Podcasts about Western blot

Dr. Gary Null provides a commentary on "Universal  Healthcare"       Universal Healthcare is the Solution to a Broken Medical System Gary Null, PhD Progressive Radio Network, March 3, 2025 For over 50 years, there has been no concerted or successful effort to bring down medical costs in the American healthcare system. Nor are the federal health agencies making disease prevention a priority. Regardless whether the political left or right sponsors proposals for reform, such measures are repeatedly defeated by both parties in Congress. As a result, the nation's healthcare system remains one of the most expensive and least efficient in the developed world. For the past 30 years, medical bills contributing to personal debt regularly rank among the top three causes of personal bankruptcy. This is a reality that reflects not only the financial strain on ordinary Americans but the systemic failure of the healthcare system itself. The urgent question is: If President Trump and his administration are truly seeking to reduce the nation's $36 trillion deficit, why is there no serious effort to reform the most bloated and corrupt sector of the economy? A key obstacle is the widespread misinformation campaign that falsely claims universal health care would cost an additional $2 trillion annually and further balloon the national debt. However, a more honest assessment reveals the opposite. If the US adopted a universal single-payer system, the nation could actually save up to $20 trillion over the next 10 years rather than add to the deficit. Even with the most ambitious efforts by people like Elon Musk to rein in federal spending or optimize government efficiency, the estimated savings would only amount to $500 billion. This is only a fraction of what could be achieved through comprehensive healthcare reform alone. Healthcare is the largest single expenditure of the federal budget. A careful examination of where the $5 trillion spent annually on healthcare actually goes reveals massive systemic fraud and inefficiency. Aside from emergency medicine, which accounts for only 10-12 percent of total healthcare expenditures, the bulk of this spending does not deliver better health outcomes nor reduce trends in physical and mental illness. Applying Ockham's Razor, the principle that the simplest solution is often the best, the obvious conclusion is that America's astronomical healthcare costs are the direct result of price gouging on an unimaginable scale. For example, in most small businesses, profit margins range between 1.6 and 2.5 percent, such as in grocery retail. Yet the pharmaceutical industrial complex routinely operates on markup rates as high as 150,000 percent for many prescription drugs. The chart below highlights the astronomical gap between the retail price of some top-selling patented pharmaceutical medications and their generic equivalents. Drug Condition Patent Price (per unit) Generic Price Estimated Manufacture Cost Markup Source Insulin (Humalog) Diabetes $300 $30 $3 10,000% Rand (2021) EpiPen Allergic reactions $600 $30 $10 6,000% BMJ (2022) Daraprim Toxoplasmosis $750/pill $2 $0.50 150,000% JAMA (2019) Harvoni Hepatitis C $94,500 (12 weeks) $30,000 $200 47,000% WHO Report (2018) Lipitor Cholesterol $150 $10 $0.50 29,900% Health Affairs (2020) Xarelto Blood Thinner $450 $25 $1.50 30,000% NEJM (2020) Abilify Schizophrenia $800 (30 tablets) $15 $2 39,900% AJMC (2019) Revlimid Cancer $16,000/mo $450 $150 10,500% Kaiser Health News (2021) Humira Arthritis $2,984/dose $400 $50 5,868% Rand (2021) Sovaldi Hepatitis C $1,000/pill $10 $2 49,900% JAMA (2021) Xolair Asthma $2,400/dose $300 $50 4,800% NEJM (2020) Gleevec Leukemia $10,000/mo $350 $200 4,900% Harvard Public Health Review (2020) OxyContin Pain Relief $600 (30 tablets) $15 $0.50 119,900% BMJ (2022) Remdesivir Covid-19 $3,120 (5 doses) N/A $10 31,100% The Lancet (2020) The corruption extends far beyond price gouging. Many pharmaceutical companies convince federal health agencies to fund their basic research and drug development with taxpayer dollars. Yet when these companies bring successful products to market, the profits are kept entirely by the corporations or shared with the agencies or groups of government scientists. On the other hand, the public, who funded the research, receives no financial return. This amounts to a systemic betrayal of the public trust on a scale of hundreds of billions of dollars annually. Another significant contributor to rising healthcare costs is the widespread practice of defensive medicine that is driven by the constant threat of litigation. Over the past 40 years, defensive medicine has become a cottage industry. Physicians order excessive diagnostic tests and unnecessary treatments simply to protect themselves from lawsuits. Study after study has shown that these over-performed procedures not only inflate costs but lead to iatrogenesis or medical injury and death caused by the medical  system and practices itself. The solution is simple: adopting no-fault healthcare coverage for everyone where patients receive care without needing to sue and thereby freeing doctors from the burden of excessive malpractice insurance. A single-payer universal healthcare system could fundamentally transform the entire industry by capping profits at every level — from drug manufacturers to hospitals to medical equipment suppliers. The Department of Health and Human Services would have the authority to set profit margins for medical procedures. This would ensure that healthcare is determined by outcomes, not profits. Additionally, the growing influence of private equity firms and vulture capitalists buying up hospitals and medical clinics across America must be reined in. These equity firms prioritize profit extraction over improving the quality of care. They often slash staff, raise prices, and dictate medical procedures based on what will yield the highest returns. Another vital reform would be to provide free medical education for doctors and nurses in exchange for five years of service under the universal system. Medical professionals would earn a realistic salary cap to prevent them from being lured into equity partnerships or charging exorbitant rates. The biggest single expense in the current system, however, is the private health insurance industry, which consumes 33 percent of the $5 trillion healthcare budget. Health insurance CEOs consistently rank among the highest-paid executives in the country. Their companies, who are nothing more than bean counters, decide what procedures and drugs will be covered, partially covered, or denied altogether. This entire industry is designed to place profits above patients' lives. If the US dismantled its existing insurance-based system and replaced it with a fully reformed national healthcare model, the country could save $2.7 trillion annually while simultaneously improving health outcomes. Over the course of 10 years, those savings would amount to $27 trillion. This could wipe out nearly the entire national debt in a short time. This solution has been available for decades but has been systematically blocked by corporate lobbying and bipartisan corruption in Washington. The path forward is clear but only if American citizens demand a system where healthcare is valued as a public service and not a commodity. The national healthcare crisis is not just a fiscal issue. It is a crucial moral failure of the highest order. With the right reforms, the nation could simultaneously restore its financial health