Fifty years ago, NASA sent Apollo 8 to the moon on December 21, 1968. The flight crew included Frank Borman, James Lovell, and William Anders. They achieved many firsts in human history, including becoming the first humans to leave Earth and orbit around the moon, taking the first pictures of the Earth from deep space, and broadcasting the first live television coverage from lunar orbit.
Who could have imagined that it was only in August 1968 that NASA decided to send astronauts to the moon in four months? Timing was of the essence because the United States wanted to beat the Soviets in the space race and pave the way for moon landings to come. It was a gutsy move. Even under the best scenario, NASA decision makers thought the Apollo 8 mission had a 50 percent chance of succeeding.
Apollo 8's marvelous historic journey took place way before I was born. Looking back on the footage, the most memorable moment for me was that on Christmas Eve, the three crew members took turns reading the first 10 verses of Genesis, "In the beginning, God created the heaven and the Earth…" According to Commander Borman's account, prior to Apollo 8's take off, he tried to find something to say to the large broadcast audience––all people on earth who could hear the crew when the time came. But everything he and his crew tried "came up trite or foolish." Eventually, a friend of a friend's wife recommended reading the verses of Genesis.
At the peak of human achievement, some people could easily become arrogant and foolish, as in "look what we can do." But these American astronauts humbly turned to the almighty God. When they looked at the beautiful and fragile earth from a distance, when they were awed by God's creation. They were right that only the words of God were appropriate.
I don't believe it is pure coincidence that the first human journey away from our planet Earth took place on Christmas Eve. I don't believe that it's sheer luck the that first humans to speak to us from outer space were Americans. I don't believe it's accidental that the first words that people on earth heard from the outer space for the first time in human history were the words of God.
Just imagine had the Soviet astronauts beat the American and reached outer space first. What would they have said, since communists don't believe in God? Thanks to comrade Stalin for his wisdom and guidance? Or would they have read something from Karl Marx's "Das Kapital": "Down with Capitalism," or "abolish private property rights"?
Thank God that didn't happen. The Almighty has an interesting way to reveal his plan. "For all those who exalt themselves will be humbled, and those who humble themselves will be exalted" (Luke 14:11).
Fifty years have passed since this historic journey, and we humans have made great strides in scientific discovery and technology development: smart phones, driverless cars, artificial intelligence (AI), to name a few. Many of these represent the best of human creativity and ingenuity. However, some of the scientific research and experiments are deeply troubling.
Although there are still so many unknowns and uncertainties, an ambitious Chinese scientist has already taken gene-editing from animals to humans. Right after this Thanksgiving, the world woke up to the shocking news that He Jiankui, a scientist of the Southern University of Science and Technology in Shenzhen, China, claimed that he had modified human embryos with the gene-editing technology to create twin girls.
He claimed he did so to prevent the twins from developing HIV. But many scientists condemn his approach as unethical and say it could affect the twins in other areas and even introduce new diseases that would be passed down through generations. He so far has been very defiant towards all the criticism. That'snot surprising from a person who only a year ago proudly claimed in a state TV interview that "Some people said we shook the global gene-sequencing industry. Right. It's me. He Jiankui. I did it." These are not the kind of words that would be uttered by a humble person.
While one Chinese scientist is determined to play God in the process of human creation, others are reaching for the sky. Back in June this year, China joined Russia in carrying out five experiments that have modified a layer of the atmosphere, about the half the size of Britain, above Vasilsursk, a small Russian town in Eastern Europe.
Chinese scientists pushed back on such criticism by saying, "We are not playing God. We are not the only country teaming up with the Russians." While it may be true, and the Russians' behavior is just as questionable as the Chinese's, Chinese scientists uniquely stand out in such controversies because they have the full backing of the Chinese government, which is determined to turn China into a global technology leader no matter what it takes. He Jiankui, for example, received "41.5 million yuan (US$5.96 million) in government funding for his research on genome sequencing – a required technology for gene-editing research" in 2015.
While China eagerly provides state funding to advance scientific discovery and technology advancement, the country employs few rules and ethical standards on what is appropriate. Even those rules and ethical standards on books are rarely enforced. International rules and standards are often ignored to make way for national interests.
In other countries, besides enforcing rules and standards, religious beliefs often play an important role, serving as a moral compass to guide people on right and wrong. But for scientists brought up in an atheist regime, since they don't believe in God, they are guided only by personal ambition and national pride. More often than not, ambition and pride can and will lead people down a dangerous path. "When pride comes, then comes disgrace, but with humility comes wisdom," says Proverbs 11:2.
Another Christmas is nearly here. As we commemorate the anniversary of Apollo 8, celebrating human achievement and the birth of our savior at the same time, it's a good time to remind ourselves and fellow human beings: be humble. Approach the unknown with care and humility.


Use the grilled scallops to top our Mixed Fresh Herb Pasta.
1. Heat grill to medium-high. In a large bowl, combine 2 tablespoons oil with the lemon juice and liqueur, if using. Add scallops, and toss to coat. Slide scallops horizontally onto small skewers (about 6 on each). Season with salt and pepper.
2. Place skewers on grill. Grill, rotating, until scallops are golden outside and just cooked through, 2 to 3 minutes. Remove from grill.
3. Cut fennel bulb in half lengthwise, and slice lengthwise again into 1/4-inch-thick pieces. Toss with remaining 1/2 tablespoon oil, and grill, turning, until fennel just wilts and starts to brown, about 5 minutes. Remove scallops from skewers. Serve warm.
If you're using wooden skewers, soak them for 30 minutes before grilling to prevent them from scorching.


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Survey Shows Canadian Parkinson's Community's Key Concerns
by Jose Marques Lopes, PhD
Long waits for diagnosis, limited access to specialized treatments and services, and out-of-pocket healthcare costs are among key concerns of Parkinson's patients, caregivers, and medical-service providers, according to a Parkinson Canada survey.
The organization created the 30-minute online survey to better understand the challenges that patients, caregivers, and providers face. It was conducted between Sept. 29 and Oct. 24, 2017.
Fifty-seven percent of the patients who participated were men, and most were aged 65-74 years. About four in five were married or living with a partner. Seven in 10 were retired.
An important finding was that patients face long waits for a diagnosis, with two out of 10 saying they waited over one year. Almost one in four patients classified their ability to gain access to medical specialists as poor or very poor. Sentiment was similar on accessing movement disorder clinics, a Parkinson's nurse specialist, and long-term care facilities.
Three in five patients said they needed additional information from Parkinson Canada or one of its support groups. Four in 10 reported depression, anxiety, stress, sadness, and loss of confidence. Access and waiting times for mental health services were a problem for many patients.
In contrast, nine of 10 patients and caregivers rated access to oral treatments and to a family doctor as good, very good or excellent.
Providers were more critical. Over half reported poor or very poor access to movement disorder clinics, and three out of 10 rated access to speech and swallowing therapy as poor or very poor.
Providers were even more negative on waiting times for specialized services and treatments, with four in 10 rating them as poor or very poor.
The patient barriers that providers mentioned included too few medical specialists, limited information and understanding of Parkinson's, scarce availability of educational resources, insufficient counseling resources, and too few support groups.
The findings also showed the toll that Parkinson's takes on caregivers. More than one in four reported that they care for a patient while having a full-time job. Seventy-seven percent of caregivers were women and most were aged 75 years or older.
Nearly half of caregivers said access to long-term care facilities was poor or very poor. They were also concerned about access to movement disorder clinics, Parkinson's nurse specialists, and mental health services.
Among the health problems that caregivers experienced were stress, exhaustion and feeling guilty that they were not doing enough. They also mentioned frustration towards the patient, helplessness and anxiety.
"People with Parkinson's often suffer from complex physical and non-motor [non-movement] health disorders, which can affect the whole family," Joyce Gordon, the CEO of Parkinson Canada, said in a press release. "We know that caregivers are heavily burdened, financially and emotionally. Our mission is to help close the gap for them and build awareness of Parkinson's."
The disease also represents a significant financial burden. Four in 10 respondents reported difficulty paying health care bills, including medication, exercise, physiotherapy, and speech and swallowing therapy.
About three-fourths of patients and care partners reported paying for exercise programs. Forty-four percent said they paid for physiotherapy and a third said they paid for at least one type of medication. Four out of 10 said paying for medication imposes a major financial burden on them if it's not reimbursed by insurers.
Three out of four care providers perceived medication and other therapies as a major burden without financial assistance
"It's important that individuals receive a correct diagnosis as early as possible and have adequate access to ongoing specialized care. Even this care is limited and follow-up visits are too far apart," said David Grimes, lead author of the "Canadian Guidelines on Parkinson's Disease."
"If the survey shows that Canadians are experiencing considerable gaps and delays to care, this hinders [providers] from effectively recommending treatments and lifestyle changes that would help patients better manage the disease," he said.
Parkinson Canada plans to share the survey results with governments and others during Parkinson Awareness Month in April. The goal is to raise awareness as a way of improving access and decreasing wait times. It will also share the information with the public and donors.
It called for more effort on research and services for Parkinson's at a time when healthcare demand is increasing as the population ages. It also called for more investment to provide medical professionals with educational opportunities and healthcare resources.
Tagged caregivers, diagnosis, financial burden, health care professionals, health services, medications, mental health, nurse specialist, out of pocket expenses, Parkinson Canada, Parkinson's, Physiotherapy, speech therapy, swallowing therapy.
Jose Marques Lopes, PhD
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redpajama_set_name: RedPajamaCommonCrawl


