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Updates found with 'impressive affects'

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Updates found with 'impressive affects'

New DNA nanorobots successfully target and kill off cancerous tumorsBY SARAH BUHRFeb 12, 2018Science fiction no more — in an article out today in Nature Biotechnology, scientists were able to show tiny autonomous bots have the potential to function as intelligent delivery vehicles to cure cancer in mice.These DNA nanorobots do so by seeking out and injecting cancerous tumors with drugs that can cut off their blood supply, shriveling them up and killing them.“Using tumor-bearing mouse models, we demonstrate that intravenously injected DNA nanorobots deliver thrombin specifically to tumor-associated blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth, ” the paper explains.DNA nanorobots are a somewhat new concept for drug delivery. They work by getting programmed DNA to fold into itself like origami and then deploying it like a tiny machine, ready for action.DNA nanorobots, Nature Biotechnology 2018The scientists behind this study tested the delivery bots by injecting them into mice with human breast cancer tumors. Within 48 hours, the bots had successfully grabbed onto vascular cells at the tumor sites, causing blood clots in the tumor’s vessels and cutting off their blood supply, leading to their death.Remarkably, the bots did not cause clotting in other parts of the body, just the cancerous cells they’d been programmed to target, according to the paper.The scientists were also able to demonstrate the bots did not cause clotting in the healthy tissues of Bama miniature pigs, calming fears over what might happen in larger animals.The goal, say the scientists behind the paper, is to eventually prove these bots can do the same thing in humans. Of course, more work will need to be done before human trials begin.Regardless, this is a huge breakthrough in cancer research. The current methods of either using chemotherapy to destroy every cell just to get at the cancer cell are barbaric in comparison. Using targeted drugs is also not as exact as simply cutting off blood supply and killing the cancer on the spot. Should this new technique gain approval for use on humans in the near future it could have impressive affects on those afflicted with the disease
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IGIB researchers partially reverse a rare disorderThe HinduR. Prasad10 FEBRUARY 2018 18:13 ISTUPDATED: 10 FEBRUARY 2018 18:14 IST The syndrome also affects about one in one lakh people, causing a range of defectResearchers at Delhi’s Institute of Genomics & Integrative Biology (CSIR-IGIB) have for the first time used zebra fish to model the rare genetic disorder — Rubinstein Taybi Syndrome (RSTS) — seen in humans. They have also used two small molecules to partially reverse some of the defects caused by the disorder in zebrafish, thus showing them to be an ideal animal model for screening drug candidates. There is currently no cure or treatment for the disorder.The Rubinstein Taybi Syndrome has a frequency of about one in one lakh people, and causes intellectual disability, growth retardation (short stature), craniofacial deformities, heart defects and broad thumbs and toes. The results were published in the journal Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease.Close to human genomeSince zebrafish genome has very close similarity to human genome and the embryonic developmental is very similar in the two, the team led by Dr. Chetana Sachidanandan at IGIB went about checking if EP300, one of the two genes that cause the disorder is present in the fish and if mutations in this gene result in a RSTS-like disease in fish.Using chemicals, the researchers inhibited the activity of the protein Ep300 to see if this resulted in the manifestation of the disorder in the brain, heart, face and pectoral fins (equivalent to forearm in humans). “Like in the case of humans, the same organs were affected in the fish when the functioning of the protein was stopped. This helped in confirming that the protein in question does the same functions in fish and humans, ” she says.Since zebrafish commonly has two copies of many human genes, the researchers first checked if one or both the genes were functional and equivalent to the human gene that causes the disorder. “We found Ep300a gene was active and functional while Ep300b was not, ” says Prof. Tapas K. Kundu from the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, the other corresponding author. The Ep300a gene is responsible for producing a protein (Ep300) that opens up the DNA.“The protein Ep300 is evolutionarily conserved from fish to humans. Though the Ep300 gene has been earlier identified in fish, its function was not known, ” says Prof. Kundu.Reversal of effectsLike in the case of fish treated with chemicals manifesting the disorder, fish mutants that lacked the Ep300a gene too exhibited defects very similar to those seen in humans.“When we introduced excess amount of a tiny portion of the Ep300a protein in the mutants, the craniofacial deformities became less severe [mutants had severed craniofacial deformities] and pectoral fins in the fish became normal, ” she says.But neuronal defects were not reversed, even partially. “It might be because only a portion of the protein was put into the fish. Probably, that potion isn’t sufficient to compensate for the loss of the whole protein, ” she explains.“It’s proof-of-concept that just a piece of the protein is sufficient to reverse some defects, even if only partially, in zebrafish, ” Dr. Sachidanandan says.Alternatively, the researchers used two small molecules to reverse the defects. If the protein Ep300 is responsible for opening the DNA, there are other proteins that are responsible for closing the DNA.The two molecules were found from a screen of compounds well known for their ability to inhibit proteins responsible for closing the DNA.Like in the case when excess amount of Ep300 protein was introduced, both the molecules could partially restore facial defects but not the neuronal defects.“Introducing excess amount of a portion of the ep300 protein showed greater rescue of deformities than the small molecules, ” says Aswini Babu from IGIB and first author of the paper. “But rescuing the deformities using small molecules is a relatively easier and better option.”
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A tool that tracks and stops bacterial blight outbreaks in ricericetoday.irri.org/a-tool-that-tracks-and-stops-bacterial-blight-outbreaks-in-rice/A new, faster, and more accurate way of identifying infectious organisms—down to their genetic fingerprint—could finally put farmers a step ahead of bacterial blight. Severe bacterial blight infection in a susceptible rice variety from West Java, Indonesia. (Photo by R. Oliva)Severe bacterial blight infection in a susceptible rice variety from West Java, Indonesia. (Photo by R. Oliva)A revolutionary tool called the PathoTracer has been developed at the International Rice Research Institute (IRRI) and it can identify the exact strain of the bacterium that causes bacterial blight present in a field in a matter of days instead of several months of laboratory work.“It’s like a paternity test that uses DNA profiling, ” said Ricardo Oliva, a plant pathologist at IRRI. “It will not only tell you that you have bacterial blight in your plant. It will tell you the particular strain of the pathogen so that we can recommend varieties resistant to it.”For more than four years, Dr. Oliva and his team worked on deciphering the genetic code of Xanthomonas oryzae pv. oryzae, the pathogen that causes bacterial blight, to develop the test. Bacterial blight is one of the most serious diseases of rice. The earlier the disease occurs, the higher the yield loss—which could be as much as 70% in vulnerable varieties.“Bacterial blight is a persistent disease in rice fields, ” said Dr. Oliva. “The epidemic builds up every season when susceptible varieties are planted. The problem is that the bacterial strains vary from one place to another and farmers don’t know which are the resistant varieties for that region. We were always behind because the pathogens always moved and evolved faster.”Identifying the strains of bacterial blight present in the field requires a lot of labor and time. You need people to collect as many samples as they can over large areas to accurately monitor the pathogen population. In addition, isolating the pathogens in the lab is laborious and it typically takes several months or even a year to determine the prevalent strains in a region.The PathoTracer can identify the local bacteria in the field using small leaf discs as samples. The samples will be sent to a certified laboratory to perform the genetic test and the results will be analyzed by IRRI.The team that developed PathoTracer. Left row: Maritess Carillaga, Cipto Nugroho, Ian Lorenzo Quibod, and Genelou Grande. Right row: Veronica Roman-Reyna, Sapphire Thea Charlene Coronejo, and Dr. Oliva. Not in photo: Eula Gems Oreiro, EiEi Aung, and Marian Hanna Nguyen. (Photo by Isagani Serrano, IRRI)The team that developed PathoTracer. Left row (front to back): Maritess Carillaga, Cipto Nugroho, Ian Lorenzo Quibod, and Genelou Grande. Right row: Veronica Roman-Reyna, Sapphire Thea Charlene Coronejo, and Dr. Oliva. Not in photo: Eula Gems Oreiro, EiEi Aung, Epifania Garcia, Ismael Mamiit, and Marian Hanna Nguyen. (Photo by Isagani Serrano, IRRI)“It takes only a few days to analyze the samples, ” Dr. Oliva explained. “With the PathoTracer, we can bring a year’s work down to probably two weeks. Because the tool can rapidly and efficiently monitor the pathogen present in each season, the information can be available before the cropping season ends.”It’s like knowing the future, and predicting what would happen the next season can empower the farmers, according to Dr. Oliva.“Recognizing the specific local bacteria present in the current season can help us plan for the next, ” he added. “We can come up with a list of recommended rice varieties that are resistant to the prevalent pathogen strains in the locality. By planting the recommended varieties, farmers can reduce the risk of an epidemic in the next season and increase their profits.”The PathoTracer was pilot tested in Mindanao in the southern part of the Philippines in April 2017. The rains came early in the region, just after the peak of the dry season, and that triggered an outbreak of bacterial blight.“We went there and took samples from different fields, ” Dr. Oliva said. “By the end of April, we had the results and we were able to come up with a list of resistant varieties that could stop the pathogen. We submitted our recommendation to give farmers a choice in reducing the risk. If the farmers planted the same rice varieties in the succeeding rainy seasons, I am 100% sure the results would be very bad.”The PathoTracer can run thousands of samples and can, therefore, easily cover large areas, making it an essential tool for extension workers of agriculture departments and private-sector rice producers, or it can be incorporated into monitoring platforms such as the Philippine Rice Information System (PRiSM) or Pest and Disease Risk Identification and Management (PRIME) to support national or regional crop health decision-making.“National breeding programs could also make more informed decisions, ” Dr. Oliva said. “If you know the pathogen population in the entire Philippines, for example, the country’s breeding program could target those strains.”IRRI is interested in expanding the genetic testing tool to include rice blast and, further down the road, all bacteria, viruses, and fungi that infect rice.The speed at which PathoTracer can identify the strains of bacterial blight present in the field can be used for recommending resistant rice varieties to farmers for planting in the next cropping season. (Photo: IRRI)The speed at which PathoTracer can identify the strains of bacterial blight present in the field can be used for recommending resistant rice varieties to farmers for planting in the next cropping season. (Photo: IRRI)The PathoTracer has been tested in other Asian countries and IRRI expects to roll it out early in 2018. When it becomes available, the expected potential impact of the PathoTracer on a devastating disease that affects rice fields worldwide would be huge.“Imagine if this tool prevented bacterial blight outbreaks every season across Asia, ” said Dr. Oliva. “It’s super cool!”For more information about bacterial blight, see Section II, Chapter 2 of IRRI’s Rice Diseases Online Resource