and deliver the kind of healthcare system its citizens have long deserved. American Healthcare: Corrupt, Broken and Lethal Richard Gale and Gary Null Progressive Radio Network, March 3, 2025 For a nation that prides itself on being the world's wealthiest, most innovative and technologically advanced, the US' healthcare system is nothing less than a disaster and disgrace. Not only are Americans the least healthy among the most developed nations, but the US' health system ranks dead last among high-income countries. Despite rising costs and our unshakeable faith in American medical exceptionalism, average life expectancy in the US has remained lower than other OECD nations for many years and continues to decline. The United Nations recognizes healthcare as a human right. In 2018, former UN Secretary General Ban Ki-moon denounced the American healthcare system as "politically and morally wrong." During the pandemic it is estimated that two to three years was lost on average life expectancy. On the other hand, before the Covid-19 pandemic, countries with universal healthcare coverage found their average life expectancy stable or slowly increasing. The fundamental problem in the U.S. is that politics have been far too beholden to the pharmaceutical, HMO and private insurance industries. Neither party has made any concerted effort to reign in the corruption of corporate campaign funding and do what is sensible, financially feasible and morally correct to improve Americans' quality of health and well-being.   The fact that our healthcare system is horribly broken is proof that moneyed interests have become so powerful to keep single-payer debate out of the media spotlight and censored. Poll after poll shows that the American public favors the expansion of public health coverage. Other incremental proposals, including Medicare and Medicaid buy-in plans, are also widely preferred to the Affordable Care Act or Obamacare mess we are currently stuck with.   It is not difficult to understand how the dismal state of American medicine is the result of a system that has been sold out to the free-market and the bottom line interests of drug makers and an inflated private insurance industry. How advanced and ethically sound can a healthcare system be if tens of millions of people have no access to medical care because it is financially out of their reach?  The figures speak for themselves. The U.S. is burdened with a $41 trillion Medicare liability. The number of uninsured has declined during the past several years but still lingers around 25 million. An additional 30-35 million are underinsured. There are currently 65 million Medicare enrollees and 89 million Medicaid recipients. This is an extremely unhealthy snapshot of the country's ability to provide affordable healthcare and it is certainly unsustainable. The system is a public economic failure, benefiting no one except the large and increasingly consolidated insurance and pharmaceutical firms at the top that supervise the racket.   Our political parties have wrestled with single-payer or universal healthcare for decades. Obama ran his first 2008 presidential campaign on a single-payer platform. Since 1985, his campaign health adviser, the late Dr. Quentin Young from the University of Illinois Medical School, was one of the nation's leading voices calling for universal health coverage.  During a private conversation with Dr. Young shortly before his passing in 2016, he conveyed his sense of betrayal at the hands of the Obama administration. Dr. Young was in his 80s when he joined the Obama campaign team to help lead the young Senator to victory on a promise that America would finally catch up with other nations. The doctor sounded defeated. He shared how he was manipulated, and that Obama held no sincere intention to make universal healthcare a part of his administration's agenda. During the closed-door negotiations, which spawned the weak and compromised Affordable Care Act, Dr. Young was neither consulted nor invited to participate. In fact, he told us that he never heard from Obama again after his White House victory.   Past efforts to even raise the issue have been viciously attacked. A huge army of private interests is determined to keep the public enslaved to private insurers and high medical costs. The failure of our healthcare is in no small measure due to it being a fully for-profit operation. Last year, private health insurance accounted for 65 percent of coverage. Consider that there are over 900 private insurance companies in the US. National Health Expenditures (NHE) grew to $4.5 trillion in 2022, which was 17.3 percent of GDP. Older corporate rank-and-file Democrats and Republicans argue that a single-payer or socialized medical program is unaffordable. However, not only is single-payer affordable, it will end bankruptcies due to unpayable medical debt. In addition, universal healthcare, structured on a preventative model, will reduce disease rates at the outset.    Corporate Democrats argue that Obama's Affordable Care Act (ACA) was a positive step inching the country towards complete public coverage. However, aside from providing coverage to the poorest of Americans, Obamacare turned into another financial anchor around the necks of millions more. According to the health policy research group KFF, the average annual health insurance premium for single coverage is $8,400 and almost $24,000 for a family. In addition, patient out-of-pocket costs continue to increase, a 6.6% increase to $471 billion in 2022. Rather than healthcare spending falling, it has exploded, and the Trump and Biden administrations made matters worse.    Clearly, a universal healthcare program will require flipping the script on the entire private insurance industry, which employed over half a million people last year.  Obviously, the most volatile debate concerning a national universal healthcare system concerns cost. Although there is already a socialized healthcare system in place -- every federal legislator, bureaucrat, government employee and veteran benefits from it -- fiscal Republican conservatives and groups such as the Koch Brothers network are single-mindedly dedicated to preventing the expansion of Medicare and Medicaid. A Koch-funded Mercatus analysis made the outrageous claim that a single-payer system would increase federal health spending by $32 trillion in ten years. However, analyses and reviews by the Congressional Budget Office in the early 1990s concluded that such a system would only increase spending at the start; enormous savings would quickly offset it as the years pass. In one analysis, "the savings in administrative costs [10 percent of health spending] would be more than enough to offset the expense of universal coverage."    Defenders of those advocating for funding a National Health Program argue this can primarily be accomplished by raising taxes to levels comparable to other developed nations. This was a platform Senator Bernie Sanders and some of the younger progressive Democrats in the House campaigned on. The strategy was to tax the highest multimillion-dollar earners 60-70 percent. Despite the outrage of its critics, including old rank-and-file multi-millionaire Democrats like Nancy Pelosi and Chuck Schumer, this is still far less than in the past. During the Korean War, the top tax rate was 91 percent; it declined to 70 percent in the late 1960s. Throughout most of the 1970s, those in the lowest income bracket were taxed at 14 percent. We are not advocating for this strategy because it ignores where the funding is going, and the corruption in the system that is contributing to exorbitant waste.    