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Cirque du Soleil's "Mad Apple" Puts Las Vegas in a New York State of Mind
It's been a minute since Cirque du Soleil debuted a brand-new show in Las Vegas.
The modern circus company opened "R.U.N" at the Luxor Las Vegas in October 2019, but hit the brakes in March 2020, when the pandemic shut down the Las Vegas Strip. While most Cirque du Soleil shows reopened in 2021, "R.U.N" and sexy older sister, "Zumanity" at New York-New York, retired to the circus in the sky.
Now, Cirque du Soleil has stepped up its game with a new show, "Mad Apple." The show debuted at New York-New York on May 26 with Vegas-style pomp, circumstance, and paparazzi on a pink carpet featuring the cast, creative team, and notable local entertainers and athletes like Vegas Golden Knights players, "Blue Man Group" cast members, "Chippendales" men, a handful of "Fantasy" girls and plaid suited magician Mac King.
There was also confetti. Lots and lots of confetti.
'the Greatest Night Out in New York'
Like the hotel where it's staged, "Mad Apple" captures the electric vibe of New York City nightlife culture and recreates it on a sparkling Las Vegas stage (from one city that never sleeps to another) with energetic musicians, dancers, comedians, magicians, and circus performers.
It's a "completely new approach to Cirque du Soleil entertainment," the company's president of resident and affiliate show divisions Eric Grilly said in a press release.
"It's an action-packed experience designed for today's audience, who increasingly want to consume entertainment as they do content – in an exciting, highly compelling, and ever-shifting manner," he said. "With 'Mad Apple,' you never know what's coming next – from vocals, off-script comedy, and live music with our horns and drum line to acrobatics – all wrapped in an incredibly fun party atmosphere that differentiates it from other shows."
Photo Credit: Denise Truscello
Co-creator and executive producer Simon Painter, creator of the "Illusionists" franchise, explained at a press conference, prior to opening, that "Mad Apple" is a departure from story-driven Cirque du Soleil shows, such as "Mystere" at Treasure Island, "O" at Bellagio and "KA" at MGM Grand.
"They're not playing characters," he explained of the 48-member global cast. "There's no storyline."
Instead, he describes "Mad Apple" as "the greatest night out in New York."
Photo Credit: Matt Beard
Concept co-creator and director Neil Dorward, an acclaimed choreographer, and director added: "The most important thing is the talent on stage" as well as "the audience experience."
Mad Apple is the first Cirque du Soleil production in creative collaboration with Painter and Dorward. Painter's company The Works was purchased by Cirque du Soleil in February 2019.
What to Expect at "Mad Apple"
"Mad Apple" takes place in the former "Zumanity" theater at New York-New York. The two-story, 1,200-seat theater and box office are conveniently located near an exit to (or entrance from) The Park Vegas outdoor mall. Tickets for the 80-minute show start at $59.
Though showtime is 7 p.m. and 9:30 p.m. Fridays through Tuesdays, the show really starts when the doors open, 45 minutes before curtain. As audience members filter into the theater, they mingle as guests at a Studio 54-style party with live music, crowds, people-watching, and upscale bars mixing specialty cocktails – such as a Proper New York Gin Martini or a Manhattan in a souvenir flask – both upstage and downstage. Bottle service is also available for $300.
Two UpStage Bars showcase nearly 600 bottles, filled with an illuminated color-changing LED liquid, while the DownStage Bar transforms before the crowd into a stage at showtime. Flair bartenders and street performers interact with the audience as they fill the theater, adding to the fun and festive atmosphere, which continues throughout the show.
Whether performers are leaping across the Brooklyn Bridge, dancing near a yellow taxi or artfully balancing by one hand atop an enormous steel Empire State Building, New York is ever-present. The set includes architectural nods to all five boroughs of NYC and its iconic skyline, crowned by a 31-foot-long Statue of Liberty-inspired tiara. An aerialist, at the top of the show, is lowered to the stage by a glittery, apple-shaped disco ball. We also spied a hot dog cart, a Central Park sign spinner, the New York Times Crossword and a failed "drug deal."
Families beware: Due to adult language and themes, children under the age of 16 are not permitted at "Mad Apple" and guests under 18 must be accompanied by an adult.
Don't Send in the Clowns
Another big difference between "Mad Apple" and other Cirque du Soleil shows: Instead of clowns, there are comedians.
"Mad Apple," which will have a rotating cast of performers, currently features stand-up comics Brad Williams and Harrison Greenbaum, and British freestyle comedy rapper Chris Turner. The funnymen were told not to hold back in their acts, adding to the authentic New York feeling.
"We're not speaking in French, were not in a boat on a pool, we just get to be ourselves," Greenbaum told the press. A New Yorker tried and true, he noted that when he first arrived at the hotel, he saw a few people in the lobby who looked homeless, "so I felt very at ease."
Lead comedian Brad Williams jokingly added: "This is an authentic New York Show. We're actually going to pipe in the smell of piss."
Along with the comedians, dancers, and acrobats, the cast includes five lead vocalists and a five-piece band, helmed by musical director Xharlie Black (real name Eddie Cole). Together the musicians perform a combination of iconic NYC hits like "New York State of Mind" and "Empire State of Mind" as well as original songs.
"We are thrilled to get the joyous Mad Apple party started in Las Vegas," Painter said in a press release. "From the moment guests enter the theater, they are whisked away to one of the most beloved cities in the world with a show that fuses hilarious acts from multiple comedians and daring acrobatics with the entertainment and eclecticism of New York City."
Get tickets for Mad Apple here, and thank us later!
By Aleza Freeman


I originally posted these Cheesy Scalloped Potatoes back in 2014, but since then I've improved the recipe a bit and updated the photos so I'm delivering a much-needed post makeover. These potatoes make a delicious side dish for any meal, any time of the year. I love making them for Easter or Christmas..
I'm always shocked when I hear people say they don't like cauliflower. Cauliflower is delicious! But if you fall into that camp of not loving its flavor, it's ok! One of the incredible things about this magical veggie is that when it is used in certain recipes, you can't even taste it. Seriously, you guys, this is total magic.
I love rice. I love it when served with a pat of butter, a few splashes of soy sauce or even completely on its own. It is so delicious when cooked to perfection. I've never been super consistent with the stove-top version of rice, but Instant Pot Rice is an entirely different story.
We go through stretches in our house when we eat tons of rice cakes and we also have rice cake droughts. I was recently gazing into our pantry and checking out a bag of rice cakes that was looking sad and lonely. My slight obsession with transforming one type of food that miiiiight be labeled as "boring"..
When I was a kid my mom made steamed artichokes all the time for us to snack on. I thought every mom prepared artichokes as snacks, but as I got older I learned that most people are deathly afraid of them. Or at least deathly afraid of cooking and preparing them.
My mom is a great cook and many of her recipes appear here on this blog (her Meatloaf and Chicken Rice Bake are two of my faves!). Something she is known for is making the creamiest, dreamiest mashed potatoes known to humankind.
My kitchen has hosted a variety of my favorite food aromas in the past month. Chili Baked Potatoes, Chili, Apple Pie and Lasagna, to name a few. I hadn't roasted a batch of tomatoes in a while and I'd forgotten how good they smell, especially when garlic shows up at the party.
So the other day a friend mentioned that baking one's own potato chips with canola oil makes them compatible with the Whole30 diet. Not that I'm following the diet, with my steady consumption of cookies as of late, but..
I've enjoyed potatoes my entire life, but it wasn't until recently that I discovered my favorite way to enjoy them. There is something about roasting potatoes, especially sweet potatoes, that is so irresistibly delicious. I tell my boys all the time that roasted sweet potatoes taste better than candy.
Are you ready for Thanksgiving? I'm not. I mean, I'm excited for it but I'm not ready. There is much to be done. By tomorrow. Did I mention that we are hosting? And that we still have boxes everywhere? And that I haven't started cleaning?