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Have You Been Diagnosed With An Autoimmune Disease? Medical medium If you or someone you love have been diagnosed with an autoimmune disease, chances are you’ve been told that your body is attacking itself. Hearing this probably stripped you of hope that you can heal and left you feeling betrayed by your own body. Today there are countless mysterious diseases that have been labeled as autoimmune conditions. But the problem is that the concept of autoimmunity is inherently flawed and this mistaken theory only hurts you and your loved ones in the end. Surprised? When we believe our own bodies are attacking themselves, we are unknowingly hindering our healing process. Our immune systems can weaken in the absence of this truth: Your body never attacks itself. It’s always working for you and loves you unconditionally. It’s never been more critical for you and your loved ones to know and feel this truth in your heart. It will greatly support you and those you care about to heal. Continue reading to learn more and check out the accompanying radio show to further immerse yourself in truth.When medical science and research do not know what is causing symptoms, such as mysterious rashes, mysterious dizziness, mysterious fatigue, mysterious aches and pains, or mysterious inflammation, it gets labeled as an autoimmune issue. This is a mere theory that was initiated in the 1950s and has since been grandfathered in to modern medicine and collectively agreed upon as law. There was a discovery of an antibody but no one had any idea of what it was or what it was for. There were theories and ideas of what it was, but they were just that: theories. One of those theories was that the antibody was there to destroy healthy cells. This theory came about because researchers didn’t have an answer as to what was causing chronic illnesses and symptoms or even an answer to what the antibody was for. Instead the blame was put on those with chronic illness and our bodies became the scapegoat. Better to blame it on our bodies than to blame it on the medical industry and research and science not having the answers. The theory of autoimmune conditions and diseases became a catch-all explanation for dozens of conditions and symptoms that medical communities don’t yet have answers for. When someone is diagnosed with Hashimoto’s, they are told their body is creating antibodies that are destroying their thyroid tissue. With eczema and psoriasis, you will likely be told your body is attacking your skin. If you have lupus, you will likely be told your body is attacking itself and that’s what creates all the inflammation. This same theory is used for countless other chronic illnesses. This is the best explanation that modern medicine has to offer about mysterious diseases today. It’s never been more important for you to know this isn’t accurate because it affects your ability to heal. Not only are you given incorrect information about your health problems, you’re also told your body has turned against you when nothing could be further from the truth. The information I am sharing with you here is meant to free you of the misguided notion that your body would ever attack itself and to reveal the true cause of mysterious diseases that have been labeled autoimmune disorders.The True Causes of Autoimmune DiseasesIf you have an illness that has been labeled as an autoimmune disease, your body truly is attacking something, but it is not attacking itself. As I said, your body is working for you every single day. It works tirelessly for you and is doing the very best it can. When you are sick and have mysterious symptoms, such as fatigue, butterfly rashes, dizziness, tremors, vertigo, aches and pains, digestive trouble, weight loss, insomnia, weight gain, memory loss, numbness, tingling, blood sugar issues, brain fog, mood swings, night sweats, loss of appetite, and many others, it means there is an invader in your body. The same is true if you have lupus, Hashimoto’s, Grave’s, Addison’s, Type 1 Diabetes, colitis, vitiligo, celiac, eczema, Graves’, chronic fatigue syndrome (CFS), myalgic encephalomyelitis (ME), rheumatoid arthritis, fibromyalgia, Lyme disease, multiple sclerosis, parkinson’s, alopecia, or any other disease that is considered to be in the autoimmune category. There is a pathogen invader in your body that it’s working hard to attack and get rid of.Modern medicine has yet to come to this conclusion about mysterious illnesses due to a few reasons. First of all, this theory about the body attacking itself has become law inside the minds of well-intentioned doctors and health professionals who are just doing their best to provide care for their patients. No one is willing to question it because they have come to believe it is the truth, even though there is no evidence backing it.Another reason is because there is no incentive to look into these mysterious diseases. When there is already an acceptable answer that seems to fit, there doesn’t appear to be a need to spend more money on research that they assume will get them nowhere. And finally, even if doctors were to seriously look for these pathogens in their patients coming into their offices with a long list of mysterious symptoms, they would come up dry. Most tests the blood lab offers cannot detect the viruses and bacteria that are invading so many people and causing so many conditions and symptoms. Many of the pathogens causing autoimmune labeled illnesses are yet to be discovered and classified; therefore, it would be quite impossible to detect. You can’t find something when you do not know what you are looking for. For example, medical communities believe there is only one Epstein-Barr virus (EBV)—the virus that causes mono—but in truth there are 61 strains of EBV that cause all kinds of different symptoms and illnesses. I revealed this truth in my book Medical Medium. You can read more about EBV and how it creates illnesses, including cancer, in my book Thyroid Healing.If the medical industry was willing to put more money into researching pathogens, it would be more likely that answers would be coming out in literature today. Viruses and bacteria would be called by name and accurate tests would be created and administered. However, the most popular answer medicine has at the moment is that the body is attacking itself and as I have shared, this is not the truth. The second most popular answer is that it’s your faulty genes causing disease, which is a theory that’s quickly on the rise and still not the answer. In fact, people are told their body is attacking itself and that their genes are the problem.The First Barrier to DestroyThe biggest barrier obstructing your path to true healing is this belief that the body is attacking itself. It may have been ingrained into you not only by your medical practitioners, but also by what you read on the Internet. You are told there is very little you can do to manage your symptom or condition, and nothing you can do to reverse it. This is a lie and something that should be discarded immediately not that you know the truth.Know this: Your body is not attacking itself. I have already said it multiple times in this article, but I must say it over and over until we all understand it deep within our beings. It’s that fundamentally important. Your body is not self-destructive. And your body is not searching out healthy blood cells to harm and kill off. In fact, your body is doing the opposite. Every day your body is working for you. It is on your side. Your immune system cannot do one thing to harm a single cell in your body. It has never been able to do that, and it never will do that. When we know this deep within our being and replace the lie with this truth, something begins to change. That barrier that was once in your way is obliterated, and your path to healing becomes clear and unhampered. It may seem like a small switch, but it is in fact very significant. When your mind is aware that it is a pathogen you are going after, a revelation clicks into place and your body begins to work even more efficiently to kill off that virus. Your immune system fires up and kicks into gear to work for you even harder. This is essential to understand. The power of knowing the truth cannot be underestimated, especially when it comes to healing from these diseases that have plagued so many people for so many years. If you know your body is working for you, it works for you even more. Recovery is finally possible when you are armed with real information that is not hiding behind funding agendas and the bureaucracy of the medical world. This simple understanding sends a message to your body to start breaking down the pathogen fast. And when you start robbing food from the pathogen, which I discuss in the section about healing foods, recovery is even faster. Triggers vs. CauseThere are many medical professionals these days that are offering advice on how to manage your autoimmune disease. Some of this advice discusses “triggers” related to your illness but ironically it’s not even the correct triggers. For example, some experts are just beginning to say that EBV is a trigger for thyroid disease. This is because the never before known information that I have released has forced experts to look at EBV as a possible concern, but this has only led these experts to say that EBV could be a trigger. But in fact, EBV is not a trigger, it is the direct cause. This is a critical distinction that must be understood.The reason we need to make a distinction between trigger and cause is because when we just manage our illness by avoiding triggers, we are not actively fighting the pathogen and addressing the root cause. And if today’s medical professionals and experts believe that EBV is possibly just a trigger but have no idea how to rid the EBV out of the body, then they’re not helping people to heal. We want to do everything we can to kill off the virus invading our body. When we simply just avoid triggers, for example gluten, the virus that’s really behind your suffering can cause further issues. And obviously, we want to do everything we can to ensure the pathogen does not proliferate and cause more symptoms again. Plus, not all triggers can be avoided. We all eventually lose a loved one for example, and the grief we experience can trigger illness if the cause hasn’t been addressed. Or we’re not choosing the right triggers we can avoid. When we recognize the true cause of our illness, we can start to move toward true and total recovery, which is something I’ve witnessed tens of thousands of people doing over the years with the information I’ve shared, and now many more.Let’s take a look at some of the illness labeled as autoimmune diseases and conditions.LupusMedical science and research are unaware that lupus is a viral condition, specifically it is the body reacting to Epstein-Barr’s byproducts and neurotoxins. Medical science and research are completely unaware of this true cause. In fact, lupus is not even diagnosable. It is only visibly diagnosed by inflammation markers. I share more about the undiscovered true cause of lupus in Thyroid Healing.Multiple Sclerosis (MS)With this autoimmune labeled condition, EBV is in the body feeding on high levels of toxic heavy metals. It is not always lesions on the brain that are causing MS symptoms. Many people have spots that show up on brain scans, and they do not have any of the symptoms related to MS. There is so much that is going on in people’s brains: calcifications, crystallizations, dark spots, white spots, heavy metal deposits, MSG deposits, and chemical solvent stains. This does not mean someone has MS. What is actually causing the MS is EBV in addition to the heavy metals—a truth that medical communities are unaware of.Hashimoto's ThyroiditisEBV is also the true cause of Hashimoto’s thyroiditis. The thyroid becomes inflamed because the virus has infected it. Your immune system is not going haywire or out to get you. It’s this virus that’s causing the damage and making you feel miserable. Your body just needs the proper support, which I describe in Thyroid Healing, to triumph over the virus.CeliacCeliac is not a genetic disease. It is inflammation in the intestinal lining from streptococcus bacteria. Medical communities believe that celiac is an autoimmune condition and that it’s limited to a sensitivity to gluten. Rather, wheat gluten is one trigger to this inflammation of the intestinal tract by feeding the strep bacteria in there.Type 1 DiabetesType 1 diabetes occurs when there is an injury to your pancreas. If you have type 1 diabetes, at one point some of your pancreatic tissue was injured from a viral condition or toxic bacteria. Your body is not attacking your pancreas.Grave's DiseaseIn Grave’s disease, a mutated variety of EBV is causing inflammation, which is scarring the thyroid. There are specific unknown varieties of EBV that prompt the thyroid gland to produce more tissue and, as a result, more thyroid hormones. Find