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Updates found with 'difference'

Genetic testing at home or from the doctor’s office – same difference?Stephany Tandy-Connor, MS, CGC March 28th, 2018There has been a lot of talk about Direct-To-Consumer (DTC) genetic testing and the caveats that come along with that type of testing, but what does it all really mean? What is the actual difference between a DTC genetic test and a genetic test ordered by a medical professional through a clinical laboratory? There are several things that make these tests different but the biggest difference to be aware of is the thoroughness of the testing. Think of your genome as a book about you. Each one of your genes is a chapter in the book. Your DNA sequence is all of the letters that make up the words in each chapter.Testing from a quality clinical laboratory will read each word in a requested chapter, and it will also make sure that large sections of the chapter are not missing or duplicated by mistake.Most DTCs do their testing with an assay called a SNP array. This type of testing reads only specific letters in a chapter but not the whole chapter. Not even a whole sentence. In addition, this type of testing does not look for large sections that are missing or duplicated at all.For example, 23andMe has recently been FDA approved to report on 3 specific alterations in BRCA1 and BRCA2 genes. These two chapters (genes) together contain roughly 16, 000 letters. The 3 alterations being analyzed and reported on involve only 4 out of the roughly 16, 000 letters in both chapters. The rest of the letters/words are not read.While DTC tests are very popular and can be entertaining, it is important for individuals to be aware of what they get with this testing and what they DON’T get – especially when dealing with health related topics that could impact an individual’s medical management.
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A new Harvard Medical School GuideReady to put a stop to the itching, burning, and discomfort of hemorrhoids?This information-packed guide reveals how you can prevent and treat hemorrhoids.Healing Hemorrhoids In Healing Hemorrhoids, you'll discover:✓ Everything you need to know about the types, causes, and symptoms of hemorrhoids✓ Symptoms that might be signs of other, more serious conditions✓ How to prevent constipation—the #1 cause of hemorrhoids✓ The differences between stool softeners, suppositories, and laxatives✓ Non-surgical, office-based hemorrhoid treatments as well as surgical procedures✓ 19 high-fiber foods that can help keep you regularRead MoreIt's the healthcare issue no one likes talking about: hemorrhoids. Yet more than 75% of people over age 45 experience hemorrhoids. If you have hemorrhoids, you know just how uncomfortable they can be. Now, with Healing Hemorrhoids, a new guide from the experts at Harvard Medical School, you'll learn how to take charge of your hemorrhoids and get back to enjoying life.Everything about hemorrhoids you were too embarrassed to askHealing Hemorrhoids gives you a complete understanding of hemorrhoids (in the comfort and privacy of your own home!). For example, you'll read about the two types of hemorrhoids—internal and external—and their causes and symptoms. (Here's some good news: hemorrhoids are not dangerous and serious complications are rare.) The guide also reveals who is more likely to get hemorrhoids, and explains how hemorrhoids are diagnosed.The #1 tip for preventing hemorrhoidsWhat's the key to preventing hemorrhoids? Preventing constipation! The guide explains in detail how constipation occurs, and what you can do to avoid it. For example, you'll learn how adding fiber to your diet, drinking plenty of water, and exercising can make a big difference in your bowel health. You also get an in-depth look at stool softeners, laxatives, prescription medicines, and other means of reducing constipation.Simple lifestyle changes that help you fight hemorrhoidsThe guide offers additional easy-to-try tips for preventing and relieving hemorrhoids. These include elevating your feet when using the toilet, sitting on soft cushions vs. hard surfaces, and "training" your bowels to stay regular.A complete overview of your treatment optionsWhen it comes to treating hemorrhoids, you have many options, depending on your particular hemorrhoid condition. Healing Hemorrhoids includes safe and easy self-help remedies such as sitz baths, fiber supplements, and topical treatments like Tucks and Preparation H. The guide also goes over non-surgical, office-based treatments for hemorrhoids, as well as surgical procedures (and what to expect after surgery, too).Don't let hemorrhoids slow you down. Get your copy of Healing Hemorrhoids today!Read MoreTo your good health, Howard E. LeWine, M.D.Chief Medical Editor, Harvard Health Publishing
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It Remains Unknown: Link between Gadolinium Exposure via MRI Exams and Health Effects in Patients with Normal Renal Function By Christoph Bahn • February 27, 2018There have been concerns in the United States recently about the possible harmful side effects from absorbing gadolinium-based contrast agents (GBCAs) into the body during some magnetic resonance imaging (MRI) exams. The popular press, patient advocacy groups, and a recent lawsuit continue to raise public anxiety about the use of GBCAs.To address some of the anxiety and concerns over this issue, Paul Jannetto, Ph.D., DABCC, FAACC, and Joshua Bornhorst, Ph.D., DABCC, FAACC, Co-Directors of the Mayo Clinic Metals Laboratory and leading experts in this field, have compiled the following list of the most up-to-date information.Why Is Gadolinium Exposure