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https://apnews.com/ACCESSWIRE/e934d5bc52f1115e60d51b7a0c0d6684
Press release content from ACCESSWIRE. The AP news staff was not involved in its creation.
ACCESSWIRE: Medical
PRESS RELEASE: Paid content from ACCESSWIRE
StageZero Life Sciences Makes ColonSentry(R) Blood Test Available to Consumers Through Online Telehealth Platform
ColonSentry® is One of the First Blood Tests to Aid in The Early Identification of Colorectal Cancer TORONTO, ON / ACCESSWIRE / November 6, 2019 / StageZero Life Sciences Ltd (TSX:SZLS) ("StageZero" or the "Company") today announced it is ...
ColonSentry® is One of the First Blood Tests to Aid in The Early Identification of Colorectal Cancer
TORONTO, ON / ACCESSWIRE / November 6, 2019 / StageZero Life Sciences Ltd (TSX:SZLS) ("StageZero" or the "Company") today announced it is offering the Company's proprietary mRNA Liquid Biopsy test for Early Colorectal Cancer Detection to U.S. consumers through its online store. ColonSentry® is a blood test that measures the risk/probability of a patient having Colorectal Cancer now1. Consumers can access the test at www.colonsentry.com.
"Everyone I speak with knows someone who has been touched by Colorectal Cancer," said James Howard-Tripp, Chairman and CEO of StageZero Life Sciences. "Unfortunately, many people are diagnosed late when outcomes are the poorest. The reasons for not getting screened are many - fear of the preparation and procedures, unpleasant stories from friends, not wanting to handle stool samples - but the data shows it saves lives."
Patients can use StageZero's telehealth network to get the test ordered via a physician, have their blood drawn at a nearby lab or in the comfort of their home, and get test results online. If their scores are elevated, they can book a follow-up call with a telehealth physician online or over the phone to discuss results. Patients are strongly encouraged to share their results with their own healthcare provider as patients with elevated scores should pursue Colonoscopy or other advanced diagnostics procedures.
"Our telehealth program puts the patient in control and provides a convenient process for getting tested," said Howard-Tripp. "We are actively reaching out to people who are not compliant with colorectal cancer screening. Any patient that we can reach and engage is a win for us and the patient."
About ColonSentry®
ColonSentry® was developed as the first liquid biopsy for Colorectal Cancer (CRC), using the Company's patented Sentinel Principle Technology Platform. The test was initially validated in 10,000 patients in a North American study1, received New York State approval, and has now been used in the US on more than 100,000 patients nationwide .
ColonSentry®, as a blood test, will indicate a person's current probability of having Colorectal Cancer. ColonSentry® is for men and women 45 years or older who are at risk for Colorectal Cancer. Studies have shown that ColonSentry can also identify both left and right sided cancers2-4, whereas stool tests lack this ability.9 Right-sided lesions appear to have a worse outcome than left-sided.10
In the US, approximately 30-40% of the population at risk for developing Colorectal Cancer are currently not compliant with testing. Data, however, shows that 70% of deaths from CRC occur in this unscreened population.6 We also know that finding Colorectal Cancer early has a marked effect on survival, with 5-year survival being nearly 90% vs 14% if found late.8
Colorectal Cancer Facts & Figures
Colorectal cancer is the second most common cause of cancer death in the U.S when men and women are combined. In 2019, an estimated 101,420 new cases of colon and 44,180 cases of rectal cancer are expected to be diagnosed and an estimated 51,020 deaths from colorectal cancer are expected to occur.7
About Telehealth
According to a report published by Fortune Business Insights, the telehealth market was valued at US$ 49.8 Bn in 2018. The surge in telehealth can be attributed to patients' desires to have more control over their healthcare, dollars spent, and convenience.
Early results from our marketing campaign to consumers includes over 1 million impressions (the times the targeted individuals have seen an ad), with over 16,000 clicks (where the individual has "clicked" on the ad for more information) to 1300 plus conversions (where the individual has given us their contact information so we can reach out to them). We can now market directly to these interested individuals and work to get the appropriate patients tested.
Patients pay for their tests online via credit card and through a secure server. Payment plans are also in place to make testing more accessible.
About StageZero Life Sciences, Ltd.
StageZero Life Sciences is dedicated to the early detection of cancer and multiple disease states through whole blood. Aristotle®, our next generation test, is a panel for simultaneously screening for 10 cancers from a single sample of blood with high sensitivity and specificity for each cancer. Aristotle is built on our proven and proprietary Sentinel Principle Technology Platform which has been validated on 10,000 patients and used to develop the first liquid biopsy for Colorectal Cancer, with further validation currently underway. In addition to building a pipeline of products for early cancer detection, the Company operates a CAP accredited and CLIA certified reference laboratory based in Richmond, Virginia that offers the ColonSentry® test as well as licensed biomarker tests for lung, breast and prostate cancers. To learn more visit www.stagezerolifesciences.com.
This press release contains forward-looking statements identified by words such as "expects", "will" and similar expressions, which reflect the Company's current expectations regarding future events. The forward-looking statements involve risks and uncertainties that could cause the Company's actual events to differ materially from those projected herein. Investors should consult the Company's ongoing quarterly filings and annual reports for additional information on risks and uncertainties relating to these forward-looking statements. The reader is cautioned not to rely on these forward-looking statements. The Company disclaims any obligation to update these forward-looking statements, except as required by law.
Company Contacts:
James R. Howard-Tripp
Tel: 1-855-420-7140 Ext. 1
Jerome Cliche
Financial Communication Advisor
SOURCE: StageZero Life Sciences, Ltd