out more about Grave’s in Thyroid Healing.Hepatitis A, B, C, DAll versions of hepatitis are viral conditions from the herpetic family, mainly the Epstein-Barr Virus. They are at different stages of what the virus is doing to the liver. Autoimmune hepatitis is a mistake all on its own because medical research doesn’t actually know the true cause of these conditions. They merely see inflammation and antibodies and make the conclusion that the antibodies are going after the liver. However, the immune system is creating these antibodies to help you go after the virus.Mysterious Skin RashingMany varieties of mysterious skin rashing is from the shingles virus. Specifically, eczema and psoriasis can be a combination of EBV and shingles, both in the liver, feeding off of high levels of copper and DDT. The reason mysterious skin rashing does not get diagnosed as shingles is because when something does not look like textbook shingles, they would never think to call it that. However, what modern medicine is unaware of is that there are over 31 varieties of the shingles virus that they have yet to discover. Therefore, it would be impossible to diagnose it as such without further research. Ehler's-Danlos SyndromeEhlers-Danlos syndrome is another example of an illness that has been labeled as autoimmune simply because medical professionals are unaware of the true cause. The truth is that this condition is caused by a virus that’s injuring connective tissue; it’s not genetic.FibromyalgiaThe aches, pains, tenderness, fatigue, and stiffness of fibromyalgia are a result of EBV’s neurotoxins creating chronic inflammation of both the central nervous system and nerves throughout the body. Knowing the true cause means that you can begin to implement the steps needed to allow for healing. You can find out more about fibromyalgia and many other autoimmune diseases and condition in Thyroid Healing.Viruses EatNow that you know that your body is not attacking itself, but rather that it is attacking a pathogen in your body, you can move onto the next step toward healing. The next thing that’s important for you to know is a truth about viruses that science has yet to discover: viruses eat. Modern medicine is still oblivious to the fact that viruses must eat in order to grow and proliferate. Without their favorite foods, viruses will die. Therefore, logically we must know which are their favorite foods and eliminate them from your diet so the virus can exit your system, leaving you free of your symptoms over time.As an example, eczema is due to a virus in the liver. And that virus feeds off certain foods, which then creates an internal dermatoxin that leaches out from the liver, goes into the bloodstream, and then surfaces through the skin. When you know this is the real cause, you can take away the foods the virus likes best.In my book Thyroid Healing I explain in depth how the Epstein-Barr virus feeds, what it feeds on, and how it excretes when it feeds. For example, EBV feeds on toxic heavy metals, as well as all pesticides, including old ones such as DDT stored in the liver, and certain foods we eat. In the book, I explain the entire process of this particular virus as it feeds, excretes, develops, and continues to feed.Viruses Love Heavy MetalsViruses love to feed off of heavy metals, such as mercury, arsenic, cadmium, lead, nickel, alloys, steel, aluminum, and copper, that are in our systems, including in our liver. Heavy metals are not the cause of your autoimmune labeled disease. They simply feed the viruses that are the cause. The good news is you can take active steps to eliminate these metals from your body by eating the five heavy metal detox foods I recommend every day within a 24-hour period. These foods are wild blueberries, Hawaiian spirulina, barley grass juice extract powder, cilantro, and Atlantic dulse. All five of these foods work together to slowly eliminate the heavy metals. Find out how you can incorporate these foods in Thyroid Healing.Many people believe chlorella is actually better for you than spirulina, but chlorella simply does not eliminate heavy metals the way spirulina does. Chlorella drops the metals instantly, while spirulina holds onto them and in tandem with the other key foods, effectively aids in the process of removing them from the body.Foods that Viruses LoveEggs may be considered the perfect food in some circles, but in reality, they are the perfect food for viruses. They essentially incubate viruses, making them stronger and more aggressive. Stay away from eggs if you have a disease that is labeled as autoimmune, especially lupus and PCOS. Unknown to medical communities, eggs are disastrous for those who have ovarian issues. Eggs feed every single virus and bacteria that create conditions that are thought to be autoimmune diseases.Even if you are eating cage-free, natural eggs, these still act as food for pathogens. Viruses look for egg material in the body to feed on. They feed on the natural hormones that are in eggs, which act as a steroid for the virus. It does not matter what kind of eggs you get because unfortunately all eggs feed viruses.In a very similar way, all dairy products feed viruses. Some doctors are knowledgeable about the allergy concern of eating dairy, but we have to recognize that simply eliminating dairy is not enough. We need to understand why we are eliminating it. It’s critical to stop eating foods like dairy and eggs if you have any symptoms or a chronic illness so that they do not continue to feed the pathogens that are causing your symptoms. It is also a good idea to stay away from all corn products and canola oil. These are in almost every packaged food out there, even organic ones, so be mindful of what is in the ingredients list. If it is intimidating to you to avoid these ingredients that seem minor, think of it this way. The more fresh fruits and vegetables you include in our diet, the more apples, sweet potatoes, salads, bananas, and squash, the less room you have in diet for processed foods that likely contain ingredients you are trying to avoid. Think of crowding out your plate with so much good stuff that the packaged foods simply do not have as much space. Also, the more fresh fruits and vegetables you eat, the more you crave them. Keeping this in mind will help you increase your whole foods intake because you know that the more you do it, the easier it will get. It’s also helpful to reduce animal protein while you’re healing because, again, we need room for the fruits, leafy greens, and vegetables, the secrets weapons, as I like to call them. If you like to eat animal protein, try not to eat it three times a day. Instead eat it once a day and find other filling options to enjoy. Instead of chicken on your salad, add black beans, avocado, sweet potato, or hummus. Instead of bacon with your toast, bake potato fries with lots of herbs of spices. And when you do eat animal proteins, choose the cleanest meats possible. Wild fish is one of the better choices. When healing from chronic illnesses and symptoms, it’s helpful to minimize added fats in the diet to allow the liver to cleanse and toxins and viruses to be most effectively purged from the body. One easy way to cut down fats is to stay away from or minimize oils, even olive oil and coconut oil. A small drizzle here or there is fine, but do not overdo the oils. The same goes for other fat sources like nuts for example. Eat just a few with lots of fresh veggies versus having a large portion of nuts.Foods that Fight PathogensThere are so many amazing powerhouse foods that you can eat in order to give your body an extra boost when fighting off pathogens. The first group we can focus on is cruciferous vegetables. There is a misconception circulating around that cruciferous vegetables, also called brassicas, are harmful because of the goitrogenic compounds in them. We are being told to stay away from kale, broccoli, cauliflower, and brussels sprouts, but this is a misguided and unproductive trend. These foods are actually incredible for anyone with an illness that has been labeled as an autoimmune disease, including thyroid conditions. Cruciferous vegetables cannot harm you. They contain phytochemicals that actually fight the bug you are trying to get rid of. These foods can be some of your greatest allies in your path to healing, and it would be wise to include them, both raw and cooked, on a daily basis.Straight cucumber juice is something that can be included daily in order to flush out toxins from the body. Straight celery juice is also important to include because its mineral salts clean up the liver, build up hydrochloric acid, and strengthen bile so you can break down bacteria and viruses in the intestinal tract that cause conditions like colitis, celiac, and crohn's disease. I share recipes for these foods and juices in my book Thyroid Healing.Include plenty of squash in your diet. There are so many delicious varieties to enjoy, such as butternut, kabocha, spaghetti, and acorn. Also, remember to eat lots of fresh herbs like cilantro, rosemary, and thyme. Eat plenty of salads with red leaf lettuce, butter lettuce, arugula, parsley, and radishes. Radishes are a miracle food when fighting off pathogens. Radish greens kill off viruses, and the radishes themselves push poisons out of the body and support the thyroid.Eating plenty of fruit is one of the best ways you can support your healing. If you are afraid of fruit, know that fruit cannot feed candida and fruit sugar isn’t bad for you. It’s the opposite—fruit is the most healing food on earth! So eat plenty of apples, bananas, oranges, and berries. Melons are wonderful for flushing out poisons that the virus creates. Bring in more tropical fruits like papaya, pineapple, and passionfruit if you are able to find them. Eat the fruits that are in season like pears and persimmons in the fall and peaches and plums in the summer. Fruit is an amazing antiviral food that is essential to include when you are fighting a pathogen such as Epstein-Barr virus that is causing your mystery illness. Bananas in particular are great for killing off pathogens in the intestinal tract. If you fear fruit, check out my chapter on Fruit Fear in my book Medical Medium.Another power food that is worth seeking out is dandelion greens. These are often sold at farmers markets and health food stores among other greens like kale and collard greens. Dandelion greens are particularly supportive for the liver because they work to push out poisons that are created from a virus or bacteria. There are so many wonderful herbal teas to include, as well. Nettle leaf and lemon balm are two particularly powerful ones that fight viruses. There is a phytochemical in lemon balm that helps you go after the virus you want to kill. Many people know that nettle leaf tea is anti-inflammatory, but they do not know why. The reason is because it’s killing the pathogens that are creating the inflammation in the first place. When you are drinking your herbal teas with this knowledge, the properties become that much more powerful.In the same way, people are aware that turmeric is anti-inflammatory, but they do not know why. Turmeric has phytochemicals that are poisonous to the bugs that are inflaming people’s joints and backs. This herb is attacking viruses and bacteria.SupplementsIncorporating supplements is critical to fighting off the viruses that are causing your autoimmune disease. As I always recommend, talk to your medical practitioner before you begin taking supplements so you can work together to find the right dosages. Bring your doctor this information and work together to fight the virus with these powerful herbs and supplements. Be sure to get the right supplements. I have a list of preferred supplements that you can find on my website. ZincZinc deficiencies are rampant and can trigger an autoimmune condition. Simply taking zinc can be a huge step toward healing and can be critical for anyone suffering from the symptoms associated with autoimmune diseases. When you take zinc sulfate, it goes directly to where the virus is and starts working on stopping it in its tracks. Additionally, zinc increases your immune system’s ability to fight. B12 with adenosylcobalamin & methylcobalaminMany people are recommending B12 today, which is good. However, there is still very little talk about the right kind of B12. When fighting a viral condition, it is essential to use a combination of both adenosylcobalamin and methylcobalamin. Including a methylfolate supplement along with this specific B12 is especially helpful. Vitamin CWhen fighting a viral condition, it’s important to include some vitamin c supplements, particularly the liposomal c. The Liposomal c I recommend on my preferred supplements page does not contain any corn, which feeds viruses, unlike other brands. Ester C is also very helpful.