Drawing So Much Attention?The increased media and regulatory attention is, in large part, due to several safety announcements in 2017, released by the U.S. Food and Drug Administration (FDA)—via its MedWatch system—regarding the use and retention of GBCAs in patients who have had an MRI. This attention particularly peaked after the announcement in December 2017, when the FDA, after review and consultation with the Medical Imaging Drugs Advisory Committee, decided to require a new class warning and other safety measures for all GBCAs used for MR imaging. (Note: GBCAs are only used in 30% to 50% of MRI exams—exposure is based on a clinical assessment of each patient’s risk and tolerance for this metal.) The new warning concerns the fact that gadolinium can be retained in patients’ bodies (e.g., the brain and other tissues) for months to years after receiving these drugs.Several research studies have shown that GBCAs may be associated with some gadolinium retention in the brain and other body tissues, despite normal renal function. However, most GBCAs are cleared in the first 96 hours after being administered to a patient. Amid all the attention, experts like Dr. Jannetto remind everyone that, “At this time, regardless of the GBCAs utilized, prolonged gadolinium retention has still not been directly linked to adverse health effects in patients with normal kidney function.”The December FDA announcement also noted that there are differences in the two types of GBCAs used (i.e., linear and macrocyclic). Of the two, linear agents result in more retention of gadolinium in patients—and retention for a longer period of time versus macrocyclic GBCAs.Regardless of the new warning, the FDA concluded that the benefits of the approved GBCAs continue to outweigh any potential risk. And the European Medicines Agency reached a similar conclusion. Even so, a number of sources continue to cast gadolinium in an unsettling light.What Gadolinium Tests Are Available, and When Should They Be Ordered?Measurement of gadolinium in clinical laboratories is not routinely conducted nor formally recommended by the FDA or any professional practice guidelines. “It should also be noted that there is no definitive or diagnostic test for gadolinium toxicity, ” says Dr. Bornhorst. “Nevertheless, Mayo Medical Laboratories currently offers gadolinium testing in serum, urine, and dermal tissue.”The test is performed by inductively coupled plasma mass spectrometry (ICP-MS), which provides a total gadolinium concentration regardless of the form used.Serum testing may be useful to monitor exposure to gadolinium. Urine testing (either a 24-hour collection reported as micrograms of gadolinium per 24 hours, or a random collection, which is reported as micrograms of gadolinium per gram of creatinine) may be useful to assess chronic exposure and monitor effectiveness of dialysis. Dermal tissue also may be used to evaluate exposure to gadolinium.What Does the Test Result Mean?Again, Drs. Jannetto and Bornhorst stress that there is no definitive or diagnostic test for gadolinium toxicity. Therefore, the presence of gadolinium in serum or urine, greater than 96 hours after the administration of a GBCA, only confirms past exposure and/or prolonged elimination of gadolinium by the renal system.Paul Jannetto, Ph.D.“Gadolinium also has been shown to be present in municipal water sources, ” says Dr. Jannetto. “And this might contribute to the observation of low concentrations of gadolinium in patients who have never been exposed to GBCAs. Ultimately, patients should consult with their health care providers to interpret any test results.”What Is the Connection involving Gadolinium, Nephrogenic Systemic Fibrosis (NSF), and Renal Dysfunction?Gadolinium is primarily eliminated by the kidneys, and impaired renal function may be a contributing reason why it can be retained in the body longer than 96 hours. Although ongoing studies continue to examine if there are any potential adverse effects of the long-term retention of gadolinium, the only known adverse health effect to date related to gadolinium retention is a rare condition called nephrogenic systemic fibrosis (NSF). “NSF is a relatively uncommon condition where fibrous plaques develop in the dermis and, often, in deeper connective tissues, ” says Dr. Bornhorst. “It’s a painful skin disease characterized by a thickening of the skin, and it can involve the joints and cause significant limitation of motion within weeks to months.”Reported cases of NSF have occurred almost exclusively in a small subgroup of patients with severe renal disease, and almost all have been associated with prior use of GBCAs. The good news is that, over the past decade, changes in clinical practice guidelines have almost completely eliminated the incidence of NSF. However, the association of NSF and observed elevated gadolinium concentrations is still not fully understood