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Microbial Immunity and Vaccines
Humoral and Cellular Immune Responses in Mice Immunized with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Producing a Pertussis Toxin-Tetanus Toxin Hybrid Protein
B. Abomoelak, K. Huygen, L. Kremer, M. Turneer, C. Locht
B. Abomoelak
Laboratoire de Microbiologie Génétique et Moléculaire, INSERM U447, Institut Pasteur de Lille, F-59019 Lille Cedex, France,and
K. Huygen
Institut Pasteur de Bruxelles, B-1180 Brussels, Belgium
L. Kremer
M. Turneer
C. Locht
The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.
Diphtheria, pertussis, and tetanus (DPT) vaccines have contributed tremendously to the decrease of childhood mortality over the past decades (31). However, despite this success, these three diseases remain among the most significant infectious childhood diseases, especially in the developing world. In addition, the safety of DPT vaccines has been questioned in several countries in recent years, especially with regard to the pertussis arm, a whole-cell vaccine component (11). As a consequence, these vaccines have suffered a decrease in acceptance, resulting in the reappearance of pertussis epidemics.
A significant improvement has recently been accomplished through the development of new, acellular pertussis vaccines. In large phase III trials, these vaccines have been shown to be highly efficacious and of negligible reactogenicity (1a, 14, 15, 37). While the acellular vaccines tested varied substantially in antigen composition, all contained at least pertussis toxin (PTX), considered to be the main protective antigen against pertussis. Although PTX-neutralizing antibodies are able to protect against Bordetella pertussisinfection (35), several observations suggest that cellular immunity may play an important role in protection against pertussis.
The role of cellular immunity against pertussis has been demonstrated in mouse models (28), and spleen cells isolated from convalescent mice produce high levels of gamma interferon (IFN-γ) and interleukin-2 (IL-2), indicative of a Th1-type response. In addition, CD4+ clones of infected human subjects also secrete mainly IFN-γ and IL-2 but little IL-4 (30). Considering that natural infection usually induces a longer-lasting protection against subsequent infection by B. pertussis than does vaccination (5, 19, 25), it is possible that a Th1-type cellular immune response rather than a Th2-type antibody response mediates long-lasting protection.
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is known to induce essentially a Th1-type response, and infection of live recombinant BCG producing foreign antigens has been shown to elicit cell-mediated immunity directed toward the heterologous antigens (2, 39). In addition to inducing a cellular immune response, recombinant BCG can also induce significant levels of antibodies against foreign antigens (23). Furthermore, BCG has been widely used as a vaccine against tuberculosis, is generally considered safe, can be administered at or any time after birth, and is substantially less expensive than most other vaccine formulations (8).
With the long-term goal to develop a combined pertussis-tetanus-tuberculosis vaccine, we therefore wished to evaluate the immune responses elicited by a recombinant BCG strain that produces a hybrid protein composed of the protective part of PTX fused to the protective part of tetanus toxin (TTX). This protein, named S1-TTC, contains at its N-terminal moiety the S1 subunit of PTX and at its C-terminal moiety fragment C of TTX (TTC). We have previously shown that it can be produced at high levels in Escherichia coliand that, in its purified form, it retains TTC-specific receptor-binding activity to the ganglioside GT1b and antigenicity to neutralizing anti-S1 antibodies which specifically recognize conformational epitopes (9). In this study, we demonstrate that S1-TTC can be produced in BCG and that the recombinant BCG is able to induce a specific T-cell response as well as an antibody response against S1-TTC which is able to neutralize TTX activity.
Animals.Eight-week-old BALB/c (H-2d) female mice (Iffa Credo, l'Arbresle, France) were used for immunization with the recombinant BCG. The mice were immunized intraperitoneally (i.p.) or intravenously (i.v.) with 5 × 106 nontransformed BCG or BCG cells producing S1-TTC.
Bacterial strains and growth conditions.All cloning steps were performed in E. coli XL1-Blue (Stratagene, La Jolla, Calif.). S1-TTC production was carried out in M. bovis BCG (vaccine strain 1173P2) and in Mycobacterium smegmatismc2 155 (38). Mycobacterial transformation and culture were done as described by Kremer et al. (21).
Plasmids and DNA manipulation.pUC18 and pUC19 were purchased from New England Biolabs (Beverly, Mass.). pUC4K (Pharmacia LKB, Uppsala, Sweden) was used to isolate the kanamycin resistance (Kanr) gene. pEC001 and pRR3ΔKan were described by Kremer et al. (21), pS1-tC encoding the S1-TTC hybrid protein was described by Boucher et al. (9), and pUC::hsp60 was described by Kremer et al. (22). Restriction enzymes, T4 DNA polymerase, Klenow fragment, and other DNA modifying enzymes were purchased from Boehringer Mannheim Corp. (Mannheim, Germany). All DNA manipulations were performed by standard protocols as described by Sambrook et al. (34).
Purification of S1-TTC.The procedure used for the purification of S1-TTC was described by Boucher et al. (9). Briefly, two liters of culture containing E. coliJM109(pS1-tC) were grown to an optical density at 600 nm of 0.4. Expression of the S1-TTC-encoding gene was then induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After an additional incubation at 37°C for 5 h, the cells were harvested by centrifugation, washed, and resuspended in 40 ml of lysis buffer (25 mM Tris-HCl [pH 7.5], 25 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride). The cells were then disrupted by sonication, and the lysate was clarified by centrifugation. The supernatant was applied onto a DEAE-cellulose column (Whatman DE-52; 2.5 by 30 cm), and after washing, the bound material was eluted with lysis buffer containing 50 mM NaCl. The fractions containing S1-TTC were pooled, adjusted to 1 M NaCl, and loaded onto a Phenyl-Sepharose (Pharmacia) column. After being washed, the protein was eluted with 10 mM Tris-HCl (pH 9.5), 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride. The fractions containing S1-TTC were pooled, immediately adjusted to pH 7.5 by the addition of 1 M Tris-HCl (pH 7.0), dialyzed overnight against several changes of phosphate-buffered saline (PBS; K2HPO4 [pH 7.5], 0.15 M NaCl), concentrated with a Millipore CX-10 concentrator to 1 mg/ml, and then stored at −80°C until further use.
Construction of expression vectors.pEN004 was constructed as follows. The promoter and signal peptide coding sequence of the antigen 85A gene were isolated from pEC001 by digesting the plasmid with HindIII, blunt ending it with Klenow fragment, and then redigesting it with BamHI. This fragment was ligated into SmaI/BamHI-digested pS1-tC, generating pEC007. The 1.3-kb HindIII fragment corresponding to the Kanr gene was isolated from pUC4K and ligated into pEC007 previously digested with HindIII and blunt ended with Klenow fragment. This yielded pEC012, which was further digested withPvuII, and the resulting 4.2-kb fragment was ligated into the blunt-ended ScaI site of pRR3ΔKan, generating the final shuttle vector, pEN004 (Fig. 1A).
Schematic representation of pEN004 and pBMX. The following relevant features of pEN004 (A) and pBMX (B) are shown. The mycobacterial and E. coli origins of replication are indicated by the black boxes labelled ori myco and ColE1, respectively. The Kanr gene is indicated by the stippled arrow labelled KmR. The black boxes labelled hsp60 and 85A denote the hsp60 promoter and the antigen 85A promoter and signal peptide coding sequence, respectively. The grey boxes correspond to the S1-TTC-coding sequence.
pBMX was constructed as follows. First, the 1.3-kb PstI fragment corresponding to the Kanr gene was isolated from pUC4K, blunt ended with T4 DNA polymerase, and cloned into pUC::hsp60, previously digested withBamHI and blunt ended with Klenow fragment. The resulting plasmid, named pUChspK, was then digested with NcoI andXbaI and blunt ended with Klenow fragment. The 1.7-kb fragment containing the Kanr gene and the hsp60promoter was cloned into the unique SmaI site of pS1-tC, giving rise to pBM02, which was in turn digested with PvuII. The resulting 3.4-kb fragment was finally inserted into theScaI site of pRR3ΔKan, yielding the shuttle vector pBMX (Fig. 1B).
Immunoblotting of recombinant proteins.Ten milliliters of each mycobacterial culture was harvested at mid-log phase by centrifugation. The cells were then resuspended in 1.5 ml of PBS and disrupted for 10 min with a Branson Sonifier 450 at half-maximal constant output. The resulting M. bovis BCG or M. smegmatis crude extracts were then subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and with a 12% polyacrylamide gel as described by Laemmli (24). After electrophoresis, the proteins were transferred onto a Hybond-C Extra membrane (Amersham France). The membrane was then saturated with 5% dry milk in PBS containing 0.1% Tween 20 (PBS-T). The antibodies used for the detection of recombinant proteins were a monoclonal antibody (1B7) directed against the S1 subunit of PTX (36) and rabbit polyclonal antibodies directed against tetanus toxoid (9), both kindly provided by H. Sato and Y. Sato (National Institute of Health, Tokyo, Japan). The immunoblots were developed with goat anti-mouse alkaline phosphatase-conjugated antibodies or goat anti-rabbit alkaline phosphatase-conjugated antibodies, purchased from Promega (Madison, Wis.).
Enzyme-linked immunosorbent assay (ELISA).The wells of polystyrene microdilution plates (catalog no. 4-39454; Nunc, Roskilde, Denmark) were coated overnight with 100 μl of the ganglioside GT1b at a concentration of 1 μg/ml in PBS. The plates were washed three times with PBS, and 50 μl of purified S1-TTC was then added at a concentration of 2 μg/ml in PBS in each well. After incubation for 2 h at room temperature, the plates were washed three times with PBS and the sera were then added in PBS-T containing 0.5% bovine serum albumin (PBS-T-BSA) in twofold serial dilutions. The plates were incubated for 2 h and washed with PBS-T-BSA, and horseradish peroxidase-conjugated anti-immunoglobulin G1 (IgG1) (1/10,000), anti-IgG2a (1/10,000), anti-IgG2b (1/12,000), or anti-IgG3 (1/10,000) was added. Alternatively, biotinylated anti-mouse IgG (catalog no. RPN.1001; Amersham) was added at a 1/500 dilution, incubated for 1 h in PBS-T, washed three times with PBS-T, and then incubated with the streptavidin-biotinylated horseradish peroxidase complex (catalog no. RPN.1051; Amersham) at a dilution of 1/1,000 for 30 min in PBS-T. The plates were finally washed three times with PBS-T and then developed with 0.4 mg ofO-phenylenediamine dihydrochloride (catalog no. P-4664; Sigma)/ml and 1 μl of hydrogen peroxide/ml in 0.1 M citrate buffer (pH 4.5) at 37°C in the dark. The plates were read with a Nunc NJ 2000 immunoreader at 490 nm.
TTX neutralization.To test for in vivo TTX-neutralizing activity of the anti-S1-TTC antisera, we used the standard techniques of the European Pharmacopoeia (1974) at the level L+/1,000, as described previously (40). The results are expressed in international units per milliliter.
Spleen cell cytokine production.Two months after i.p. immunization of mice with recombinant BCG producing S1-TTC, the mice were sacrificed by cervical dislocation and the spleens were removed aseptically. Spleen cells were isolated by using a loosely fitting Dounce homogenizer, washed, adjusted to a concentration of 4 × 106 per ml, and grown in flat-bottomed microwell plates (Nunc) in RPMI 1640 medium supplemented with HEPES, glutamine, 5 × 10−5 M 2-mercaptoethanol, antibiotics, and 10% heat-inactivated fetal calf serum. The purified S1-TTC or PTX, purchased from List Biologicals (Campbell, Calif.), was added to the cultures at 10 μg/ml and 1 μg/ml, respectively. The cells were incubated at 37°C in a humidified incubator at 5% CO2. After 24 h the supernatants were harvested for the determination of IL-2 production as described previously (17). Each experiment was repeated at least three times, and the results of one representative experiment are shown.
Production of S1-TTC under the control of the antigen 85A promoter and signal peptide.We have previously shown that the antigen 85A promoter and signal peptide can be used for heterologous expression and protein secretion in mycobacteria (21). In a first attempt, therefore, we used these expression and secretion signals for the production of S1-TTC in BCG by constructing pEN004, as described in Materials and Methods. In addition to the S1-TTC expression cassette, this vector contains a mycobacterial origin of replication and a Kanr gene (Fig. 1A).
M. smegmatis and BCG were transformed with pEN004, and the recombinant mycobacteria were selected on solid Middlebrook medium containing 25 μg of kanamycin/ml. Kanamycin-resistant colonies were tested for their plasmid content by the electroduction method described by Baulard et al. (6). Recombinant mycobacteria containing the expected plasmid were then grown in liquid medium containing 25 μg of kanamycin/ml for approximately 5 days in the case of M. smegmatis and for 2 weeks in the case of BCG. Mycobacterial cell extracts were then prepared and analyzed by immunoblotting with anti-S1 monoclonal antibody 1B7 and an anti-TTC polyclonal antibody. As shown in Fig. 2, only the recombinant BCG containing pEN004 produced an immunoreactive band, corresponding to an approximately 75-kDa protein. The size of the protein was as expected, and it was recognized by both anti-S1 and anti-TTC antibodies, indicating that it contained both antigenic determinants. In addition to the 75-kDa protein, a smaller, approximately 37-kDa protein was also detected by the anti-TTC antibodies. This protein was not recognized by 1B7, suggesting that it is a proteolytic breakdown product containing essentially the C-terminal portion of the hybrid protein. Similar results were obtained with M. smegmatis (not shown).
Immunoblot analysis of recombinant BCG(pEN004). Recombinant BCG(pEN004) (lanes 3 to 5) was analyzed with anti-S1 monoclonal antibody 1B7 (lanes 1 to 4) and anti-TTC polyclonal antibodies (lane 5). Both whole-cell extracts (lanes 4 and 5) and culture supernatant (lane 3) were analyzed. Lane 1 contains a whole-cell extract, and lane 2 contains culture supernatant of nonrecombinant BCG as a control. The sizes of the molecular mass markers are given in the right margin.