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Harvard Health LetterBuild a better bladderExercises, lifestyle change, medications, and procedures can alleviate incontinence and give you your life back.Published: February, 2016A leaky bladder or a sudden urge to go to the bathroom is uncomfortable andembarrassing. But you can take steps to alleviate the problem. "Some people tell me they would have sought treatment sooner if they'd known it was this simple, " says Dr. Anurag Das, director of the Center for Neurourology and Continence at Harvard-affiliated Beth Israel Deaconess Medical Center.Lifestyle changesOne of the first lines of defense is pill-free and costs nothing: lifestyle change. For urge incontinence (see "Types of incontinence"), you can try timed voiding (urinating on a schedule) and bladder guarding, which teaches you to cope with triggers that set off the urge to go, such as washing dishes or hearing water. "You squeeze your muscles to hold in urine before a trigger, which sends a message to the brain that this is not the time to go, " says Dr. Das. Other lifestyle changes include watching fluid intake; quitting smoking, to reduce coughing and pressure on the bladder; and minimizing bladder irritants such as caffeine, alcohol, and carbonated drinks.Pelvic floor rehabThe pelvic floor muscles aid control of your bladder and bowels. Strengthening these muscles can be helpful to people with stress incontinence (see "Types of incontinence") as well as those with urge incontinence. This is done with Kegel exercises, which involve squeezing and releasing the muscles you use to hold in urine. A physical therapist can help you learn how to do the exercises properly. "The majority of people with urge incontinence will improve with rehab. It may not make it 100% better, but even 75% may be acceptable to many people, " says Dr. Das.MedicationsWhen pill-free measures aren't enough to curb incontinence, medications may help. The most commonly prescribed drugs for urge incontinence are anticholinergics, such as oxybutynin (Ditropan). Side effects can include dry mouth and eyes, headache, constipation, and confusion.Some drugs relieve stress incontinence, such as tricyclics like imipramine (Tofranil) and alpha-adrenergic agonists like pseudoephedrine (Sudafed), which are often found in cold medicines. However, these, too, are limited by side effects, and their effect declines over time. When an enlarged prostate causes overflow incontinence (see "Types of incontinence"), alpha blockers such as doxazosin (Cardura) may help by relaxing the smooth muscle of the prostate. It may take trial and error to find the best drugs with the fewest side effects.ProceduresInjections of botulinum toxin (Botox), a muscle relaxant, are used to treat urge incontinence. "The downside is a higher incidence of urine retention and risk of bladder infection. Some patients won't be able to urinate and may need to use a catheter, " says Dr. Das. Injections of calcium hydroxylapatite (Coaptite) are used to treat stress incontinence by tightening the urethra.A procedure called sacral neuromodulation can help people with urge incontinence. A small device called Interstim is implanted in the lower back to stimulate the sacral nerve, improving both bladder and bowel problems.Surgery sometimes is used for stress incontinence. This involves creating a sling that wraps around a person's urethra. "You don't have to use artificial mesh materials for the sling; you can use the patient's own fascia (muscle lining) if preferred, " says Dr. Das. There is no surgery for overflow incontinence if the bladder muscle does not work. However, in cases of blockage from the prostate, prostate surgery may help.Types of incontinenceStress incontinence occurs mostly in women, and it's often the result of a weakened or stretched pelvic floor from childbirth. The telltale symptom is leakage with pressure or stress on the bladder, such as when laughing or coughing. Stress incontinence is less common in men unless they've had an injury or had their prostate removed.Urge incontinence (overactive bladder) is characterized by an inability to get to the bathroom in time. It's caused by overactive contractions of the bladder muscle that may be related to an enlarged prostate in men; changes to the bladder lining or bladder muscle in postmenopausal women; or a chronic neurological condition, such as multiple sclerosis.Overflow incontinence occurs when there's no room for additional urine because the bladder is not emptying, either because it is blocked or because it isn't working properly because of a neurological disorder or a medication side effect. Overflow incontinence primarily affects men with enlarged prostates. Symptoms include frequent leakage or a feeling of lower-belly fullness. The condition increases the risk for bladder infections because the urine pools in the bladder for long periods of time
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Harvard Medical SchoolProper posture the tai chi waytai chi postureIf you're like most people, your posture could use some improvement. But how do you do that? The classic advice is to stand straight, with your head up, shoulders back, and belly in. While these are reasonable adjustments, tai chi takes a different approach that may be more effective. It aims to align the body in ways that afford safe, unstrained, and graceful postures—not the military-like, one-size-fits all, rigid stances many think of as good postureLiving Better, Living Longer Mind-body exercises, such as tai chi and yoga, have been gaining popularity over the past few decades. This is not surprising, given the increasing number of studies on the positive effects of these gentler forms of exercise—everything from lowering blood pressure and managing depression to building strength and improving balance. There is even evidence that tai chi may help you live a longer, more vital life.Instead, in tai chi, good posture centers around the principle of verticality. That means the head is centered over the torso, the torso rests over the hips, and the hips are centered over the legs and feet, your base of support. Here's how to do it.The spine is literally the backbone of verticality, so let's start there. Tai chi classics say, "The spine should be like a necklace of pearls hanging from heaven." Your goal should be to elongate the spine while still allowing for its natural curves. This can reduce wear and tear on the discs between your vertebrae. In this position, your head will also sit squarely on top of your spine instead of jutting forward— a common profile that you will see if you take the time to observe others. Your head weighs about 10 to 12 pounds, but just a slight tilt forward more than doubles the amount of strain on your neck muscles. Get into typical texting position, and your muscles must work five times as hard. No wonder your neck and upper back are tight and achy. Proper alignment helps prevent that.The next area to focus on comprises the waist and pelvic region, which connect the upper body and lower body. In tai chi classics, this area is called the "commander." It's the central, coordinating hub where all movements originate. Muscle imbalances in this area have been associated with pain in the back, knee, and even neck, along with affecting your gait and balance. Maintaining tone and flexibility in the muscles and other soft tissues of the hip and pelvic area facilitates good posture and movement patterns and reduces pain.At the foundation of good posture are your feet, but you rarely hear about them in traditional posture advice. If alignment is off at your feet, though, the imbalance travels up the body, possibly causing painful problems along the way. Western thinking usually attempts to correct these misalignments with products like shoe inserts, but tai chi works to naturally correct imbalances and improve range of motion. While specifics vary with different tai chi movements, in general you should keep your weight centered over the balls of your feet and all of your toes, and point your feet in the direction of your kneecaps. The result is a healthier posture that works for you without forcing your body into uncomfortable positions.So, the next time you want to improve your posture, skip the stiff stance and focus on your spine, pelvic region, and feet.To learn more about tai chi, its health benefits and how to learn its movements, check out Introduction to Tai Chi from Harvard Medical School.The active ingredients of tai chieight active ingredients of tai chiWhen Peter M. Wayne, medical editor of Introduction to Tai Chi from Harvard Medical School, began conducting scientific studies on the health benefits of tai chi, he began noticing that tai chi works in a variety of ways, not just one. Whereas most drugs have a single active ingredient, he observed that tai chi was more like a multidrug combination that uses different components to produce a variety of effects. He formulated the idea of the "eight active ingredients" of tai chi, which he and his colleagues now use as a conceptual framework to help evaluate the clinical benefits of tai chi, explore the underlying mechanisms that produce these effects, and shape the way tai chi is taught to participants in clinical trials (and to teachers). While different styles of tai chi emphasize different ingredients, these therapeutic factors are interwoven and synergistic. Here's a summary of one of the active ingredients.Structural integration. Tai chi looks at the body as an interconnected system, not as a collection of individual parts. As a result, when practicing tai chi, you won't do one exercise for your biceps and another for your glutes. Instead, tai chi integrates the upper body with the lower body, the right side with the left side, and the extremities with the core. Alignment and posture are part of this structural integration, and tai chi trains you to find alignments that are safe and unstrained, allowing you to perform graceful movements. You move more efficiently—not just during your tai chi practice, but throughout your day. The result is less stress and load on your joints and better balance. Similarly, improved posture has benefits that extend well beyond your tai chi class. When you walk or sit with your shoulders rounded and your torso hunched over, it is hard to take deep breaths. But when you straighten your back, roll your shoulders back and down, and open your chest, you breathe more deeply and efficiently. Not only does this integration improve your ability to move without pain, but it also affects your mental health. In two different studies, people who sat or walked more upright during the experiments had a more positive outlook afterward than those who slouched while sitting or walking. To learn more about the other active ingredients of tai chi, its health benefits, and how to learn the movements, read Introduction to Tai Chi from Harvard Medical School.