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A Molecular Diagnostic Test That Made All the DifferenceDan C. is a guest blogger for My Colon Cancer Coach. Diagnosed at the age of 43, he is a 5 year colon cancer survivor. A policeman, husband, and father of four, Dan writes about his experience as a colon cancer patient and the path he followed to make decisions about diagnostic tests and treatment.Getting the DiagnosisDan and WifeI was just 43 years old when I was diagnosed with Stage 2 colon cancer. As a husband, father of four kids, and a police officer, the word “cancer” was the last thing on my mind. I led a relatively healthy lifestyle, so when the results of my colonoscopy came back, I couldn’t believe I was talking to my doctor about a tumor in my colon. I didn’t know how to act – I was shocked. I was scared for my own future and the future of my family, and I wondered if and how my life would change. My cancer diagnosis scared me, and I knew I had to get as much information as possible about my cancer and treatment options.Taking the First Step: Talking to Your DoctorWhen you’re first diagnosed with cancer, you panic. You don’t know what to do and you fear the unknown, which is why it’s critical to take the time to learn about your individual diagnosis and make a treatment decision that’s right for you.After my surgery, I talked to my doctor about my options and next steps. Because I had been diagnosed with stage II colon cancer and had not yet undergone chemotherapy, I was a suitable candidate for the Oncotype DX® colon cancer test. The test would tell me and my doctor the likelihood of recurrence, or whether or not my cancer would return.My doctor felt this was the best option because it would provide us with more facts based on the unique biology of my tumor. With information specific to my own cancer, we’d have a better idea of whether or not additional treatment would be needed. It’s important to ask your doctor about diagnostic tests that can help with your treatment decisions.Value of an Individualized DiagnosisThe process itself was actually quite simple. Following surgery to remove my tumor, a small piece of the tumor was sent to the Genomic Health lab for analysis. I was relieved when my doctor told me that the results showed that my cancer was not likely to return. This test score played a tremendous role in determining whether or not I’d benefit from chemotherapy post-surgery, and we ultimately decided not to move forward with chemo.I’m grateful my doctor took the time to talk to me about my specific diagnosis and options for tackling this disease, including diagnostic tests. Oncotype DX provided information about my individual cancer diagnosis and helped me and my doctor develop a treatment plan that was right for me. I avoided the harsh side effects of chemotherapy, and my score gave me a piece of mind about my decision and my future.Benefits of a Personalized Treatment PlanEveryone’s cancer is different, and it’s so important for each patient to develop a treatment plan tailored to his or her individual needs. I am confident my doctor and I made the best decisions and plan based on specific information about my cancer. It’s allowed me to get back to my normal life and my family and children, and I look forward to maintaining a long, healthy life.
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Gene Editing for GoodHow CRISPR Could Transform Global DevelopmentBy Bill GatesToday, more people are living healthy, productive lives than ever before. This good news may come as a surprise, but there is plenty of evidence for it. Since the early 1990s, global child mortality has been cut in half. There have been massive reductions in cases of tuberculosis, malaria, and HIV/AIDS. The incidence of polio has decreased by 99 percent, bringing the world to the verge of eradicating a major infectious disease, a feat humanity has accomplished only once before, with smallpox. The proportion of the world’s population in extreme poverty, defined by the World Bank as living on less than $1.90 per day, has fallen from 35 percent to about 11 percent.Continued progress is not inevitable, however, and a great deal of unnecessary suffering and inequity remains. By the end of this year, five million children under the age of five will have died—mostly in poor countries and mostly from preventable causes. Hundreds of millions of other children will continue to suffer needlessly from diseases and malnutrition that can cause lifelong cognitive and physical disabilities. And more than 750 million people—mostly rural farm families in sub-Saharan Africa and South Asia—still live in extreme poverty, according to World Bank estimates. The women and girls among them, in particular, are denied economic opportunity.Some of the remaining suffering can be eased by continuing to fund the development assistance programs and multilateral partnerships that are known to work. These efforts can help sustain progress, especially as the world gets better at using data to help guide the allocation of resources. But ultimately, eliminating the most persistent diseases and causes of poverty will require scientific discovery and technological innovations.That includes CRISPR and other technologies for targeted gene editing. Over the next decade, gene editing could help humanity overcome some of the biggest and most persistent challenges in global health and development. The technology is making it much easier for scientists to discover better diagnostics, treatments, and other tools to fight diseases that still kill and disable millions of people every year, primarily the poor. It is also accelerating research that could help end extreme poverty by enabling millions of farmers in the developing world to grow crops and raise livestock that are more productive, more nutritious, and hardier. New technologies are often met with skepticism. But if the world is to continue the remarkable progress of the past few decades, it is vital that scientists, subject to safety and ethics guidelines, be encouraged to continue taking advantage of such promising tools as CRISPR.FEEDING THE WORLDEarlier this year, I traveled to Scotland, where I met with some extraordinary scientists associated with the Centre for Tropical Livestock Genetics and Health at the University of Edinburgh. I learned about advanced genomic research to help farmers in Africa breed more productive chickens and cows. As the scientists explained, the breeds of dairy cows that can survive in hot, tropical