Production of S1-TTC under the control of the hsp60promoter.A second expression system used in this study was driven by the M. bovis BCG hsp60 promoter to produce intracellular S1-TTC via the construction of pBMX. This plasmid had characteristics similar to those of pEN004, except that the antigen 85A promoter and signal-peptide coding sequence were replaced by the BCGhsp60 promoter (Fig. 1B).
After transformation and selection of recombinant M. smegmatis and BCG, cell extracts were prepared and analyzed by immunoblotting with the monoclonal antibody 1B7 and the polyclonal anti-TTC antibodies. With either reagent, an approximately 75-kDa protein was again readily detectable in extracts of BCG containing pBMX (Fig. 3). Similar results were obtained for M. smegmatis (not shown). This protein was not present in the BCG control extracts, indicating that the pBMX expression system also yielded an immunoreactive hybrid protein containing both the PTX and TTX antigenic determinants. Expression levels appeared to be significantly higher than in BCG(pEN004) extracts, and several smaller peptides were detected with anti-TTC antibodies in BCG(pBMX) extracts, whereas a single major breakdown product was found in BCG(pEN004) lysates. When compared on a quantitative basis by densitometric scanning of the immunoblots, BCG(pBMX) was found to produce approximately 10-fold more S1-TTC than BCG(pEN004) (approximately 5 ng of S1-TTC/μg of total protein versus approximately 0.4 ng of S1-TTC/μg of total protein). As expected, no extracellular S1-TTC was detected in culture supernatants of BCG(pBMX).
Immunoblot analysis of recombinant BCG(pBMX). Recombinant BCG(pBMX) (lanes 2 and 3) was analyzed with anti-S1 monoclonal antibody 1B7 (lane 3) and anti-TTC polyclonal antibodies (lane 2). Lane 1 contains a whole-cell extract of nonrecombinant BCG as a control. The sizes of the molecular mass markers are given in the right margin.
Antibody responses after immunization with BCG(pEN004) or BCG(pBMX).Groups of five BALB/c mice were immunized with 5 × 106 BCG(pEN004), BCG(pBMX), or nonrecombinant control BCG cells either i.v. or i.p. Two months later, the mice were boosted with 5 × 106 cells of the same BCG strains. At different time intervals after the boost, sera were collected, pooled per group of mice, and then tested for the presence of anti-BCG and anti-S1-TTC antibodies by immunoblotting and ELISA. All groups of mice produced anti-BCG antibodies at similar levels (data not shown). However, only those mice that had been immunized i.p. with BCG(pBMX) produced antibodies directed against S1-TTC (Fig.4). These antibodies did not react with purified S1, suggesting that they were directed against the TTC part of the protein. No such antibodies were found in the sera of mice that were immunized i.p. with BCG(pEN004) or with the control BCG or those that were immunized i.v. with any strain.
Immunoblot analysis of sera of mice immunized with BCG(pBMX). Ten weeks after the first immunization, the sera of mice immunized with BCG(pBMX) (lanes 2 and 3) or with nonrecombinant BCG (lane 1) was analyzed by immunoblotting with purified S1-TTC (lanes 1 and 3) or recombinant S1d (lane 2) as an antigen. Lane 4 contains the molecular mass markers, the sizes of which are given in the right margin.
As shown in Fig. 5, the anti-S1-TTC antibody titers of the mice that had received BCG(pBMX) stayed high for at least 26 weeks after the boost. The analysis of the isotype profile indicated that specific anti-S1-TTC IgG1, IgG2a, and IgG2b were produced (Fig. 6), suggestive of a mixed antibody response. The antibodies were exclusively directed against the TTC part of the hybrid protein, since no anti-PTX antibodies were detected by ELISA (not shown).
Kinetics of anti-S1-TTC antibody titers of mice immunized with BCG(pBMX). The total anti-S1-TTC IgG titers of mice immunized with BCG(pBMX) were determined at 0, 10, 14, 18, and 26 weeks after the first immunization, as indicated. The titers correspond to the reciprocals of the highest serum dilutions giving optical density values that are at least threefold over background levels.
Isotype profile of anti-S1-TTC antibodies of mice immunized with BCG(pBMX). Pooled sera of mice immunized with BCG(pBMX) were analyzed for their anti-S1-TTC IgG1, IgG2a, IgG2b, and IgG3 titers. The titers were estimated 18 weeks after the first immunization and correspond to the reciprocals of the highest serum dilutions giving optical density values that are at least threefold over background levels.
To test for the functional activities of these anti-TTX antibodies, the in vivo toxin-neutralizing activities of the sera were measured by using the reference method of the European Pharmacopoeia. At 2 weeks after the first immunization with BCG(pBMX) the sera contained 0.01 to 0.02 IU/ml. This level of neutralizing antibodies rose to 0.06 to 0.08 IU/ml after the boost, whereas the neutralizing activities of sera of mice immunized with nonrecombinant BCG remained below detectable levels. Since levels above 0.01 IU/ml are considered protective in humans, a single immunization with the recombinant strain thus induced protective levels of anti-TTX antibodies.
IL-2 production after immunization with BCG(pBMX).The spleens of BALB/c mice immunized and boosted with either BCG(pEN004), BCG(pBMX), or nonrecombinant BCG were tested for the secretion of IL-2 by using purified S1-TTC for stimulation of the spleen cells. As shown in Fig. 7A, high levels of S1-TTC-specific IL-2 secretion were detected only for spleen cells isolated from mice that were immunized with BCG(pBMX), whereas BCG-specific IL-2 secretion was also detected for cells isolated from nonrecombinant BCG-infected mice.
Antigen-specific IL-2 production in mice immunized with BCG(pBMX). The IL-2 contents in culture supernatants of splenocytes isolated from mice immunized with nonrecombinant BCG (left) or BCG(pBMX) (right) were estimated as described in Materials and Methods either after stimulation with purified S1-TTC (A) or with PTX (B). The error bars indicate the standard deviations.
When PTX was used to stimulate the spleen cells, IL-2 secretion was also detected only when the spleen cells were isolated from mice immunized with BCG(pBMX) (Fig. 7B).
BCG is one of the most widely used vaccines available today and is the only live bacterial vaccine recommended for use in children by the World Health Organization. Although its efficacy against tuberculosis still remains a matter of considerable debate (12), BCG has a remarkably low incidence of severe side effects. This makes it one of the most promising bacterial live vectors for the production of heterologous antigens to develop cost-effective vaccines able to protect against several diseases simultaneously. Consequently, several mycobacterial expression systems have been developed over the last few years (2, 23, 29, 39). In some instances protection against challenge has been achieved against the heterologous pathogen when recombinant BCG was given as a vaccine (13, 16, 26, 27). Different laboratories have used different expression systems based on several promoters. The most widely used promoter is that of thehsp60 gene. However, other promoters have also been used, such as the pAN promoter from Mycobacterium paratuberculosis(29) or the promoters of the genes encoding the antigen 85 complex (20, 21). The antigen 85 complex contains several major mycobacterial secreted proteins, named 85A, 85B, and 85C. The use of the promoter of the 85B gene controlling the production of the hybrid protein antigen 85B fused to the V3 epitope of the human immunodeficiency virus led to a recombinant BCG strain that was able to induce protective immune responses in small animals (16). In a comparative study with the same reporter gene, we have recently shown that the 85A promoter is stronger than the 85B promoter (1). The 85A promoter has been used successfully for the expression of several heterologous antigens (7, 21).
In this study, we compared the immunogenicity of recombinant BCG strains producing a PTX-TTX hybrid protein (S1-TTC) under the control of either the hsp60 promoter or the 85A promoter and signal peptide. The recombinant protein was readily detected in both expression systems. When the chimeric gene was expressed under the control of the hsp60 promoter, the expression level was approximately 10-fold higher than when the 85A promoter and signal peptide were used. The comparison of the anti-S1-TTC immune responses indicated that only the BCG strain producing the antigen under the control of the hsp60 promoter induced anti-S1-TTC antibodies. No immune response was detected with the 85A expression system, even after three immunizations. This finding was somewhat surprising, since, although the hsp60 promoter induced stronger expression of the foreign gene, the difference with the 85A expression system was only about 10-fold in vitro. However, it may be conceivable that the expression levels are much more divergent in vivo. In Salmonella the hsp gene is induced in vivo (10). When chromosome-borne, the BCG hsp60promoter is also induced by stress; however, this regulation is apparently lost when the hsp60 promoter is plasmid-borne (39). Although there is no evidence yet that the antigen 85 complex genes are regulated in vivo, mice or humans infected byM. tuberculosis show a strong immune response against the antigen 85 complex (18), indicating that these proteins are produced in vivo.
Another difference between the two expression systems described here is the presence of the signal peptide in the 85A system. Although this system has been shown to allow for secretion of recombinant proteins (7, 21), no S1-TTC was found in the culture supernatant of the recombinant BCG strains. It is most likely that this is due to the relatively large size (75 kDa) of the antigen compared to antigens successfully secreted in the previous studies. Indeed, progressive shortening of the S1-TTC hybrid protein by the 3′ truncation of its gene gradually increased its secretion efficiency (7a). Nevertheless, it can be assumed that in the 85A expression system S1-TTC is located at the external side of the membrane, since the estimated size is consistent with that of the mature protein after removal of the signal peptide. Because of its relatively large size, the antigen is probably trapped in the cell wall. Since it has been shown by others that secretion of the foreign antigen by recombinant BCG may increase its immune response, we are currently constructing new vectors combining the hsp60 promoter with the 85A signal peptide coding region in an effort to distinguish between the role of secretion or hsp60-dependent expression in the induction of antibody responses against S1-TTC.
Anti-S1-TTC antibody titers remained high for at least 6 months after immunization with the recombinant BCG producing the antigen under the control of the hsp60 promoter. Isotyping indicated that the mice produced IgG1, IgG2a, and IgG2b antibodies, suggestive of a mixed response. These antibodies, however, were mostly directed against the TTC part of the molecule, since no antibodies reactive against PTX were detected in the mouse sera. Instead, the mice immunized with this recombinant BCG strain mounted a weak but significant cellular immune response against PTX, as evidenced by the secretion of antigen-specific IL-2. This finding is particularly interesting with respect to the development of a combined vaccine against tetanus and pertussis, since recent evidence strongly suggests that the mechanism of protective immunity against pertussis in mice involves IL-2-secreting T cells (28). In addition, Th1 CD4+ cells probably also play a major role in protective immunity in humans, since convalescent patients with whooping cough produce B. pertussis-specific T cells that secrete high levels of IFN-γ and IL-2 but very little IL-5 or IL-4 (32, 33), and it is well known that infection withB. pertussis provides better and longer-lasting protection against whooping cough than does vaccination. Since immunization with recombinant BCG producing S1-TTC induces anti-TTX antibodies and anti-PTX-specific T cells of the Th1 subpopulation, this may constitute a promising approach for the development of a novel, effective, and safe combined vaccine against tetanus and whooping cough, affordable even for the developing world.
However, several points remain to be addressed. Disseminated BCG infections have been reported in patients with AIDS, and the use of recombinant BCG may be particularly counterindicated in areas where the human immunodeficiency virus prevalence is high. Recent efforts to genetically engineer BCG strains so that they can be used in immunocompromised hosts have provided some encouraging initial results (3), and it will be interesting to evaluate the S1-TTC construct for its immunogenicity in some of those strains. In addition, the immune response to the PTX part of the molecule induced after administration of the recombinant BCG strain is rather weak, and future constructs may help to increase its immunogenicity, either by the coexpression of cytokines or by simply redesigning the hybrid protein. Using similar S1-TTC constructs expressed in Salmonella typhi vaccine strains, Barry et al. (4) have shown that the S1 subunit fused to the N terminus of TTC was less effective than S1 fused to the C terminus in generating anti-PTX antibodies. Finally, antipertussis and antitetanus vaccines are usually administered together with antidiphtheria vaccines. We are therefore constructing recombinant BCG strains coexpressing protective diphtheria toxin domains together with the S1-TTC hybrid protein.
We thank H. and Y. Sato for antibodies, A. Baulard for helpful discussion in the initial stages of the project, and E. Fort for photography.
The work was supported by INSERM, Institut Pasteur de Lille, Région Nord-Pas de Calais et Ministère de l'Enseignement Supérieur et de la Recherche. B.A. holds a fellowship from CNOUS.
Editor: R. N. Moore
Received 22 March 1999.
Returned for modification 10 May 1999.
Accepted 20 July 1999.
Abomoelak, B., et al. Unpublished data.
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You are going to email the following Humoral and Cellular Immune Responses in Mice Immunized with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Producing a Pertussis Toxin-Tetanus Toxin Hybrid Protein