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Decoding the chemistry of fearSalk team charts pathway for fear in worms to reveal more about human anxietyLA JOLLA—Ask a dozen people about their greatest fears, and you’ll likely get a dozen different responses. That, along with the complexity of the human brain, makes fear—and its close cousin, anxiety—difficult to study. For this reason, clinical anti-anxiety medicines have mixed results, even though they are broadly prescribed. In fact, one in six Americans takes a psychiatric drug.A team of investigators from the Salk Institute uncovered new clues about the mechanisms of fear and anxiety through an unlikely creature: the tiny nematode worm. By analyzing the responses of worms exposed to chemicals secreted by its natural predator and studying the underlying molecular pathways, the team uncovered a rudimentary fear-like response that has parallels to human anxiety. Such insights may eventually help refine prescriptions for current anti-anxiety drugs and enable the development of new drugs to treat conditions like PTSD and panic disorder.In this illustration, a C. elegans worm (lower right) exposed to sulfolipid chemicals from one of its natural predators, a worm called P. pacificus, quickly reverses direction in a response analogous to human fear.“For the past 30 or 40 years, scientists have used simpler animals to figure out how fear might work in humans, ” says Sreekanth Chalasani, associate professor in Salk’s Molecular Neurobiology Laboratory and senior author of the paper, published in Nature Communications on March 19, 2018. “The idea has been that if you could figure out which underlying signals in the brain are related to fear and anxiety, you could develop better drugs to block them.”The team at Salk started with a simple creature, the microscopic worm called Caenorhabditis elegans. C. elegans, which contains only 302 neurons, has a natural predator—another worm called Pristionchus pacificus, which bites and kills C. elegans. The researchers discovered that by exposing C. elegans to chemicals that are excreted by P. pacificus, they could elicit a fear-like response. When it encounters these predator-excreted chemicals, C. elegans rapidly reverses direction and crawls away.They found that this fear-inducing chemical, a new class of molecules called sulfolipids, could activate four redundant brain circuits that led to this behavior. Additionally, C. elegans continued to change its behavior even after the fear-chemical was removed. This is analogous to behavior in mice, who express fear when exposed to the scent of cat urine, even if a cat is nowhere nearby.“For years, we thought that only advanced brains like those of mammals would have this complex reaction, ” Chalasani says. “But our study is showing that a simple animal expresses something very much like fear.”In the experiment, coauthor and UC San Diego graduate student Amy Pribadi soaked C. elegans in a solution containing the sulfolipid for 30 minutes. The worms failed to lay eggs, even for an hour after they had been removed from the solution—an indicator of acute stress as well as a longer-term response akin to anxiety. Further research showed that the signaling pathways activated during the worms’ response are similar to the pathways activated when more complex animals experience fear.When the worms were soaked in a solution containing Zoloft (a human anti-anxiety drug), however, these fear- and anxiety-like responses were not observed. This suggested that at least some of the pathways that the drug acts on to eliminate anxiety in mammals have been preserved by evolution.Also intriguingly, the team found that Zoloft acted on the worms’ GABA signaling in a neuron that affects the animal’s sleep. Whether this is also the case in humans is not yet known, but points to a potential pathway to understand why Zoloft works in some people and not others. The research eventually could lead to a change in how these drugs are prescribed.“We hope the findings from this paper will contribute to the field by providing a broader picture of some of these signaling activities, ” Chalasani says. “Our findings suggest that fear and anxiety are ancient and evolved much earlier than we originally thought. The pathways, nerves, circuits and genes that we’ll now be able to study in the worm should inform us about this process in humans.”In addition, he says, understanding which chemicals may repel nematodes could have implications for developing new kinds of pesticides, potentially ones that are even nontoxic. “C. elegans is not a pathogen, but many other types of nematodes can do severe damage to crops, ” he explains. “Biology research can go in many different directions, and you never know what you’re going to uncover.”The paper’s other authors were Zheng Liu, Maro J. Kariya, Christopher D. Chute, Sarah G. Leinwand, Ada Tong, and Kevin P. Curran of Salk; Neelanjan Bose and Frank C. Schroeder of Cornell University; and Jagan Srinivasan of Worcester Polytechnic Institute.This work was funded by the W. M. Keck Foundation, the National Institutes of Health and a Salk Alumni Fellowship.PUBLICATION INFORMATION JOURNALNature CommunicationsTITLEPredator-secreted sulfolipids induce defensive responses in C. elegansAUTHORSZheng Liu, Maro J. Kariya, Christopher D. Chute, Amy K. Pribadi, Sarah G. Leinwand, Ada Tong, Kevin P. Curran, Neelanjan Bose, Frank C. Schroeder, Jagan Srinivasan and Sreekanth H. Chalasani
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What is Tourette syndrome? Rani Mukerji plays a patient with rare disorder in HichkiTourette Syndrome, the neurological condition Rani Mukerji’s character has in her forthcoming film Hichki, cannot be cured but it can be controlled. In her forthcoming film Hichki, Rani Mukherjee plays a woman with Tourette Syndrome who wants to be a teacher.Chances are that you may not have heard of the Tourette Syndrome till you saw the just-released trailer of Rani Mukerji’s forthcoming film Hichki. For those who still haven’t seen it, here’s a quick round up.In the film, Rani Mukerji plays Naina Mathur – a woman with Tourette Syndrome who wants to be a teacher. She seems bright, enthusiastic and devoted, but there is just one problem. Her rare disorder comes with an involuntary vocal tic (she frequently makes a loud noise like two hiccups in a row), which would make her a soft target for ridicule especially in a class full of adolescents.But surely that is not all there is to Tourette Syndrome, named after French physician and neurologist Gilles de la Tourette who did pioneering research on the disorder in 1885. Here’s what we know.Happens because…Tourette Syndrome manifests in adolescence (before age 18) and is attributed to genetic and environmental factors.“It is a neurobehavioural disorder that occurs because of dysfunction in the areas of the brain involved in movement and behaviour – basal ganglia, cerebral cortex and the thalamus, ” says Dr Anshu Rohatgi, senior consultant neurologist at Sir Ganga Ram Hospital in Delhi.While the exact cause for Tourette Syndrome is not known, genetics plays a major role. “If someone has Tourette, there is a 50% chance that it may be passed onto to the children, ” says Dr Rohatgi.How to identify…There is no test for Tourette Syndrome. It can be identified only through a clinical diagnosis.The syndrome is characterised by motor tics, which vary from person to person and may manifest as blinking, jerking head, arm or shoulder, clearing of throat, scrunching of face and vocal tics like shouting, grunting. In some cases, the vocal tics can occur as involuntary barking, swearing, or repeating your own words or things spoken by other people, which can be embarrassing.“In addition to the motor and vocal tics, you see a lot of behavioural changes such as hyperactivity, obsession, depression, mood changes, aggression, ” says Dr Rohatgi. “Besides a neurologist, most patients also see a psychiatrist, ” he adds.Appearance of motor and vocal tics are essential to identify a condition as Tourette Syndrome. If a person just suffers from compulsive motor tics (like shrugging shoulders or shaking leg) it could also be due to anxiety, which can be treated with behavioural therapy.“Tourette Syndrome is quite rare, ” says Dr Rachna Sehgal, associate professor, paediatrics and paediatric neurologist at Safdarjung Hospital in Delhi. “In my 20 years of practice as a paediatrician and 8 years as a neurologist, I have seen two children with Tourette, ” she says. Treatable but not curable…Tourette Syndrome cannot be cured, say doctors but it can be controlled. “The motor tics can be treated with medication, ” says Dr Rohatgi, “And there are anti-psychotic medicines to control the behavioural disorders, which are harder to control.”Patients with Tourette have the intelligence and work output of a regular person, says Dr Sehgal, but the stigma their condition invokes affects their day-to-day functioning. “People around them don’t understand they can’t control the tics. Not just the patients, but even their parents become stigmatised, ” she says.“People with Tourette become objects of ridicule in social situations, ” says Dr Rohatgi. “People don’t like to interact with them and tend to avoid them.”Given the stigma and ridicule this difficult condition invites, Naina Mathur’s dream of becoming a teacher may seem like a tall order. It remains to be seen whether the disorder gets a realistic treatment in the film. Let’s wait and watch.Hindustan times
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A breakthrough in our understanding of how red blood cells developPosted on March 15, 2018 by Kat J. McAlpine Posted in People, Science, Therapeutics By taking a deep dive into the molecular underpinnings of Diamond-Blackfan anemia, scientists have made a new discovery about what drives the development of mature red blood cells from the earliest form of blood cells, called hematopoietic (blood-forming) stem cells.For the first time, cellular machines called ribosomes — which create proteins in every cell of the body — have been linked to blood stem cell differentiation. The findings, published today in Cell, have revealed a potential new therapeutic pathway to treat Diamond-Blackfan anemia. They also cap off a research effort at Boston Children’s Hospital spanning nearly 80 years and several generations of scientists.Diamond-Blackfan anemia — a severe, rare, congenital blood disorder — was first described in 1938 by Louis Diamond, MD, and Kenneth Blackfan, MD, of Boston Children’s. The disorder impairs red blood cell production, impacting delivery of oxygen throughout the body and causing anemia. Forty years ago, David Nathan, MD, of Boston Children’s determined that the disorder specifically affects the way blood stem cells become mature red blood cells.Then, nearly 30 years ago, Stuart Orkin, MD, also of Boston Children’s, identified a protein called GATA1 as being a key factor in the production of hemoglobin, the essential protein in red blood cells that is responsible for transporting oxygen. Interestingly, in more recent years, genetic analysis has revealed that some patients with Diamond-Blackfan have mutations that block normal GATA1 production.Now, the final pieces of the puzzle — what causes Diamond-Blackfan anemia on a molecular level and how exactly ribosomes and GATA1 are involved — have finally been solved by another member of the Boston Children’s scientific community, Vijay Sankaran, MD, PhD, senior author of the new Cell paper.“Much of the history of how this disorder works was written at Boston Children’s, ” says Sankaran, who is a hematologist/oncologist and a principal investigator at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center. “Now, we can move on to the next era of research — what we can do to treat it.”Learning from an error of naturePrevious studies have found that many patients with Diamond-Blackfan anemia have mutated ribosomal protein genes. But the question has remained: Do these mutations have something to do with GATA1 and why do they only impair the maturation of red blood cells? In Diamond-Blackfan, other mature blood cells — such as platelets, T cells and B cells — still fare well despite mutations of ribosomal protein or GATA1 genes.EPO molecule which is linked to production of red blood cellsLast year, Sankaran’s research on Diamond-Blackfan anemia led to a discovery that saved an infant’s life.“There has been controversy over whether a ribosomal protein mutation changes the composition of the ribosomes or the quantity of the ribosomes, ” Sankaran says. “We know now that it is the latter.”By closely examining human cell samples from patients with Diamond-Blackfan anemia, Sankaran and his team of collaborators found that the quantity of ribosomes within blood cell precursors directly influences their ability to produce effective levels of GATA1, which, if you remember, is needed for hemoglobin production and also for red blood cell production.Now, finally tying all the pieces together, Sankaran and his team have definitively found that a reduced number of ribosomes slashes the output of GATA1 proteins inside blood stem cells, therefore impairing their differentiation into mature red blood cells.An opportunity for gene therapyTheir finding supports the hypothesis that the presence of GATA1 proteins in early blood stem cells helps prime them for differentiation into red blood cells. Without enough ribosomes to produce enough GATA1 proteins, these early cells simply never receive the signal to become red blood cells.“This raises the question of whether we can design a gene therapy to overcome the GATA1 deficiency, ” Sankaran says. “We now have a tremendous interest in this approach and believe it can be done.”Although a bone marrow transplant from a matched donor can treat Diamond-Blackfan anemia, Sankaran says that a gene therapy would be advantageous since it would use a patient’s own engineered cells, avoiding dangerous risks associated with graft versus host disease.“I think what’s great is that we can learn about developmental biology just by looking at our own patients very carefully, ” says Sankaran. “Genetic errors can give us the chance to pick apart the complex pieces of health and discover how they relate to one another.”In addition to Sankaran, additional authors of the new paper are Rajiv K. Khajuria, Mathias Munschauer, Jacob C. Ulirsch, Claudia Fiorini, Leif S. Ludwig, Sean K. McFarland, Nour J. Abdulhay, Harrison Specht, Hasmik Keshishian, D.R. Mani, Marko Jovanovic, Steven R. Ellis, Charles P. Fulco, Jesse M. Engreitz, Sabina Schütz, John Lian, Karen W. Gripp, Olga K. Weinberg, Geraldine S. Pinkus, Lee Gehrke, Aviv Regev, Eric S. Lander, Hanna T. Gazda, Winston Y. Lee, Vikram G. Panse and Steven A. Carr.This research was supported by the National Institutes of Health (DK103794, R33 HL120791 and T32 HL007574), a Manton Center Endowed Scholar Award, a DBA Foundation and March of Dimes Basil O’Connor Scholar Award, a Boehringer Ingelheim MD Fellowship, the Swiss National Science Foundation, Novartis Foundation, Olga Maybenfisch Stiftung and the European Research Council (EURIBIO260676).