But Democratic supporters of the ACA who oppose a universal healthcare plan ignore the additional taxes Obama levied to pay for the program. These included surtaxes on investment income, Medicare taxes from those earning over $200,000, taxes on tanning services, an excise tax on medical equipment, and a 40 percent tax on health coverage for costs over the designated cap that applied to flexible savings and health savings accounts. The entire ACA was reckless, sloppy and unnecessarily complicated from the start.    The fact that Obamacare further strengthened the distinctions between two parallel systems -- federal and private -- with entirely different economic structures created a labyrinth of red tape, rules, and wasteful bureaucracy. Since the ACA went into effect, over 150 new boards, agencies and programs have had to be established to monitor its 2,700 pages of gibberish. A federal single-payer system would easily eliminate this bureaucracy and waste.    A medical New Deal to establish universal healthcare coverage is a decisive step in the correct direction. But we must look at the crisis holistically and in a systematic way. Simply shuffling private insurance into a federal Medicare-for-all or buy-in program, funded by taxing the wealthiest of citizens, would only temporarily reduce costs. It will neither curtail nor slash escalating disease rates e. Any effective healthcare reform must also tackle the underlying reasons for Americans' poor state of health. We cannot shy away from examining the social illnesses infecting our entire free-market capitalist culture and its addiction to deregulation. A viable healthcare model would have to structurally transform how the medical economy operates. Finally, a successful medical New Deal must honestly evaluate the best and most reliable scientific evidence in order to effectively redirect public health spending.    For example, Dr. Ezekiel Emanuel, a former Obama healthcare adviser, observed that AIDS-HIV measures consume the most public health spending, even though the disease "ranked 75th on the list of diseases by personal health expenditures." On the other hand, according to the American Medical Association, a large percentage of the nation's $3.4 trillion healthcare spending goes towards treating preventable diseases, notably diabetes, common forms of heart disease, and back and neck pain conditions. In 2016, these three conditions were the most costly and accounted for approximately $277 billion in spending. Last year, the CDC announced the autism rate is now 1 in 36 children compared to 1 in 44 two years ago. A retracted study by Mark Blaxill, an autism activist at the Holland Center and a friend of the authors, estimates that ASD costs will reach $589 billion annually by 2030. There are no signs that this alarming trend will reverse and decline; and yet, our entire federal health system has failed to conscientiously investigate the underlying causes of this epidemic. All explanations that might interfere with the pharmaceutical industry's unchecked growth, such as over-vaccination, are ignored and viciously discredited without any sound scientific evidence. Therefore, a proper medical New Deal will require a systemic overhaul and reform of our federal health agencies, especially the HHS, CDC and FDA. Only the Robert Kennedy Jr presidential campaign is even addressing the crisis and has an inexpensive and comprehensive plan to deal with it. For any medical revolution to succeed in advancing universal healthcare, the plan must prioritize spending in a manner that serves public health and not private interests. It will also require reshuffling private corporate interests and their lobbyists to the sidelines, away from any strategic planning, in order to break up the private interests' control over federal agencies and its revolving door policies. Aside from those who benefit from this medical corruption, the overwhelming majority of Americans would agree with this criticism. However, there is a complete lack of national trust that our legislators, including the so-called progressives, would be willing to undertake such actions.    In addition, America's healthcare system ignores the single most critical initiative to reduce costs - that is, preventative efforts and programs instead of deregulation and closing loopholes designed to protect the drug and insurance industries' bottom line. Prevention can begin with banning toxic chemicals that are proven health hazards associated with current disease epidemics, and it can begin by removing a 1,000-plus toxins already banned in Europe. This should be a no-brainer for any legislator who cares for public health. For example, Stacy Malkan, co-founder of the Campaign for Safe Cosmetics, notes that "the policy approach in the US and Europe is dramatically different" when it comes to chemical allowances in cosmetic products. Whereas the EU has banned 1,328 toxic substances from the cosmetic industry alone, the US has banned only 11. The US continues to allow carcinogenic formaldehyde, petroleum, forever chemicals, many parabens (an estrogen mimicker and endocrine hormone destroyer), the highly allergenic p-phenylenediamine or PBD, triclosan, which has been associated with the rise in antibiotic resistant bacteria, avobenzone, and many others to be used in cosmetics, sunscreens, shampoo and hair dyes.   Next, the food Americans consume can be reevaluated for its health benefits. There should be no hesitation to tax the unhealthiest foods, such as commercial junk food, sodas and candy relying on high fructose corn syrup, products that contain ingredients proven to be toxic, and meat products laden with dangerous chemicals including growth hormones and antibiotics. The scientific evidence that the average American diet is contributing to rising disease trends is indisputable. We could also implement additional taxes on the public advertising of these demonstrably unhealthy products. All such tax revenue would accrue to a national universal health program to offset medical expenditures associated with the very illnesses linked to these products. Although such tax measures would help pay for a new medical New Deal, it may be combined with programs to educate the public about healthy nutrition if it is to produce a reduction in the most common preventable diseases. In fact, comprehensive nutrition courses in medical schools should be mandatory because the average physician receives no education in this crucial subject.  In addition, preventative health education should be mandatory throughout public school systems.   Private insurers force hospitals, clinics and private physicians into financial corners, and this is contributing to prodigious waste in money and resources. Annually, healthcare spending towards medical liability insurance costs tens of billions of dollars. In particular, this economic burden has taxed small clinics and physicians. It is well past the time that physician liability insurance is replaced with no-fault options. Today's doctors are spending an inordinate amount of money to protect themselves. Legions of liability and trial lawyers seek big paydays for themselves stemming from physician error. This has created a culture of fear among doctors and hospitals, resulting in the overly cautious practice of defensive medicine, driving up costs and insurance premiums just to avoid lawsuits. Doctors are forced to order unnecessary tests and prescribe more medications and medical procedures just to cover their backsides. No-fault insurance is a common-sense plan that enables physicians to pursue their profession in a manner that will reduce iatrogenic injuries and costs. Individual cases requiring additional medical intervention and loss of income would still be compensated. This would generate huge savings.    