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of earth turned from season to season.
by the scents of centuries.
Commended in the Stanza Poetry Competition 2018 on the theme of 'Tradition', judged by Penelope Shuttle.
Lesley was born in Yorkshire but now lives in Ledbury. She was a late-starter, but has a Diploma in CW from the Open University and a Masters from the University of Gloucestershire, with a final thesis on 'Ekphrasis as Translation'. Her first collection, Scumbled (Cinnamon), was published in 2015 and her poems have appeared in anthologies and online & print journals. She has been an IT Business/Systems Analyst, Customs officer, VAT collector, ESL teacher, French Gite owner and is Stanza rep for Herefordshire. She photographs walls, floors & ceilings and has yet to find the right free WordPress site to share them properly - although does not expect a huge following. She loves liquorice and flat lands and a perfect 25 degree temperature.


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The data retrieved from the cache file.
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Thomas Bjørn and Jim Furyk have recreated an iconic moment in history by hitting golf balls off the famous Eiffel Tower, 41 years after the legendary Arnold Palmer became the first – and only previous person – to do so.
What an incredible morning for Europe, as they continued to build on the momentum from Friday afternoon, when they trounced America 4- nil.
Galvin Green, the premium performance golf apparel brand based in Sweden, is supporting this year's European Ryder Cup Team with the most advanced waterproof and windproof garments ever put into play…and making them available to all golfers across Europe from this month.