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Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killingSAGE journal Nikita H Trivedi, Jieh-Juen Yu, Chiung-Yu Hung, ...First Published February 26, 2018 Research Article AbstractMacrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guẻrin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24–48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.Keywords Francisella, BCG, co-infection, macrophage, IL-4IntroductionOur understanding of host–pathogen interaction is largely based on the host response to a single pathogen. This approach has provided valuable information about the action(s) taken by the host against invasion by specific microbes and the mechanisms employed to subvert the host immune system.1 However, many human infections are now known to be polymicrobial in nature (see the review by Bakaletz2). In such an environment, immune responses triggered by one pathogen may influence a host reaction to co-infecting pathogens.1During the initial stage of a host–pathogen interaction, the pathogen encounters strategically placed phagocytic cells, e.g., macrophages that target and eliminate infectious agents. Because macrophages may encounter a variety of microbes, they are exposed to a multitude of stimulatory and suppressive signals that may have a profound effect on pathogen control. For example, persistent infection with respiratory syncytial virus in macrophages was shown to reduce the phagocytic capacity to engulf Haemophilus influenzae.3 Moreover, interaction with acidified Coxiella burnetii phagolysosomes resulted in reduced growth of Mycobacterium tuberculosis, which normally inhibits phagolysosomal fusion.4 Additionally, resident M0 macrophages can be activated classically by Th1 cytokines (e.g., IFN-γ) or alternatively by Th2 cytokines (e.g., IL-4 and IL-13) to become phenotypically and functionally distinct (M1 and M2, respectively) in response to different pathogens and environmental insults, and such M1 and M2 macrophage populations may vary under different health conditions. For example, a significant increase of M2 macrophages has been reported in allergen-exposed mouse lungs, 5 the lungs of asthmatic patients, 6 and of individuals exposed to cigarette smoke.7Thus, this paper seeks to provide a model to address the question of whether the outcome of multi-bacterial infection is different in M0 and M2 dominant microenvironments. For this purpose, we used two well-characterized intracellular bacteria, M. bovis BCG (Bacillus Calmette Guẻrin) and Francisella tularensis live vaccine strain (LVS) as model organisms because of different outcomes in the phagosome, i.e. BCG mainly resides in membrane-bound phagosomes while LVS rapidly escapes to the cytosol.8 Both Mycobacterium and Francisella can cause pulmonary infection and cervical lymphadenopathy. Although not the purpose of this study, a recent survey of 1170 tuberculosis cervical lymphadenitis patients in Turkey (a country with emergent endemic tularemia and epidemic tuberculosis) by Karabay et al. found that ≈7% of these patients were seropositive for F. tularensis.9Previously, we have shown a novel IL-4 dependent pathway that aids in reducing F. tularensis replication in macrophages.10, 11 IL-4-activated M2 macrophages, subsequently infected with F. tularensis, exhibited increased ATP levels and an associated increase in phagosomal acidification that led to a decrease in F. tularensis phagosomal escape and reduced replication.10, 11 In contrast, Mycobacterium infection results in a dominant M1 polarization of alveolar macrophages during the first 3 wk of infection.12 Taking advantage of this established LVS-infected macrophage system, and the well-characterized inhibitory mechanisms in M0 and M2 macrophages, we studied the influence of BCG on LVS infection in these two distinctly different macrophages. While both BCG and LVS target and replicate inside macrophages, they possess different survival mechanisms. F. tularensis is a facultative, intracellular, Gram-negative bacterium that causes the human disease tularemia.13 The LVS is a human attenuated strain derived from F. tularensis subsp. holarctica14 that is often used as an experimental alternative in lieu of the more virulent subsp. tularensis15 and which, like F. tularensis, escapes from phagosomes and replicates in high numbers in the cytosol.16 In contrast, BCG is an acid fast bacterium and vaccine strain derived from multiple in vitro passages of M. bovis, which arrests phagosomal maturation and fusion with lysosomes and replicates within the phagosome.17 This phagosomal maturation arrest is crucial for the persistence and replication of mycobacteria in macrophages.18Considering that LVS replicates much faster than BCG and can kill infected macrophages between 48 and 72 h post-infection, we focused on studying the effects of pre-BCG infection-induced host modulation on the subsequent endocytic trafficking and replication of LVS. Distinct LVS killing mechanisms mediated by BCG pre-infection in non-polarized and M2 polarized macrophages are discussed and modeled.Materials and methodsMiceAll animal experiments were performed in compliance with the Animal Welfare Act, the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Research Council. All animal work was done in accordance with the guidelines set forth by the University of Texas at San Antonio Institutional Animal Care and Use Committee, which specifically approved this study under protocol IS00000029. Animals were euthanized in a closed chamber with CO2 followed by cervical dislocation and all tissues were collected post-mortem. Specific pathogen-free 4–8-wk-old mice were used for all procedures. C57BL/6 mice were purchased from the National Cancer Institute. TLR2−/−19 and TLR4−/−20 mice were provided by Dr M. T. Berton (UT Health San Antonio).BacteriaF. tularensis LVS (obtained from Dr R. Lyons, University of New Mexico and Dr Karen Elkins, Food and Drug Administration) and F. tularensis Schu S4 (obtained from the Centers for Disease Control, CDC) were grown in tryptic soy broth supplemented with L-cysteine.21 Experiments using Schu S4 were conducted in a CDC-registered and annually certified Animal Biosafety Level 3 (ABSL-3) facility. M. bovis BCG was obtained from Heartland National TB Center and grown in Middlebrook 7H9 broth supplemented with Middlebrook Albumin Dextrose Complex enrichment and 0.05% Tween 80.Generation of IL-4-expressing J774 cellsStandard molecular cloning methods were employed to insert the mouse IL-4-encoding nucleotide sequence into a pRetroX-Tight-Pur vector using the Retro-XTM Tet-On® Advanced Expression System, according to the manufacturer’s recommendation (Clontech). The vector containing the IL-4 gene or vector alone was used to transfect J774A.1 cells, and the resulting stable cell lines containing either the IL-4 gene or vector alone were designated as J774.IL4 and J774.vec, respectively. IL-4 production in J774.IL4 cells is minimal but can be induced with the addition of doxycycline. To determine optimal induction, J774.IL4 cells were exposed to increasing concentrations (25–100 ng/ml) of doxycycline for 4–12 h (data not shown). Based on the kinetics of IL-4 production, we observed that a minimum of 50 ng/ml doxycycline was required for maximal IL-4 induction by 12 h, and thus this induction condition was used throughout the study.Cell culture and generation of primary cells for infectionCells (J774, J774.vec and J774.IL4) were cultured at 37℃ in complete DMEM with 4.5 g/l Gluc, L-glutamine, sodium pyruvate and 10% FBS. Primary macrophages were derived from C57BL/6 wild type (WT), TLR2−/− and TLR4−/− mouse bone marrow as previously described.10 Mincle−/−22, caspase recruitment domain family member 9-deficient (CARD9−/−23) and MyD88−/− bone marrow were gifts from Dr Garry Cole, University of Texas at San Antonio. For infection, J774, J774.vec and J774.IL4 cells were counted and seeded (5 × 105/well in 24-well plates) in the presence of 50 ng/ml doxycycline for a period of 12 h. After 12 h, cells were infected with 10 MOI BCG for 48 h. At the end of the BCG infection period, supernatants were collected and filtered. BCG-infected macrophages were washed with DMEM and co-infected with 10 MOI LVS suspended in the filtered supernatants. LVS uptake and replication were then measured at 3 and 24 h post-LVS inoculation, by lysing infected macrophages with 0.2% deoxycholate and determining the number of viable LVS by serial dilution in sterile PBS and plating on supplemented Tryptic Soy Agar plates.Quantification of cytokines, NO and arginase activityCell supernatants were collected at the indicated time points for assessment of IL-4 concentrations by ELISA, according to the manufacturer’s recommendations (BD Bioscience). Cells were lysed with 0.2% deoxycholate in the presence of protease inhibitor (Roche Diagnostics) for arginase activity measurement using the QuantiChrom Arginase Assay Kit (Gentaur) and reported as U/g (Units per gram of cell lysate protein). NO was detected in culture supernatants using Griess reagent.24 In some experiments, the NO inhibitor NG-monomethyl-L-arginine acetate salt (L-NMMA, 1.0 mM) was added at the time of doxycycline addition and throughout the infection.25Quantitation of cytosolic and total intracellular LVSIn order to assess escape of LVS from phagosomes to the cytosol, we employed a differential membrane permeabilization method using digitonin and saponin.16, 26 Briefly, macrophages were seeded (5 × 105cells/well) onto cover slips, induced with 50 ng/ml doxycycline for 12 h and subsequently infected with 5 × 106 CFU BCG, followed 48 h later by infection with LVS (5 × 106 CFU). Cover slips were fixed with 2% paraformaldehyde at indicated time points and treated with 50 µg/ml digitonin for 5 min at room temperature (approx. 