulatory molecules, e.g., CD80 and CD86 (23–25). T cells that receive signals only via TCR by engagement with MHC-peptide complex, without receiving the second signal from costimulatory ligands, are destined to undergo anergy (26). Moreover, the local cytokine niche determines the type of T cell response generated. For example, IL-12 and IFN-γ favor a T helper 1 immune response, while IL-10 and transforming growth factor-β favor a regulatory T cell immune response (27, 28). Furthermore, APCs can be activated to express MHC II, costimulatory molecules, and pro-inflammatory cytokines by exposing them to various pathogen-associated molecular patterns (23). Therefore, it is important that APCs are exposed to vaccine Ags in the context of appropriate APC activation signals (29). In this investigation, iFts derived from growth in MHB medium exhibited a superior ability to activate APCs versus iFt-BHI. Specifically, MHB-derived iFt exhibited an increased ability to induce CD80 and CD86 on BMDCs. iFt-MHB also induced higher pro-inflammatory cytokine production in BMDCs. Moreover, we also observed higher levels of Ag presentation/T cell activation induced by BMDCs exposed to iFt-MHB versus to iFt-BHI. Notably, iFt-MHB also exhibited comparatively greater binding to BMDCs than iFt-BHI. The increased binding may explain the higher levels of pro-inflammatory cytokine stimulation and Ag presentation by iFt-MHB (Figure 7). Observations from other investigators are consistent with our observations (10, 11) that iFt-MHB can induce higher pro-inflammatory cytokines compared to iFt-BHI. Thus, collectively, it is clear that iFt-MHB exhibits a superior ability to impact the nature and magnitude of adaptive immune response, thereby enhancing its protective efficacy. The apparent differences in APC engagement between iFt-MHB and iFt-BHI may be a reflection of their structural differences. In this regard, we have demonstrated that BHI-grown Ft LVS expresses additional layers of capsular polysaccharides, which have been implicated in hindering Fts' ability to bind with complement and Abs (9). Capsular polysaccharides have also been implicated in masking TLR2 and Tul4 ligands on the surface of Ft (9). Furthermore, Singh et al demonstrated that Ft grown in MHB exhibited increased engagement of cytosolic DNA sensors, possibly due to structural defects resulting in an increased release of DNA into cytosol after uptake by APCs, thereby inducing AIM 2-dependent cytokine production (11). The LPS of Ft LVS has a peculiar structure that minimizes its interaction with TLR4. Also, recently it has been shown that genetic engineering of microbes that express E. coli LPS significantly enhances their TLR4 engagement with bone marrow-derived macrophage (30). Thus, in future, one potential means to improve Fts' ability to engage and activate APCs may be to genetically engineer Ft to express modified TLR agonists such as E. coli LPS.
It is also important to note that environmental conditions such as temperature, metal ions present, pH, and other media ingredients used for in vitro growth can significantly influence antigenic expression by bacterial pathogens (8). Various investigations have demonstrated the significance of growth conditions on bacterial recognition by Abs (1–3). In this investigation, we demonstrate that the two growth media employed for preparation of iFt LVS whole cell vaccines significantly altered the vaccine-induced immune response and protection against cognate pathogen. Recently, BCG vaccine exhibited differential protective efficacy based on the presence or absence of detergents in the growth medium. The latter was associated with differential expression of capsular polysaccharides, the presence of capsule specific Ab, splenic IL-17 and IFN-γ production, and multifunctional CD4+ T cell responses (31). We have also observed differential protective efficacy of BHI- and MHB-grown live Ft LVS vaccine, where BHI-grown Ft LVS exhibited better protection against lethal infection with Ft SchuS4 (32). This contrasting observation may be due to the distinct immunological requirements for protection against Ft LVS and Ft SchuS4 or live versus inactivated vaccine. Thus, we are further investigating this distinction in our laboratory. Nevertheless, these studies strongly emphasize the critical importance of growth conditions when developing whole cell vaccines.
When we started this investigation, the goal was to improve the efficacy of Ft LVS-based whole cell vaccines. Our assumption was that by exposing the host to an antigenic repertoire more similar to that of the infecting agent in vivo (iFt-BHI), a superior protective immune response would be generated as compared to iFt-MHB (7). However, as depicted in Figure 12, we observed that iFt-MHB elicited a superior ability to stimulate the immune response and protection, as compared to iFt-BHI. In conclusion, our observations indicate that to endow hosts with protective immune response, vaccine formulations should simultaneously deliver a variety of protective Ags as well as APC activation components. This investigation also emphasizes that in vitro growth conditions, which influence Ag expression as well as APC activation capacity, are critical variables that must be considered when developing whole cell-based vaccines.
Figure 12. A model comparing observations when utilizing inactivated Francisella tularensis (iFt)-Mueller Hinton Broth (MHB) versus iFt-Brain-Heart Infusion (BHI) as immunogen. iFt-MHB in comparison to iFt-BHI engages and activates antigen (Ag)-presenting cells (APCs) including significantly higher binding, expression of costimulatory molecules (CD80 and CD86) and secretion of pro-inflammatory cytokines [interleukin (IL)-1b, IL-6, and TNF]. iFt-MHB Ags are also presented to Ft-specific T cells more efficiently compared to iFt-BHI. Collectively, superior activation of APCs by iFt-MHB results in higher IgG response and protection against lethal Ft challenge.
This study was carried out in accordance with the recommendations of the Institutional Animal Care and Use Committee (IACUC) of Albany Medical Center, Albany, NY, USA. The protocol was approved by the IACUC of Albany Medical Center, Albany, NY, USA.
EG, KH, and SK conceptualized and designed the study. SK, RS, GP, BF, and SR performed the experiments and acquired and analyzed the data. SK drafted the manuscript. EG and KH critically revised the manuscript. All the authors approved the publication of the manuscript and agreed to be accountable for all aspects of the work.
The authors thank Yili Lin and Li Barry, Manager, Immunology core facility, Department of Immunology and Microbial Diseases, Albany Medical College, for their assistance in acquiring flow cytometry data.
This work was supported by grants from the National Institutes of Health (R01AI100138—KH & EG; RO1AI123129-KROH; P01AI056320—EG and KH). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Supplementary Material for this article can be found online at https://www.frontiersin.org/article/10.3389/fimmu.2016.00677/full#supplementary-material.
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Keywords: APC, whole cell-based vaccines, Francisella tularensis, costimulatory molecules, growth factors
Citation: Kumar S, Sunagar R, Pham G, Franz BJ, Rosa SJ, Hazlett KRO and Gosselin EJ (2017) Differential Cultivation of Francisella tularensis Induces Changes in the Immune Response to and Protective Efficacy of Whole Cell-Based Inactivated Vaccines. Front. Immunol. 7:677. doi: 10.3389/fimmu.2016.00677
Lee Mark Wetzler, Boston University School of Medicine, USA
John S. Gunn, Ohio State University, USA
María Marcela Barrio, Fundación Cáncer FUCA, Argentina
Arun Kumar, GlaxoSmithKline (GSK) Vaccines, Italy
Copyright: © 2017 Kumar, Sunagar, Pham, Franz, Rosa, Hazlett and Gosselin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Edmund J. Gosselin, gossele@mail.amc.edu