No other nation suffers from the scourge of excessive drug price gouging like the US. After many years of haggling to lower prices and increase access to generic drugs, only a minute amount of progress has been made in recent years. A 60 Minutes feature about the Affordable Care Act reported an "orgy of lobbying and backroom deals in which just about everyone with a stake in the $3-trillion-a-year health industry came out ahead—except the taxpayers.” For example, Life Extension magazine reported that an antiviral cream (acyclovir), which had lost its patent protection, "was being sold to pharmacies for 7,500% over the active ingredient cost. The active ingredient (acyclovir) costs only 8 pennies, yet pharmacies are paying a generic maker $600 for this drug and selling it to consumers for around $700." Other examples include the antibiotic Doxycycline. The price per pill averages 7 cents to $3.36 but has a 5,300 percent markup when it reaches the consumer. The antidepressant Clomipramine is marked up 3,780 percent, and the anti-hypertensive drug Captopril's mark-up is 2,850 percent. And these are generic drugs!    Medication costs need to be dramatically cut to allow drug manufacturers a reasonable but not obscene profit margin. By capping profits approximately 100 percent above all costs, we would save our system hundreds of billions of dollars. Such a measure would also extirpate the growing corporate misdemeanors of pricing fraud, which forces patients to pay out-of-pocket in order to make up for the costs insurers are unwilling to pay.    Finally, we can acknowledge that our healthcare is fundamentally a despotic rationing system based upon high insurance costs vis-a-vis a toss of the dice to determine where a person sits on the economic ladder. For the past three decades it has contributed to inequality. The present insurance-based economic metrics cast millions of Americans out of coverage because private insurance costs are beyond their means. Uwe Reinhardt, a Princeton University political economist, has called our system "brutal" because it "rations [people] out of the system." He defined rationing as "withholding something from someone that is beneficial." Discriminatory healthcare rationing now affects upwards to 60 million people who have been either priced out of the system or under insured. They make too much to qualify for Medicare under Obamacare, yet earn far too little to afford private insurance costs and premiums. In the final analysis, the entire system is discriminatory and predatory.    However, we must be realistic. Almost every member of Congress has benefited from Big Pharma and private insurance lobbyists. The only way to begin to bring our healthcare program up to the level of a truly developed nation is to remove the drug industry's rampant and unnecessary profiteering from the equation.     How did Fauci memory-hole a cure for AIDS and get away with it?   By Helen Buyniski   Over 700,000 Americans have died of AIDS since 1981, with the disease claiming some 42.3 million victims worldwide. While an HIV diagnosis is no longer considered a certain death sentence, the disease looms large in the public imagination and in public health funding, with contemporary treatments running into thousands of dollars per patient annually.   But was there a cure for AIDS all this time - an affordable and safe treatment that was ruthlessly suppressed and attacked by the US public health bureaucracy and its agents? Could this have saved millions of lives and billions of dollars spent on AZT, ddI and failed HIV vaccine trials? What could possibly justify the decision to disappear a safe and effective approach down the memory hole?   The inventor of the cure, Gary Null, already had several decades of experience creating healing protocols for physicians to help patients not responding well to conventional treatments by the time AIDS was officially defined in 1981. Null, a registered dietitian and board-certified nutritionist with a PhD in human nutrition and public health science, was a senior research fellow and Director of Anti-Aging Medicine at the Institute of Applied Biology for 36 years and has published over 950 papers, conducting groundbreaking experiments in reversing biological aging as confirmed with DNA methylation testing. Additionally, Null is a multi-award-winning documentary filmmaker, bestselling author, and investigative journalist whose work exposing crimes against humanity over the last 50 years has highlighted abuses by Big Pharma, the military-industrial complex, the financial industry, and the permanent government stay-behind networks that have come to be known as the Deep State.   Null was contacted in 1974 by Dr. Stephen Caiazza, a physician working with a subculture of gay men in New York living the so-called “fast track” lifestyle, an extreme manifestation of the gay liberation movement that began with the Stonewall riots. Defined by rampant sexual promiscuity and copious use of illegal and prescription drugs, including heavy antibiotic use for a cornucopia of sexually-transmitted diseases, the fast-track never included more than about two percent of gay men, though these dominated many of the bathhouses and clubs that defined gay nightlife in the era. These patients had become seriously ill as a result of their indulgence, generally arriving at the clinic with multiple STDs including cytomegalovirus and several types of herpes and hepatitis, along with candida overgrowth, nutritional deficiencies, gut issues, and recurring pneumonia. Every week for the next 10 years, Null would counsel two or three of these men - a total of 800 patients - on how to detoxify their bodies and de-stress their lives, tracking their progress with Caiazza and the other providers at weekly feedback meetings that he credits with allowing the team to quickly evaluate which treatments were most effective. He observed that it only took about two years on the “fast track” for a healthy young person to begin seeing muscle loss and the recurrent, lingering opportunistic infections that would later come to be associated with AIDS - while those willing to commit to a healthier lifestyle could regain their health in about a year.    It was with this background that Null established the Tri-State Healing Center in Manhattan in 1980, staffing the facility with what would eventually run to 22 certified health professionals to offer safe, natural, and effective low- and no-cost treatments to thousands of patients with HIV and AIDS-defining conditions. Null and his staff used variations of the protocols he had perfected with Caiazza's patients, a multifactorial patient-tailored approach that included high-dose vitamin C drips, intravenous ozone therapy, juicing and nutritional improvements and supplementation, aspects of homeopathy and naturopathy with some Traditional Chinese Medicine and Ayurvedic practices. Additional services offered on-site included acupuncture and holistic dentistry, while peer support groups were also held at the facility so that patients could find community and a positive environment, healing their minds and spirits while they healed their bodies.   “Instead of trying to kill the virus with antiretroviral pharmaceuticals designed to stop viral replication before it kills patients, we focused on what benefits could be gained by building up the patients' natural immunity and restoring biochemical integrity so the body could fight for itself,” Null wrote in a 2014 article describing the philosophy behind the Center's approach, which was wholly at odds with the pharmaceutical model.1   Patients were comprehensively tested every week, with any “recovery” defined solely by the labs, which documented AIDS patient after patient - 1,200 of them - returning to good health and reversing their debilitating conditions. Null claims to have never lost an AIDS patient in the Center's care, even as the death toll for the disease - and its pharmaceutical standard of care AZT - reached an all-time high in the early 1990s. Eight patients who had opted for a more intensive course of treatment - visiting the Center six days a week rather than one - actually sero-deconverted, with repeated subsequent testing showing no trace of HIV in their bodies.   As an experienced clinical researcher himself, Null recognized that any claims made by the Center would be massively scrutinized, challenging as they did the prevailing scientific consensus that AIDS was an incurable, terminal illness. He freely gave his protocols to any medical practitioner who asked, understanding that his own work could be considered scientifically valid only if others could replicate it under the same conditions. After weeks of daily observational visits to the Center, Dr. Robert Cathcart took the protocols back to San Francisco, where he excitedly reported that patients were no longer dying in his care.    Null's own colleague at the Institute of Applied Biology, senior research fellow Elana Avram, set up IV drip rooms at the Institute and used his intensive protocols to sero-deconvert 10 patients over a two-year period. While the experiment had been conducted in secret, as the Institute had been funded by Big Pharma since its inception half a century earlier, Avram had hoped she would be able to publish a journal article to further publicize Null's protocols and potentially help AIDS patients, who were still dying at incredibly high rates thanks to Burroughs Wellcome's noxious but profitable AZT. But as she would later explain in a 2019 letter to Null, their groundbreaking research never made it into print - despite meticulous documentation of their successes - because the Institute's director and board feared their pharmaceutical benefactors would withdraw the funding on which they depended, given that Null's protocols did not involve any patentable or otherwise profitable drugs. When Avram approached them about publication, the board vetoed the idea, arguing that it would “draw negative attention because [the work] was contrary to standard drug treatments.” With no real point in continuing experiments along those lines without institutional support and no hope of obtaining funding from elsewhere, the department she had created specifically for these experiments shut down after a two-year followup with her test subjects - all of whom remained alive and healthy - was completed.2   While the Center was receiving regular visits by this time from medical professionals and, increasingly, black celebrities like Stokely Carmichael and Isaac Hayes, who would occasionally perform for the patients, the news was spreading by word of mouth alone - not a single media outlet had dared to document the clinic that was curing AIDS patients for free. Instead, they gave airtime to Anthony Fauci, director of the National Institute of Allergies and Infectious Diseases, who had for years been spreading baseless, hysteria-fueling claims about HIV and AIDS to any news outlet that would put him on. His claim that children could contract the virus from “ordinary household conduct” with an infected relative proved so outrageous he had to walk it back,3 and he never really stopped insisting the deadly plague associated with gays and drug users was about to explode like a nuclear bomb among the law-abiding heterosexual population. Fauci by this time controlled all government science funding through NIAID, and his zero-tolerance approach to dissent on the HIV/AIDS front had already seen prominent scientists like virologist Peter Duesberg stripped of the resources they needed for their work because they had dared to question his commandment: There is no cause of AIDS but HIV, and AZT is its treatment. Even the AIDS activist groups, which by then had been coopted by Big Pharma and essentially reduced to astroturfing for the toxic failed chemotherapy drug AZT backed by the institutional might of Fauci's NIAID,4 didn't seem to want to hear that there was a cure. Unconcerned with the irrationality of denouncing the man touting his free AIDS cure as an  “AIDS denier,” they warned journalists that platforming Null or anyone else rejecting the mainstream medical line would be met with organized demands for their firing.    Determined to breach the institutional iron curtain and get his message to the masses, Null and his team staged a press conference in New York, inviting scientists and doctors from around the world to share their research on alternative approaches to HIV and AIDS in 1993. To emphasize the sound scientific basis of the Center's protocols and encourage guests to adopt them into their own practices, Null printed out thousands of abstracts in support of each nutrient and treatment being used. However, despite over 7,000 invitations sent three times to major media, government figures, scientists, and activists, almost none of the intended audience members showed up. Over 100 AIDS patients and their doctors, whose charts exhaustively documented their improvements using natural and nontoxic modalities over the preceding 12 months, gave filmed testimonials, declaring that the feared disease was no longer a death sentence, but the conference had effectively been silenced. Bill Tatum, publisher of the Amsterdam News, suggested Null and his patients would find a more welcoming audience in his home neighborhood of Harlem - specifically, its iconic Apollo Theatre. For three nights, the theater was packed to capacity. Hit especially hard by the epidemic and distrustful of a medical system that had only recently stopped being openly racist (the Tuskegee syphilis experiment only ended in 1972), black Americans, at least, did not seem to care what Anthony Fauci would do if he found out they were investigating alternatives to AZT and death.    PBS journalist Tony Brown, having obtained a copy of the video of patient testimonials from the failed press conference, was among a handful of black journalists who began visiting the Center to investigate the legitimacy of Null's claims. Satisfied they had something significant to offer his audience, Brown invited eight patients - along with Null himself - onto his program over the course of several episodes to discuss the work. It was the first time these protocols had received any attention in the media, despite Null having released nearly two dozen articles and multiple documentaries on the subject by that time. A typical patient on one program, Al, a recovered IV drug user who was diagnosed with AIDS at age 32, described how he “panicked,” saw a doctor and started taking AZT despite his misgivings - only to be forced to discontinue the drug after just a few weeks due to his condition deteriorating rapidly. Researching alternatives brought him to Null, and after six months of “detoxing [his] lifestyle,” he observed his initial symptoms - swollen lymph nodes and weight loss - begin to reverse, culminating with sero-deconversion. On Bill McCreary's Channel 5 program, a married couple diagnosed with HIV described how they watched their T-cell counts increase as they cut out sugar, caffeine, smoking, and drinking and began eating a healthy diet. They also saw the virus leave their bodies.   