25℃) or with 2% saponin for 30 min at room temperature. Cover slips without detergent treatment were used to determine surface LVS. Cover slips were subsequently blocked using 1% BSA, 0.3 M glycine in PBS for 30 min at room temperature and then incubated with rat anti-LVS primary Ab (generated in our lab) for 2 h. Digitonin permeabilizes the plasma membrane and allows Ab binding to the LVS only in the cytosol. In contrast, saponin permeabilizes all the membranes, including phagosomal membranes, allowing access to all intracellular LVS. Alexa Fluor 488-conjugated goat anti-rat IgG (H + L) (Life Technologies) was used as a secondary Ab to label LVS for 1 h. Cover slips were mounted using FluroSave reagent (Calbiochem) and the presence of the Alexa Fluor 488-labeled LVS was visualized using a Zeiss LSM 510 confocal microscope. Total intracellular LVS was calculated by subtracting surface LVS counts (sum of 25 randomly selected macrophages) from saponin-treated samples (sum of 25 randomly selected macrophages). Similarly, cytosolic LVS was calculated by subtracting surface LVS from LVS counts of digitonin-treated samples.StatisticsData were analyzed by Student’s t–test between the two examined groups. A P value of 0.05 or less was considered statistically significant. Data are representative of experiments repeated at least twice.ResultsLVS replication is reduced in alternatively activated (M2) J774 cellsPreviously, we have demonstrated that mast cells inhibit F. tularensis replication in macrophages via IL-4 secretion.10, 11 In order to study this bacterial inhibition independent of mast cells, we generated the J774.IL4 macrophage cell line, which is capable of producing IL-4 upon doxycycline induction. As shown in Figure 1a, IL-4 was produced in the J774.IL4 cells after 12 h doxycycline (50 ng/ml) induction but not in its absence. In contrast, doxycycline did not induce IL-4 production in J774 or J774.vec (J774 transfected with an empty vector) cells. Increased arginase activity in J774.IL4 cells following doxycycline induction (Figure 1b) further suggested that these cells behaved in a similar fashion to that of an activated M2 phenotype.27 Although there was no marked difference in cell morphology among uninfected doxycycline-treated J774, J774.vec and J774.IL4 cells (Figure 1c, upper panels), J774 and J774.vec cells exhibited a more (38 and 13%, respectively) amoeboid-like shape with multiple pseudopodia following 24 h LVS infection (Figure 1c, lower panels) compared to infected J774.IL4 cells (less than 1% elongated cells), which were found to be more spherical in shape, resembling non-infected macrophages. Similar observations were reported in LVS-infected bone marrow-derived macrophages (BMMØ), which exhibited an increase in surface area, loss of sphericity, elongation and decrease of volume due to apoptosis;10, 11 while the presence of IL-4 (addition of exogenous rIL-4 or co-culture with mast cells) led to increased intramacrophage killing of LVS with restoration of spherical morphology. To assess the inhibitory effect of M2 J774.IL4 cells on LVS replication, macrophages were grown for 12 h in the presence of doxycycline, culture medium removed, and the cells infected with LVS (10 MOI) for 3 h (bacterial uptake) and 24 h (replication) without doxycycline. As shown in Figure 2a, bacterial uptake was comparable among the three macrophage types; however, LVS replication was significantly reduced (approximately 1.5 log) in the J774.IL4 compared to J774 and J774.vec cells. Enhanced LVS inhibition in J774.IL4 cells correlated with increased IL-4 production (Figure 2b) and arginase activity (Figure 2c), consistent with the M2 macrophage activation phenotype. Similarly, reduced growth of the human virulent strain F. tularensis Schu S4, associated with increased IL-4 levels, was observed in M2 J774.IL4 macrophages (Figure 2d and e).figureFigure 1. Alternative activation of J774.IL-4 macrophages. J774, J774.vec or J774.IL4 cells were grown for 12 h in the presence (50 ng/ml) or absence of DOX. IL-4 secretion (a) in culture media was measured by ELISA, cellular arginase activity (b) was determined using the QuantiChrom Arginase Assay Kit, and cellular morphology (c) was visualized (400× phase contrast microscopy) 24 h after LVS inoculation (10 MOI). *P < 0.05 between indicated groups. ǂP < 0.05 between the indicated J774.IL4 and respective J774 and J774.vec groups.BCG: Bacillus Calmette Guẻrin; DOX: doxycycline; LVS: live vaccine strain.figureFigure 2. F. tularensis replication in J774 cell lines. J774, J774.vec and J774.IL4 cells (5 × 105 per well) were incubated with doxycycline (50 ng/ml) for 12 h and subsequently infected with 5 × 106 LVS ((a) to (c)) or Schu S4 ((d) and (e)). Bacterial uptake and replication were measured 3 h and 24 h post-inoculation ((a) and (d)). Additionally, the IL-4 concentration in culture media was measured ((b) and (e)) and the cellular arginase activity was determined (C). ǂP < 0.05 between the indicated J774.IL4 and respective J774 and J774.vec groups.LVS: live vaccine strain.BCG-mediated control of intramacrophage LVS replicationIn order to study how bacterial superinfection affects LVS replication, macrophages were infected with BCG 48 h prior to LVS infection. Using a GFP-expressing BCG28, 29 and an mCherry-expressing LVS30 for image flow cytometry assays (the Imagestream MKII, Amnis, EMD Millipore), 31 we estimated that 62% of J774 cells were co-infected with BCG and LVS at 4 h post-LVS inoculation. In addition, 4 and 23% of J774 cells harbored LVS and BCG alone, respectively (Figure 3a to d). These results suggest that > 90% of LVS-infected cells were pre-infected by BCG. It also was noted that uptake of LVS by J774 cells, with or without pre-BCG infection, was comparable (Figure 3e). Similarly, comparable rates (> 90%) of LVS infection were associated with BCG pre-infection among doxycycline-treated J774, J774.vec and J774.IL4 cells. Representative images from flow cytometric assays of these BCG–LVS co-infected cells are shown in Figure 3f. A twofold increase in cellular GFP intensity was observed in BCG-infected J774.IL4, compared to J774 (and J774.vec), cells, suggesting that M2 macrophages are more susceptible to BCG infection, which is consistent with other reports.32, 33figureFigure 3. Uptake of BCG and LVS by J774 macrophage-like cells. Untreated J774 cells ((a) to (e)) or 50 ng/ml doxycycline-treated J774, J774.vec and J774.IL4 cells (f) were seeded (5 × 105/well in 24-well plates) for 12 h followed by infection with GFP-expressing BCG (10 MOI) for 48 h, and subsequent inoculation with mCherry-expressing LVS (10 MOI). After 4 h incubation, co-infection with BCG and LVS was visualized and frequency analyzed using the Imagestream MKII (Amnis, EMD Millipore). (a) The dot-plot depicts the GFP and mCherry intensity of each cell and three gated cell populations: (b) mCherryhiGFPlow, (c) mCherryhiGFPhi and (d) mCherrylowGFPhi. The representative cell images of these three gated populations are shown in (b) LVS-infected J774 cells, (c) BCG–LVS co-infected cells (cellular localization of bacteria within this population was 93% using the Internalization Index analysis, IDEAS®) and (d) BCG-infected cells. (e) Uptake of mCherry LVS by J774 cells was comparable between LVS alone (pink area) and BCG + LVS co-infection (green line). (F) Shown are representative J774, J774.vec and J774.IL4 cells co-infected with BCG and LVS.BCG: Bacillus Calmette Guẻrin; BF: Bright Field; LVS: live vaccine strain.It has been well documented that LVS escapes from phagosomes within 30–60 min following phagocytosis with resulting replication in the cytosol.13 Our data are consistent with these observations, indicating that in both J774 and J774.vec cells the cytosolic LVS numbers (macrophages permeabilized with digitonin) were similar to the total intracellular LVS counts (macrophages permeabilized with saponin) following 1 and 4 h of LVS infection, respectively, suggesting that most LVS had escaped from phagosomes by 1 h (Figure 4a). In contrast, approximately 50% of LVS remained in the phagosomes of doxycycline-activated J774.IL4 (M2) cells at 4 h post-infection (Figure 4a). Interestingly, pre-BCG infection (48 h) did not prevent LVS escape from phagosomes in J774 and J774.vec cells within 4 h (Figure 4b), but pre-infection with BCG did result in the reduction of LVS replication at 24 h in macrophages compared to LVS infection alone (Figure 4c). This BCG-mediated LVS inhibition appears to be associated with NO production (Figure 4d, J774 and J774.vec). Infection with BCG 12 h prior to LVS infection (time 0, Figure 4d, J774 and J774.vec) induced a significant amount of NO, which had minimal effect on uptake of LVS (Figure 4c, 3 h). However, increased NO production (Figure 4d, comparing BCG + LVS to LVS alone) correlated with decreased LVS replication (Figure 4c, 24 h). In contrast, in M2 polarized J774.IL4 cells, pre-BCG infection did not reduce arginase production (data not shown) nor induce NO production (Figure 4d), and had little effect on LVS escape (Figure 4a and b) and replication (Figure 4c, LVS vs BCG + LVS).figureFigure 4. BCG and LVS co-infection in J774 cell lines. J774 cell lines (5 × 105) were seeded in wells on cover slips and IL-4 expression induced with 50 ng/ml doxycycline for 12 h. Cells were then infected with either (a) LVS (5 × 106) or (b) BCG (5 × 106) for 24 h, followed by LVS (5 × 106). At 1 and 4 h post-LVS inoculation, cells were fixed, treated with either Dig or Sap, and stained for LVS. The cytosolic (LVS-Dig and BCG + LVS-Dig) and total intracellular (LVS-Sap and BCG + LVS-Sap) LVS quantifications were determined as described in the Materials and methods. Concurrently, J774 cell lines (5 × 105 per well) were induced with doxycycline, infected with BCG or mock treated with medium for 48 h, and infected with LVS. Uptake and replication of LVS were measured at 3 and 24 h post-LVS inoculation (c), and NO levels in culture medium were measured prior to and 24 h after LVS inoculation using the Griess reagent (d). *P < 0.05 between indicated groups. ǂP < 0.05 between the indicated J774.IL4 and respective J774 and J774.vec groups.BCG: Bacillus Calmette Guẻrin; Dig: digoxin; LVS: live vaccine strain; Sap: saponin.Inhibition of intramacrophage LVS replication by BCG was associated with NO productionTo confirm the essential role of NO in BCG-mediated LVS inhibition, macrophages were treated with the NO inhibitor L-NMMA. As shown in Figure 5a, BCG infection significantly elevated NO production in J774 and J774.vec cells as well as in BMMØs 48 h post-inoculation, while addition of L-NMMA significantly abrogated NO production. We further assessed the role of NO in suppression of LVS intramacrophage replication with LVS infection alone and pre-infection with BCG. We observed that LVS infection alone induced minimal NO in all three J774 cell lines and in BMMØs (Figure 5b, upper panels). Consistent with previous observations, LVS replication was significantly reduced in the M2 J774.IL4 macrophages in contrast to M0 J774 and J774.vec cells, and addition of the NO inhibitor L-NMMA had no effect on LVS replication in all three cell lines or in BMMØs (Figure 5b, lower panels). However, when pre-infected with BCG, LVS replication was inhibited in J774, J774.vec and BMMØs (Figure 5b, lower panels), and this inhibition correlated with marked NO production (Figure 5b, upper panels).figureFigure 