text:
A Matter of Life and Death is a stage adaptation by Tom Morris and Emma Rice of Powell and Pressburger's 1946 film of the same name for the company Kneehigh Theatre. Its first run at the National Theatre lasted from 3 May 2007 to 21 June 2007.

Cast & crew
Taken from the programme of the performance at the Olivier Theatre, London, 12 May 2007.

Production team
 Director: Emma Rice
 Set Designer: Bill Mitchell
 Costume Designer: Vicki Mortimer
 Lighting Designer: Mark Henderson
 Choreographer: Debra Batton & Emma Rice
 Music: Stu Barker
 Sound Designer: Gareth Fry
 Projection Designers: Jon Driscoll & Gemma Carrington

Cast

Differences from the film

Though the plot of the adaptation was broadly similar, there are some differences. In the play June, the radio operator, with whom Peter falls in love, was British rather than American, since the company "felt that it would distract attention from the central story and towards the different issues of Anglo-American relations today".  However, this meant that the courtroom scenes could not include the arguments about Britain, its historical place in world events and how it is perceived by the rest of the world, and had to be replaced with different arguments about war and the effects of war. For the denouement, Peter's fate was decided by the toss of a coin, rather than by June's offer to change places with him; the end thus varied from performance to performance.

Production
The production itself included many coups de theatre to represent things like the camera obscura, the table tennis game frozen in time and the Stairway to Heaven.

Most of the major reviewers seem to have seen it on a night when the toss of the coin determined that Peter would die. This does tend to negate much of what has gone before, all of his struggles to stay alive. Those that saw it on a night when Peter lived usually give a more positive report.

Controversy
Though individual assessments varied from Nicholas de Jongh's wholly negative account in the Evening Standard to Susannah Clapp's enthusiastic review in The Observer, critical reaction to the play was generally poor. This prompted an attack by National Theatre director Nicholas Hytner on the major London critics, whom he described as "dead white men". His charges of misogyny and prejudice against female directors and new styles of theatre were hotly rebutted by his targets.

Other productions
A production was staged at The Space, an arts and community centre on the Isle of Dogs in London, from 14 to 16 June 2012.

References

External links
National Theatre
Guardian newspaper article
Interview with actor Douglas Hodge