environments tend to produce far less milk than do Holsteins—which fare poorly in hot places but are extremely productive in more moderate climates, thanks in part to naturally occurring mutations that breeders have selected for generations. The scientists in Scotland are collaborating with counterparts in Ethiopia, Kenya, Nigeria, Tanzania, and the United States. They are studying ways to edit the genes of tropical breeds of cattle to give them the same favorable genetic traits that make Holsteins so productive, potentially boosting the tropical breeds’ milk and protein production by as much as 50 percent. Conversely, scientists are also considering editing the genes of Holsteins to produce a sub-breed with a short, sleek coat of hair, which would allow the cattle to tolerate heat.This sort of research is vital, because a cow or a few chickens, goats, or sheep can make a big difference in the lives of the world’s poorest people, three-quarters of whom get their food and income by farming small plots of land. Farmers with livestock can sell eggs or milk to pay for day-to-day expenses. Chickens, in particular, tend to be raised by women, who are more likely than men to use the proceeds to buy household necessities. Livestock help farmers’ families get the nutrition they need, setting children up for healthy growth and success in school.Similarly, improving the productivity of crops is fundamental to ending extreme poverty. Sixty percent of people in sub-Saharan Africa earn their living by working the land. But given the region’s generally low agricultural productivity—yields of basic cereals are five times higher in North America—Africa remains a net importer of food. This gap between supply and demand will only grow as the number of mouths to feed increases. Africa’s population is expected to more than double by 2050, reaching 2.5 billion, and its food production will need to match that growth to feed everyone on the continent. The challenge will become even more difficult as climate change threatens the livelihoods of smallholder farmers in Africa and South Asia.Gene editing to make crops more abundant and resilient could be a lifesaver on a massive scale. The technology is already beginning to show results, attracting public and private investment, and for good reason. Scientists are developing crops with traits that enhance their growth, reduce the need for fertilizers and pesticides, boost their nutritional value, and make the plants hardier during droughts and hot spells. Already, many crops that have been improved by gene editing are being developed and tested in the field, including mushrooms with longer shelf lives, potatoes low in acrylamide (a potential carcinogen), and soybeans that produce healthier oil.Improving the productivity of crops is fundamental to ending extreme poverty.For a decade, the Bill & Melinda Gates Foundation has been backing research into the use of gene editing in agriculture. In one of the first projects we funded, scientists from the University of Oxford are developing improved varieties of rice, including one called C4 rice. Using gene editing and other tools, the Oxford scientists were able to rearrange the cellular structures in rice plant leaves, making C4 rice a remarkable 20 percent more efficient at photosynthesis, the process by which plants convert sunlight into food. The result is a crop that not only produces higher yields but also needs less water. That’s good for food security, farmers’ livelihoods, and the environment, and it will also help smallholder farmers adapt to climate change.Such alterations of the genomes of plants and even animals are not new. Humans have been doing this for thousands of years through selective breeding. Scientists began recombining DNA molecules in the early 1970s, and today, genetic engineering is widely used in agriculture and in medicine, the latter to mass-produce human insulin, hormones, vaccines, and many drugs. Gene editing is different in that it does not produce transgenic plants or animals—meaning it does not involve combining DNA from different organisms. With CRISPR, enzymes are used to target and delete a section of DNA or alter it in other ways that result in favorable or useful traits. Most important, it makes the discovery and development of innovations much faster and more precise.ENDING MALARIA In global health, one of the most promising near-term uses of gene editing involves research on malaria. Although insecticide-treated bed nets and more effective drugs have cut malaria deaths dramatically in recent decades, the parasitic disease still takes a terrible toll. Every year, about 200 million cases of malaria are recorded, and some 450, 000 people die from it, about 70 percent of them children under five. Children who survive often suffer lasting mental and physical impairments. In adults, the high fever, chills, and anemia caused by malaria can keep people from working and trap families in a cycle of illness and poverty. Beyond the human suffering, the economic costs are staggering. In sub-Saharan Africa, which is home to 90 percent of all malaria cases, the direct and indirect costs associated with the disease add up to an estimated 1.3 percent of GDP—a significant drag on countries working to lift themselves out of poverty.With sufficient funding and smart interventions using existing approaches, malaria is largely preventable and treatable—but not completely. Current tools for prevention, such as spraying for insects and their larvae, have only a temporary effect. The standard treatment for malaria today—medicine derived from artemisinin, a compound isolated from an herb used in traditional Chinese medicine—may relieve symptoms, but