5. NO-mediated control of LVS replication. J774, J774.vec and J774.IL-4 cells (1 × 106 per well) were induced with doxycycline (50 ng/ml, 12 h). BMMØs derived from C57BL/6 mice were infected with BCG (1 × 107) in the presence or absence of NO inhibitor L-NMMA, and NO levels in culture media measured 24 h post-inoculation (a). In a similar study, BMMØs and doxycycline-induced J774 cell lines were infected with BCG, or mock treated with medium in the presence or absence of L-NMMA for 48 h, and infected with LVS. NO concentration ((b), upper panel) and LVS replication ((b), lower panel) were measured 24 h post-LVS inoculation. *P < 0.05 between indicated groups. ǂP < 0.05 between the indicated J774.IL4 and respective J774 and J774.vec groups.BCG: Bacillus Calmette Guẻrin; BMMØ: bone marrow-derived macrophages; L-NMMA: NG-monomethyl-L-arginine acetate salt; LVS: live vaccine strain.Additionally, inhibition of LVS was abrogated in the presence of the NO inhibitor L-NMMA. These results strongly suggest that BCG-mediated LVS inhibition is NO-dependent in M0 macrophages. However, BCG and NO have a minimal role in the control of LVS replication in M2 J774.IL4 cells (Figure 5b, lower left panel).Activation of Mincle and TLR2 signaling is critical for the control of LVS replication by BCG pre-infectionBCG has an atypical cell wall that contains ligands for various PRRs. For example, lipoproteins, phosphatidylinositol mannans and lipomannan are all TLR2 ligands that are present on the surface of BCG.34 The adaptor molecule for TLR2 signaling is MyD88, which plays a central role in activation of the NF-kB pathway and the production of pro-inflammatory cytokines.35 BCG also contains an unique glycolipid, trehalose 6, 6'-dimycolate (TDM), which binds to C-type lectin receptors such as Mincle that signal through the adaptor protein CARD9 to stimulate inflammatory responses.36 In order to determine if these PRR signaling components are essential for BCG-mediated NO-dependent LVS inhibition, we generated BMMØs from C57BL/6 Mincle, CARD9, TLR2 and MyD88 knockout mice. We also used C57BL/6 WT and TLR4−/− BMMØs for comparison. In contrast to TLR2, TLR4 has been shown to play a minimal role in macrophage activation by BCG.37 We infected BMMØs with BCG for 48 h, measured NO levels in culture media, and observed that deficiency of Mincle, CARD9, TLR2 and MyD88, but not TLR4, resulted in a significant reduction of NO production (Figure 6a), suggesting that activation of Mincle-CARD9 and TLR2-MyD88 signaling was critical for BCG-induced NO production in macrophages. Similar reductions of NO levels were observed in BCG + LVS superinfected Mincle-, CARD9-, TLR2- and MyD88-deficient BMMØs (Figure 6b), and these NO reductions correlated with loss of BCG-mediated LVS inhibition in these four cell types (Figure 6c). In contrast, TLR4 was not essential in BCG-mediated LVS inhibition.figureFigure 6. Innate signaling is required for BCG-mediated LVS inhibition during BCG–LVS superinfection. BMMØs prepared from WT C57BL6 and various gene-deficient mice, including Mincle, CARD9, TLR2, TLR4 and MyD88, were infected with BCG for 48 h and NO production was measured (a). Similarly prepared BMMØs were infected with LVS for 24 h with (BCG + LVS) or without (mock + LVS) prior BCG (48 h) infection, and the NO levels in culture medium (b) and viable LVS within macrophages (c) were analyzed. *P < 0.05 between indicated groups. ǂP < 0.05 between the indicated gene-deficiency and WT groups.BCG: Bacillus Calmette Guẻrin; BMMØ: bone marrow-derived macrophages; CARD9: caspase recruitment domain family member 9; LVS: live vaccine strain; WT, wild type.DiscussionIn this study, we investigated the influence of pre-BCG exposure in M0 or M2 polarized macrophages on subsequent LVS infection. Based upon our published observations of IL-4-mediated LVS inhibition10, 11 and data obtained from this study, we propose a model (Figure 7) exhibiting the two distinct defense mechanisms by which macrophages control LVS infection. When the M0 J774 and BMMØ cells are infected with BCG, their surface PPRs, i.e. TLR2 and Mincle, may recognize respective ligands (e.g. liproproteins and TDM) from BCG and subsequently activate NF-κB through the MyD88 and CARD9 adaptors. Activation of NF-κB may then up-regulate inducible NO synthase (iNOS) gene transcription and protein expression, resulting in increased NO production. Subsequent LVS infection of BCG-activated macrophages allows most LVS to escape from phagosomes followed by elimination via NO-mediated killing. In contrast, in M2 J774.IL4 macrophages, IL-4 may upregulate arginase (ARG1) gene transcription and protein expression via IL-4 R binding and STAT6 phosphorylation.38 The induced arginase converts arginine to ornithine, a precursor of polyamines and hydroxyproline that induces cell proliferation and collagen production.39 Because of the high ARG1 activity in M2-polarized J774.IL4 cells, minimal arginine, the common substrate for iNOS and ARG1, is available for conversion to L-hydroxy-arginine by BCG induced iNOS, with resulting minimal NO production following BCG infection. During subsequent LVS infection in M2 macrophages with prior BCG exposure, fewer LVS escape from the phagosome into the cytosol, and the macrophages control LVS growth by enhancing ATP production and phagosomal acidification as previously reported.11 In contrast, reduction of LVS replication in M2 J774.IL4 is independent of BCG activation during BCG–LVS superinfection.figureFigure 7. Working model for BCG-mediated LVS inhibition during BCG–LVS superinfection. Shown in the proposed model are distinct LVS inhibition mechanisms following BCG–LVS superinfection in M0 J774/BMMØs and M2 J774.IL4 cells. In the M0 macrophage, BCG infection activates TLR2-MyD88 and Mincle-CARD9 signal pathways leading to NO production for subsequent LVS killing. In contrast, BCG has minimal effect on IL-4 mediated LVS killing in J774.IL4 M2 macrophages.BCG: Bacillus Calmette Guẻrin; BMMØ: bone marrow-derived macrophages; iNOS: inducible NO synthase; CARD9: caspase recruitment domain family member 9; LVS: live vaccine strain; M0: non-activated.Both BCG and LVS can evade the endosomal–lysosomal degradation pathway, but by different mechanisms. Following phagocytosis, typical pathogen-containing phagosomes acquire markers such as Rab5 (a small GTPase) and EEA1 (early endosomal antigen 1), which directs the fusion of phagosomes with early endosomal vesicles.40 Endosomes continue to mature into late endosomes by replacing Rab5 with Rab7, and become acidified after the acquisition of vacuolar proton-ATPase molecules, eventually fusing with lysosomes for ultimate pathogen degradation. In the case of F. tularensis uptake by macrophages, the bacterium-containing phagosome matures into early and late endosomes, but fails to become acidified. Non-acidified late endosome-like phagosomes containing F. tularensis do not fuse with lysosomes and allow the bacteria to escape into the cytoplasm following gradual disruption of the vesicle.8 However, in J774.IL4 cells, IL-4 activation most likely enhances endosomal acidification, as suggested by our previous studies, 11 improving LVS killing. Conversely, BCG phagosomes acquire some endosomal markers such as Rab 5, but do not mature into late endosomes by preventing Rab5/Rab7 conversion, and BCG replicates in these arrested vesicles.17, 41 However, pre-infection with BCG appears to have little effect on LVS escape from the endosomes in both J774 and BMMØs.It should be noted that the results presented here were obtained from a population of macrophages pre-infected with BCG and then infected with LVS. We have not specifically investigated the macrophages during simultaneous co-infection with both BCG and LVS. However, > 90% of LVS-infected cells were pre-infected with BCG (Figure 3). Thus, the observed LVS replication was a collective outcome from BCG pre-infected macrophages and single LVS-infected macrophages activated by BCG-induced secretory factors. A mixture of singly and multi-infected macrophages may exist in naturally occurring co-infection. Furthermore, our results imply a complexity of disease outcome via pre-infection due to the plasticity and dynamics of macrophage phenotypes in the infection compartment. It also needs to be noted that experiments conducted in this study also used BMMØ but not the more disease relevant alveolar macrophages (ALVM); however, we have compared rat BMMØ and ALVM and demonstrated comparable Francisella replication in these two types of macrophages.42 Additional studies using mouse ALVM are required to confirm the similar mechanisms of BCG-mediated inhibition of LVS replication that were observed in BMMØ and J774 cells.In summary, we have provided evidence of BCG-mediated suppression of LVS replication using an in vitro macrophage infection model and have further characterized the mechanisms of LVS killing by M0, M1 (equivalent to BCG infection alone) and M2 macrophage phenotypes. Additionally, we have answered the question of whether an M1 polarizing pathogen influences the outcome of secondary infection in already M2 polarized macrophages by altering the bacterial killing mechanism(s). However, the effect of BCG on pneumonic tularemia in animals following superinfection remains to be elucidated. In this regard, mycobacterium-mediated protection against lethal malaria infection in a co-infection mouse model has been demonstrated.43AcknowledgementsWe thank Mr Srikanth Manam and Dr Jilani Chaudry for their technical support for the construction of the J774.vec and J774.IL4 cell lines. We also thank Dr Chinnaswamy Jagannath at the University of Texas Health Science Center at Houston who kindly provided the GFP-expressing BCG under support from his National Institutes of Health (NIH) grant (AI-78420). Image flow analyses were conducted in the University of Texas at San Antonio Immune Defense Core (supported by Research Centers in Minority Institutions, NIH grant G12MD007591).Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.FundingThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funding from the Army Research Office of the Department of Defense under contract no. W911NF-11-1-0136, and by the Jane and Roland Blumberg Professorship in Biology, to BPA.References1. Lijek, RS, Weiser, JN. Co-infection subverts mucosal immunity in the upper respiratory tract. Curr Opin Immunol 2012; 24: 417–423. Google Scholar, Crossref, Medline2. Bakaletz, LO. Developing animal models for polymicrobial diseases. Nat Rev Microbiol 2004; 2: 552–568. Google Scholar, Crossref, Medline, ISI3. 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