2007 plays
Plays based on films
British plays
Existentialist plays

redpajama_set_name: RedPajamaWikipedia


text:
Media distrust in Wyoming: A Q&A with Rod Hicks
Howard Schneider, executive director of the Center for News Literacy at Stony Brook University, leads a discussion on media bias with Casper Project participants on May 7, 2019. (Rod Hicks)
October 15, 2019 by Katie Klingsporn 9 Comments
Media trust in America has sunk to alarming lows. From an acme of some 70% of Americans who reported having a great deal or fair amount of trust in the media in 1976, that number had plummeted to 32% by 2016.
The Society of Professional Journalists, a nonprofit membership organization dedicated to perpetuating a strong free press, set out to get to the bottom of this troubling erosion. The organization in 2017 tapped veteran editor and journalist Rod Hicks — a former editor with the Associated Press, St. Louis Post-Dispatch and elsewhere — to pilot a project aimed at better understanding, and potentially reversing, the trend.
Rod Hicks
The quest landed him in Casper, which studies have identified as a ground zero of sorts for media distrust.
There, he launched The Casper Project, a 6-month-long series of community discussions aimed at gaining a deep understanding of the causes behind distrust. During the project, some three dozen Casper residents — including 16 conservatives, 12 liberals and seven moderates — gathered every few Tuesdays. During sessions, participants aired concerns about media bias, learned about the functions of newsrooms and took in panels and presentations from media professionals.
At times, things grew tense. There were occasional outbursts. And in the end, minds weren't drastically changed. But, Hicks said, it was a valuable and eye-opening experience that contained important lessons both for journalists and news consumers.
"Going into this, I assumed that we would not change most people's minds," Hicks said. "I wanted them to get something out of it even if it didn't change their minds. So, we now have three dozen people who are more savvy news consumers, and I think that is noteworthy."
It was also personally illuminating for Hicks, who had never set foot in Wyoming before the project. Hicks, who presented his final findings to participants and the public last week in Casper, sat down with WyoFile to talk about the project, why it's time for media outlets to take a hard look in the mirror and his first encounter with real-life cowboys.
WF: Why did you pick Casper for this project?
RH: I did not want the selection of the place that we went to be totally arbitrary. I went to some organizations that do polling, particularly polling of the media, like the Pew Research Center and Gallup. When I talked to Gallup, they told me that just the year before, a survey they had released identified the top states with the highest level of media distrust. And Wyoming was at the top of the list.
Now, you have to put it into perspective. Everybody hates the press. So it's not like Wyoming is way up here and the rest of the country is way down there. Wyoming is slightly above everybody else.
And [I chose] Casper because it's centrally located, has a good population, it has the biggest newspaper and the only newspaper that is published seven days a week, plus it has some other local media options in television, radio and online.
Did you have much experience visiting Wyoming or spending time here?
I had never been to Wyoming before in my life, and I've been to like 36 states.
It is very unfamiliar to me, but I think it's nice. One of the things I thought was so cool was seeing the mountains in the distance and the landscape. And I was intrigued by the culture there. I went to restaurants on the weekends, and the guys had on their jeans and boots and plaid shirts and cowboy hats. That's how they dress up to go out. It's not just the stereotype. I was intrigued by that.
Joshua Wolfson (left), editor of the Casper Star-Tribune, addresses participants of The Casper Project during a session on June 4, 2019. Other local journalists who participated include, left to right, Nick Learned, news director, K2 Radio; Halle Jones, anchor, KTWO-TV; and Trevor Trujillo, editor, Oil City News. (Rod Hicks)
What were you looking for in participants of the project?
I wanted people who were going to be thoughtful and who really wanted to let us know legitimate concerns that they had with the media. I wanted there to be above all else some legitimate concern with how the media works and some interest in seeing it improve.
What would you say were your major takeaways from the sessions?
One of the things that people kept saying, throughout the entire project, was that the media is biased. This even started before the sessions started because we gave everyone a survey as part of their registration to participate, and one of the questions was, "what's the biggest problem with the news media?" And bias was the winner, by a long shot. I mean, overwhelmingly. So that theme kept coming up. And one of the takeaways for me is that there just has to be some legitimacy to that. I really do believe that journalists need to look for that in the copy. Before I had this job, I worked at the Associated Press for almost 10 years as an editor. If I were to go back to the AP editing copy again, I think that I would do it with a different eye. I would be looking for things like that.
You also noted in your report a difference in perspectives of local versus national news outlets, particularly when it related to anything about President Trump. Can you talk about that?
One of the things that the people in the group did was go on a tour of the newspaper and the TV station. The editor at the newspaper said he was expecting people to come there complaining that, you know, "you give this high school sport more coverage than this one." But all they wanted to talk about was national politics, specifically how Donald Trump is treated in the press.
I was a little surprised by that. I went in knowing that local media enjoys a higher level of trust than national media. So I expected them to have more complaints about the national news than local. But I don't know that I expected them to largely focus on national news.
Dean Miller, editor of The Jefferson County & Port Townsend Leader in Washington state, leads a discussion with Casper Project participants on March 19, 2019, about how to distinguish news from other types of information. (Rod Hicks)
And it sounds like no participant changed his or her mind in drastic ways regarding their feelings on the press. Despite that, do you think it was a valuable exercise, and why?
I think it was valuable. We were trying to accomplish two things. One, we wanted to educate people. We wanted them to become more discerning news consumers. We wanted them to better understand how the media works, and what goes into the types of decisions that news organizations have to make. I wanted them to better discern between news and other types of information — propaganda, advertising, satire even.
The other side of it is that we wanted to really understand what is the root of this distrust. What is the press doing wrong that makes people feel that it is overwhelmingly biased. And the reason we wanted that was because we wanted to figure out what can we do differently to win these people back.
If you had one recommendation for media outlets to help salvage and rebuild this, what would it be?
If I only had to say one, it would be to turn on your bias antenna when you're reading copy or writing your news product. Look for it. Question whether this can be construed as biased. Because I don't think you're going to catch it unless you are actively looking for it. We've become immune to it.
Another thing I know you were stressing to media is better engagement.
Yes. We need to demystify this process that we go through. I make a mention of Trustingnews.org. I really hope that journalists go to the website and look at the examples of what newsrooms are actually doing. They are explaining controversial decisions that they make, they are opening up to their audiences, looking for opportunities for engagement. I just think you can't do that enough.
You have to be really transparent and really talk to your audiences. Try to get to know them, try to get to know what they want. And respect them.
The Casper Project's logo (Courtesy Rod Hicks)
Another interesting thing you brought up is that professional journalists know all the intricacies of the job and maybe take for granted that many people don't.
We have our own jargon in our business. We think that everybody knows what a cutline, a headline, a dateline, all these lines are. And they don't! We talk about anonymous sources, and we expect that people know what that means. People do not know what that means.
We have to understand that we are so close to this that we forget that the general population doesn't know all this stuff.
There has been a lot of negative news about the decline of journalism in the last decade. Despite that, can you talk about its role in a healthy democracy? Why is journalism important enough to try to bolster with efforts like this project?
We asked people in this project in their registration form, "How important is a free press to a democracy?" Everybody indicated it was important. And they are right. I hope people also recognize that if the press is essential to a democracy, the fact that they don't trust it is a problem.
Support WyoFile with a tax-deductible donation today.
In these times, we really, really, need a free press. We need to put a check on power. That's one of the most important things we can do as journalists. And that's the way it was intended. We're in the Constitution for a reason. And so, the press needs to do the best that it can to be fair and to be unbiased and to be accurate.
This transcript has been lightly edited for length and clarity.
This story has been updated to clarify the meaning of the first sentence. —ED.
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Filed Under: Featured, People, Places, Politics
About Katie Klingsporn
Katie Klingsporn is WyoFile's managing editor. She is a journalist and word geek who has been writing about life in the West for 15 years. Her pieces have appeared in Adventure Journal, National Geographic Adventure and Patagonia's Cleanest Line blog.
Paul Butler says
Terrific to hear that Wyo folks are more intelligent than average……….even though I suspected as much.
The other good quality for Wyomingites is their refusal to follow media like sheep. Media has become extremely corrupted agenda driven drivel.
City: cheyenne
State of Residence: Wyoming
Dave Racich says
We as human beings have bias built into our DNA. Unfortunately we let that bias take hold of our critical thinking process. "You can fool all the people some of the time and some of the people all the time, but you cannot fool all the people all the time."
Thank goodness for journalist who are trying to bring balance to all venues of journalism
City: Pinedale
Robb Gould says
Media bias? Ya think DiNozzo?! THANK YOU for bringing the topic out into the light! I'm eager to see where it goes.
Years ago, there was a segment at the end of the news (can't remember the show or network) called "Point – Counterpoint" where two individuals would each take a few minutes to present opposite sides of an issue. This mini-debate brought out points for the viewer to consider. We could use more of that style in media.
City: Cody
Dewey Vanderhoff says
Robb- As one who has been in journalism since age 16 ( I'm 68 now ) , I've learned that nearly every person who swears the news media is biased is a person who has no idea how newsgathering really works and the methodology of the journalistic process. They default to : I don;t agree with that , so it must be flawed somehow… which became bias …and finally devolved to the hue and cry of " Fake News ! " which frankly I only heard spoke out loud maybe five times in the five decades before Trump was elected, and now I hear it five times a day. Then there's this : While it is technically an unbreakable rule of the 4D Universe that every story has at least two sides ( nothing detectable by us can be 1-dimensional ) too many people make the mistake that each ' side' must be given equal space or air time, a 50/50 balance of information. So when you go to a public meeting on a topic, and 9 people speak for something and one speaks against, is it realistic to expect next week's Cody Emptyprize to have 10 column inches on each side of the issue ? Of course not. It all needs to be apportioned and weighted. Example : 98+ percent of the world's scientists qualified to give an informed opinion on Climate Change will tell us it is real and mankind is driving it faster and further. But that Counterpoint needs to be adjusted to scale. It's a tough call.
Heisenberg's Uncertainty Principle in physics that says among other things you can't interact with or measure something without altering it ( e.g. a thermometer has to steal some of the heat it is reading ) also applies to a lot of other things. All news at some point is condensed , distilled down , editted , and cut to fit before it's presented. That's the journalistic process at work , But it's not bias or censorship. Very very few ordinary citizens can discern the difference between editting, biasing, and censoring these days.
Harvey Reading says
I've read too many misleading stories covering subjects with which I was familiar to fully trust in noozepapers. I've found that using my own brain gives me a better notion of what is truly going on locally, or around the world.
City: Shoshoni
Martha Braymance says
A blind trust in the veracity of the reporting in a small town rag can be equally dangerous. Our local weekly (the News Letter Journal) is as good an example as any of the problems that can and do arise as a result of the symbiotic (sometimes parasitic) relationship between advertisers and conventional print media outlets. The overwhelming bulk of ink on any page of the NLJ is devoted to the hawking of local wares, not local news. As a general rule, any controversial issues in Newcastle seem to be avoided like the plague, and as we all know all too well, potential conflicts of interest are often unavoidable, and by nature or by necessity are always rife in small towns. I'm not saying that it is in a community 's best interest to air every scrap of dirty laundry, but if something is filthy I believe it should get a good running through the wringer even if it risks agitating the loyal customers as well. Too often what passes for journalism around here is a heaping helpin' of front page feel good quilting bee coverage brought to you by your hometown purveyor of fine and affordable crisp clean line ns. Pretty pieces neatly stitched together. Far from the real article. Far from the unbiased truth.
City: Newcastle
Paul Cook says
Kudos to Klingsporn. A light to the future.
Keith Collins says
Your first sentence in the article is worded wrong. You meant to say, "Media TRUST in America has sunk to alarming lows." Anyway, the problem could be relieved if the public thought you were trying to tell both sides of a story. Instead, you twist everything to advocate changing everything about America.
Robert Hoskins says
Unfortunately, too often the perception of bias is nothing more than the refusal or inability to acknowledge the (hard) truth.
City: Dubois
John Brown on Lummis, Eathorne responses to insurrection are delusional
Pat Carpenter on Week 43: The pandemic in Wyoming from Jan. 2-8
Harvey Reading on Lummis, Eathorne responses to insurrection are delusional