it may also leave behind in the human body a form of the malaria parasite that can still be spread by mosquitoes. To make matters worse, the malaria parasite has begun to develop resistance to drugs, and mosquitoes are developing resistance to insecticides.Efforts against malaria must continue to make use of existing tools, but moving toward eradication will require scientific and technological advances in multiple areas. For instance, sophisticated geospatial surveillance systems, combined with computational modeling and simulation, will make it possible to tailor antimalarial efforts to unique local conditions. Gene editing can play a big role, too. There are more than 3, 500 known mosquito species worldwide, but just a handful of them are any good at transmitting malaria parasites between people. Only female mosquitoes can spread malaria, and so researchers have used CRISPR to successfully create gene drives—making inheritable edits to their genes—that cause females to become sterile or skew them toward producing mostly male offspring. Scientists are also exploring other ways to use CRISPR to inhibit mosquitoes’ ability to transmit malaria—for example, by introducing genes that could eliminate the parasites as they pass through a mosquito’s gut on their way to its salivary glands, the main path through which infections are transmitted to humans. In comparable ways, the tool also holds promise for fighting other diseases carried by mosquitoes, such as dengue fever and the Zika virus.It will be several years, however, before any genetically edited mosquitoes are released into the wild for field trials. Although many questions about safety and efficacy will have to be answered first, there is reason to be optimistic that creating gene drives in malaria-spreading mosquitoes will not do much, if any, harm to the environment. That’s because the edits would target only the few species that tend to transmit the disease. And although natural selection will eventually produce mosquitoes that are resistant to any gene drives released into the wild, part of the value of CRISPR is that it expedites the development of new approaches—meaning that scientists can stay one step ahead.THE PATH FORWARDLike other new and potentially powerful technologies, gene editing raises legitimate questions and understandable concerns about possible risks and misuse. How, then, should the technology be regulated? Rules developed decades ago for other forms of genetic engineering do not necessarily fit. Noting that gene-edited organisms are not transgenic, the U.S. Department of Agriculture has reasonably concluded that genetically edited plants are like plants with naturally occurring mutations and thus are not subject to special regulations and raise no special safety concerns.The benefits of emerging technologies should not be reserved only for people in developed countries.Gene editing in animals or even humans raises more complicated questions of safety and ethics. In 2014, the World Health Organization issued guidelines for testing genetically modified mosquitoes, including standards for efficacy, biosafety, bioethics, and public participation. In 2016, the National Academy of Sciences built on the WHO’s guidelines with recommendations for responsible conduct in gene-drive research on animals. (The Gates Foundation co-funded this work with the National Institutes of Health, the Foundation for the National Institutes of Health, and the Defense Advanced Research Projects Agency.) These recommendations emphasized the need for thorough research in the lab, including interim evaluations at set points, before scientists move to field trials. They also urged scientists to assess any ecological risks and to actively involve the public, especially in the communities and countries directly affected by the research. Wherever gene-editing research takes place, it should involve all the key stakeholders—scientists, civil society, government leaders, and local communities—from wherever it is likely to be deployed.Part of the challenge in regulating gene editing is that the rules and practices in different countries may differ widely. A more harmonized policy environment would prove more efficient, and it would probably also raise overall standards. International organizations, especially of scientists, could help establish global norms. Meanwhile, funders of gene-editing research must ensure that it is conducted in compliance with standards such as those advanced by the WHO and the National Academy of Sciences, no matter where the research takes place.When it comes to gene-editing research on malaria, the Gates Foundation has joined with others to help universities and other institutions in the regions affected by the disease to conduct risk assessments and advise regional bodies on experiments and future field tests. The goal is to empower affected countries and communities to take the lead in the research, evaluate its costs and benefits, and make informed decisions about whether and when to apply the resulting technology.Finally, it’s important to recognize the costs and risks of failing to explore the use of new tools such as CRISPR for global health and development. The benefits of emerging technologies should not be reserved only for people in developed countries. Nor should decisions about whether to take advantage of them. Used responsibly, gene editing holds the potential to save millions of lives and empower millions of people to lift themselves out of poverty. It would be a tragedy to pass up the opportunity.
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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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