For HIV-positive viewers surrounded by fear and negativity, watching healthy-looking, cheerful “AIDS patients” detail their recovery while Null backed up their claims with charts must have been balm for the soul. But the TV programs were also a form of outreach to the medical community, with patients' charts always on hand to convince skeptics the cure was scientifically valid. Null brought patients' charts to every program, urging them to keep an open mind: “Other physicians and public health officials should know that there's good science in the alternative perspective. It may not be a therapy that they're familiar with, because they're just not trained in it, but if the results are positive, and you can document them…” He challenged doubters to send in charts from their own sero-deconverted patients on AZT, and volunteered to debate proponents of the orthodox treatment paradigm - though the NIH and WHO both refused to participate in such a debate on Tony Brown's Journal, following Fauci's directive prohibiting engagement with forbidden ideas.    Aside from those few TV programs and Null's own films, suppression of Null's AIDS cure beyond word of mouth was total. The 2021 documentary The Cost of Denial, produced by the Society for Independent Journalists, tells the story of the Tri-State Healing Center and the medical paradigm that sought to destroy it, lamenting the loss of the lives that might have been saved in a more enlightened society. Nurse practitioner Luanne Pennesi, who treated many of the AIDS patients at the Center, speculated in the film that the refusal by the scientific establishment and AIDS activists to accept their successes was financially motivated. “It was as if they didn't want this information to get out. Understand that our healthcare system as we know it is a corporation, it's a corporate model, and it's about generating revenue. My concern was that maybe they couldn't generate enough revenue from these natural approaches.”5   Funding was certainly the main disciplinary tool Fauci's NIAID used to keep the scientific community in line. Despite the massive community interest in the work being done at the Center, no foundation or institution would defy Fauci and risk getting itself blacklisted, leaving Null to continue funding the operation out of his pocket with the profits from book sales. After 15 years, he left the Center in 1995, convinced the mainstream model had so thoroughly been institutionalized that there was no chance of overthrowing it. He has continued to counsel patients and advocate for a reappraisal of the HIV=AIDS hypothesis and its pharmaceutical treatments, highlighting the deeply flawed science underpinning the model of the disease espoused by the scientific establishment in 39 articles, six documentaries and a 700-page textbook on AIDS, but the Center's achievements have been effectively memory-holed by Fauci's multi-billion-dollar propaganda apparatus.     FRUIT OF THE POISONOUS TREE   To understand just how much of a threat Null's work was to the HIV/AIDS establishment, it is instructive to revisit the 1984 paper, published by Dr. Robert Gallo of the National Cancer Institute, that established HIV as the sole cause of AIDS. The CDC's official recognition of AIDS in 1981 had done little to quell the mounting public panic over the mysterious illness afflicting gay men in the US, as the agency had effectively admitted it had no idea what was causing them to sicken and die. As years passed with no progress determining the causative agent of the plague, activist groups like Gay Men's Health Crisis disrupted public events and threatened further mass civil disobedience as they excoriated the NIH for its sluggish allocation of government science funding to uncovering the cause of the “gay cancer.”6 When Gallo published his paper declaring that the retrovirus we now know as HIV was the sole “probable” cause of AIDS, its simple, single-factor hypothesis was the answer to the scientific establishment's prayers. This was particularly true for Fauci, as the NIAID chief was able to claim the hot new disease as his agency's own domain in what has been described as a “dramatic confrontation” with his rival Sam Broder at the National Cancer Institute. After all, Fauci pointed out, Gallo's findings - presented by Health and Human Services Secretary Margaret Heckler as if they were gospel truth before any other scientists had had a chance to inspect them, never mind conduct a full peer review - clearly classified AIDS as an infectious disease, and not a cancer like the Kaposi's sarcoma which was at the time its most visible manifestation. Money and media attention began pouring in, even as funding for the investigation of other potential causes of AIDS dried up. Having already patented a diagnostic test for “his” retrovirus before introducing it to the world, Gallo was poised for a financial windfall, while Fauci was busily leveraging the discovery into full bureaucratic empire of the US scientific apparatus.   While it would serve as the sole basis for all US government-backed AIDS research to follow - quickly turning Gallo into the most-cited scientist in the world during the 1980s,7 Gallo's “discovery” of HIV was deeply problematic. The sample that yielded the momentous discovery actually belonged to Prof. Luc Montagnier of the French Institut Pasteur, a fact Gallo finally admitted in 1991, four years after a lawsuit from the French government challenged his patent on the HIV antibody test, forcing the US government to negotiate a hasty profit-sharing agreement between Gallo's and Montagnier's labs. That lawsuit triggered a cascade of official investigations into scientific misconduct by Gallo, and evidence submitted during one of these probes, unearthed in 2008 by journalist Janine Roberts, revealed a much deeper problem with the seminal “discovery.” While Gallo's co-author, Mikulas Popovic, had concluded after numerous experiments with the French samples that the virus they contained was not the cause of AIDS, Gallo had drastically altered the paper's conclusion, scribbling his notes in the margins, and submitted it for publication to the journal Science without informing his co-author.   After Roberts shared her discovery with contacts in the scientific community, 37 scientific experts wrote to the journal demanding that Gallo's career-defining HIV paper be retracted from Science for lacking scientific integrity.8 Their call, backed by an endorsement from the 2,600-member scientific organization Rethinking AIDS, was ignored by the publication and by the rest of mainstream science despite - or perhaps because of - its profound implications.   That 2008 letter, addressed to Science editor-in-chief Bruce Alberts and copied to American Association for the Advancement of Science CEO Alan Leshner, is worth reproducing here in its entirety, as it utterly dismantles Gallo's hypothesis - and with them the entire HIV is the sole cause of AIDS dogma upon which the contemporary medical model of the disease rests:   On May 4, 1984 your journal published four papers by a group led by Dr. Robert Gallo. We are writing to express our serious concerns with regard to the integrity and veracity of the lead paper among these four of which Dr. Mikulas Popovic is the lead author.[1] The other three are also of concern because they rely upon the conclusions of the lead paper .[2][3][4]  In the early 1990s, several highly critical reports on the research underlying these papers were produced as a result of governmental inquiries working under the supervision of scientists nominated by the National Academy of Sciences and the Institute of Medicine. The Office of Research Integrity of the US Department of Health and Human Services concluded that the lead paper was “fraught with false and erroneous statements,” and that the “ORI believes that the careless and unacceptable keeping of research records...reflects irresponsible laboratory management that has permanently impaired the ability to retrace the important steps taken.”[5] Further, a Congressional Subcommittee on Oversight and Investigations led by US Representative John D. Dingell of Michigan produced a staff report on the papers which contains scathing criticisms of their integrity.[6]  Despite the publically available record of challenges to their veracity, these papers have remained uncorrected and continue to be part of the scientific record.  What prompts our communication today is the recent revelation of an astonishing number of previously unreported deletions and unjustified alterations made by Gallo to the lead paper. There are several documents originating from Gallo's laboratory that, while available for some time, have only recently been fully analyzed. These include a draft of the lead paper typewritten by Popovic which contains handwritten changes made to it by Gallo.[7] This draft was the key evidence used in the above described inquiries to establish that Gallo had concealed his laboratory's use of a cell culture sample (known as LAV) which it received from the Institut Pasteur.  These earlier inquiries verified that the typed manuscript draft was produced by Popovic who had carried out the recorded experiment while his laboratory chief, Gallo, was in Europe and that, upon his return, Gallo changed the document by hand a few days before it was submitted to Science on March 30, 1984. According to the ORI investigation, “Dr. Gallo systematically rewrote the manuscript for what would become a renowned LTCB [Gallo's laboratory at the National Cancer Institute] paper.”[5]  This document provided the important evidence that established the basis for awarding Dr. Luc Montagnier and Dr. Francoise Barré-Sinoussi the 2008 Nobel Prize in Medicine for the discovery of the AIDS virus by proving it was their samples of LAV that Popovic used in his key experiment. The draft reveals that Popovic had forthrightly admitted using the French samples of LAV renamed as Gallo's virus, HTLV-III, and that Gallo had deleted this admission, concealing their use of LAV.  However, it has not been previously reported that on page three of this same document Gallo had also deleted Popovic's unambiguous statement that, "Despite intensive research efforts, the causative agent of AIDS has not yet been identified,” replacing it in the published paper with a statement that said practically the opposite, namely, “That a retrovirus of the HTLV family might be an etiologic agent of AIDS was suggested by the findings.”  It is clear that the rest of Popovic's typed paper is entirely consistent with his statement that the cause of AIDS had not been found, despite his use of the French LAV. Popovic's final conclusion was that the culture he produced “provides the possibility” for detailed studies. He claimed to have achieved nothing more. At no point in his paper did Popovic attempt to prove that any virus caused AIDS, and it is evident that Gallo concealed these key elements in Popovic's experimental findings.  It is astonishing now to discover these unreported changes to such a seminal document. We can only assume that Gallo's alterations of Popovic's conclusions were not highlighted by earlier inquiries because the focus at the time was on establishing that the sample used by Gallo's lab came from Montagnier and was not independently collected by Gallo. In fact, the only attention paid to the deletions made by Gallo pertains to his effort to hide the identity of the sample. The questions of whether Gallo and Popovic's research proved that LAV or any other virus was the cause of AIDS were clearly not considered.  Related to these questions are other long overlooked documents that merit your attention. One of these is a letter from Dr. Matthew A. Gonda, then Head of the Electron Microscopy Laboratory at the National Cancer Institute, which is addressed to Popovic, copied to Gallo and dated just four days prior to Gallo's submission to Science.[8] In this letter, Gonda remarks on samples he had been sent for imaging because “Dr Gallo wanted these micrographs for publication because they contain HTLV.” He states, “I do not believe any of the particles photographed are of HTLV-I, II or III.” According to Gonda, one sample contained cellular debris, while another had no particles near the size of a retrovirus. Despite Gonda's clearly worded statement, Science published on May 4, 1984 papers attributed to Gallo et al with micrographs attributed to Gonda and described unequivocally as HTLV-III.  In another letter by Gallo, dated one day before he submitted his papers to Science, Gallo states, “It's extremely rare to find fresh cells [from AIDS patients] expressing the virus... cell culture seems to be necessary to induce virus,” a statement which raises the possibility he was working with a laboratory artifact. [9]  Included here are copies of these documents and links to the same. The very serious flaws they reveal in the preparation of the lead paper published in your journal in 1984 prompts our request that this paper be withdrawn. It appears that key experimental findings have been concealed. We further request that the three associated papers published on the same date also be withdrawn as they depend on the accuracy of this paper.  For the scientific record to be reliable, it is vital that papers shown to be flawed, or falsified be retracted. Because a very public record now exists showing that the Gallo papers drew unjustified conclusions, their withdrawal from Science is all the more important to maintain integrity. Future researchers must also understand they cannot rely on the 1984 Gallo papers for statements about HIV and AIDS, and all authors of papers that previously relied on this set of four papers should have the opportunity to consider whether their own conclusions are weakened by these revelations.      Gallo's handwritten revision, submitted without his colleague's knowledge despite multiple experiments that failed to support the new conclusion, was the sole foundation for the HIV=AIDS hypothesis. Had Science published the manuscript the way Popovic had typed it, there would be no AIDS “pandemic” - merely small clusters of people with AIDS. Without a viral hypothesis backing the development of expensive and deadly pharmaceuticals, would Fauci have allowed these patients to learn about the cure that existed all along?   Faced with a potential rebellion, Fauci marshaled the full resources under his control to squelch the publication of the investigations into Gallo and restrict any discussion of competing hypotheses in the scientific and mainstream press, which had been running virus-scare stories full-time since 1984. The effect was total, according to biochemist Dr. Kary Mullis, inventor of the polymerase chain reaction (PCR) procedure. In a 2009 interview, Mullis recalled his own shock when he attempted to unearth the experimental basis for the HIV=AIDS hypothesis. Despite his extensive inquiry into the literature, “there wasn't a scientific reference…[that] said ‘here's how come we know that HIV is the probable cause of AIDS.' There was nothing out there like that.”9 This yawning void at the core of HIV/AIDS “science" turned him into a strident critic of AIDS dogma - and those views made him persona non grata where the scientific press was concerned, suddenly unable to publish a single paper despite having won the Nobel Prize for his invention of the PCR test just weeks before.  10   DISSENT BECOMES “DENIAL”   While many of those who dissent from the orthodox HIV=AIDS view believe HIV plays a role in the development of AIDS, they point to lifestyle and other co-factors as being equally if not more important. Individuals who test positive for HIV can live for decades in perfect health - so long as they don't take AZT or the other toxic antivirals fast-tracked by Fauci's NIAID - but those who developed full-blown AIDS generally engaged in highly risky behaviors like extreme promiscuity and prodigious drug abuse, contracting STDs they took large quantities of antibiotics to treat, further running down their immune systems. While AIDS was largely portrayed as a “gay disease,” it was only the “fast track” gays, hooking up with dozens of partners nightly in sex marathons fueled by “poppers” (nitrate inhalants notorious for their own devastating effects on the immune system), who became sick. Kaposi's sarcoma, one of the original AIDS-defining conditions, was widespread among poppers-using gay men, but never appeared among IV drug users or hemophiliacs, the other two main risk groups during the early years of the epidemic. Even Robert Gallo himself, at a 1994 conference on poppers held by the National Institute on Drug Abuse, would admit that the previously-rare form of skin cancer surging among gay men was not primarily caused by HIV - and that it was immune stimulation, rather than suppression, that was likely responsible.11 Similarly, IV drug users are often riddled with opportunistic infections as their habit depresses the immune system and their focus on maintaining their addiction means that healthier habits - like good nutrition and even basic hygiene - fall by the wayside.    Supporting the call for revising the HIV=AIDS hypothesis to include co-factors is the fact that the mass heterosexual outbreaks long predicted by Fauci and his ilk in seemingly every country on Earth have failed to materialize, except - supposedly - in Africa, where the diagnostic standard for AIDS differs dramatically from those of the West. Given the prohibitively high cost of HIV testing for poor African nations, the WHO in 1985 crafted a diagnostic loophole that became known as the “Bangui definition,” allowing medical professionals to diagnose AIDS in the absence of a test using just clinical symptoms: high fever, persistent cough, at least 30 days of diarrhea, and the loss of 10% of one's body weight within two months. Often suffering from malnutrition and without access to clean drinking water, many of the inhabitants of sub-Saharan Africa fit the bill, especially when the WHO added tuberculosis to the list of AIDS-defining illnesses in 1993 - a move which may be responsible for as many as one half of African “AIDS” cases, according to journalist Christine Johnson. The WHO's former Chief of Global HIV Surveillance, James Chin, acknowledged their manipulation of statistics, but stressed that it was the entire AIDS industry - not just his organization - perpetrating the fraud. “There's the saying that, if you knew what sausages are made of, most people would hesitate to sort of eat them, because they wouldn't like what's in it. And if you knew how HIV/AIDS numbers are cooked, or made up, you would use them with extreme caution,” Chin told an interviewer in 2009.12   With infected numbers stubbornly remaining constant in the US despite Fauci's fearmongering projections of the looming heterosexually-transmitted plague, the CDC in 1993 broadened its definition of AIDS to include asymptomatic (that is, healthy) HIV-positive people with low T-cell counts - an absurd criteria given that an individual's T-cell count can fluctuate by hundreds within a single day. As a result, the number of “AIDS cases” in the US immediately doubled. Supervised by Fauci, the NIAID had been quietly piling on diseases into the “AIDS-related” category for years, bloating the list from just two conditions - pneumocystis carinii pneumonia and Kaposi's sarcoma - to 30 so fast it raised eyebrows among some of science's leading lights. Deeming the entire process “bizarre” and unprecedented, Kary Mullis wondered aloud why no one had called the AIDS establishment out: “There's something wrong here. And it's got to be financial.”13   Indeed, an early CDC public relations campaign was exposed by the Wall Street Journal in 1987 as having deliberately mischaracterized AIDS as a threat to the entire population so as to garner increased public and private funding for what was very much a niche issue, with the risk to average heterosexuals from a single act of sex “smaller than the risk of ever getting hit by lightning.” Ironically, the ads, which sought to humanize AIDS patients in an era when few Americans knew anyone with the disease and more than half the adult population thought infected people should be forced to carry cards warning of their status, could be seen as a reaction to the fear tactics deployed by Fauci early on.14   It's hard to tell where fraud ends and incompetence begins with Gallo's HIV antibody test. Much like Covid-19 would become a “pandemic of testing,” with murder victims and motorcycle crashes lumped into “Covid deaths” thanks to over-sensitized PCR tests that yielded as many as 90% false positives,15 HIV testing is fraught with false positives - and unlike with Covid-19, most people who hear they are HIV-positive still believe they are receiving a death sentence. Due to the difficulty of isolating HIV itself from human samples, the most common diagnostic tests, ELISA and the Western Blot, are designed to detect not the virus but antibodies to it, upending the traditional medical understanding that the presence of antibodies indicates only exposure - and often that the body has actually vanquished the pathogen. Patients are known to test positive for HIV antibodies in the absence of the virus due to at least 70 other conditions, including hepatitis, lupus, rheumatoid arthritis, syphilis, recent vaccination or even pregnancy. (https://www.chcfl.org/diseases-that-can-cause-a-false-positive-hiv-test/) Positive results are often followed up with a PCR “viral load” test, even though the inventor of the PCR technique Kary Mullis famously condemned its misuse as a tool for diagnosing infection. Packaging inserts for all three tests warn the user that they cannot be reliably used to diagnose HIV.16 The ELISA HIV antibody test explicitly states: “At present there is no recognized standard for establishing the presence and absence of HIV antibody in human blood.”17   That the public remains largely unaware of these and other massive holes in the supposedly airtight HIV=AIDS=DEATH paradigm is a testament to Fauci's multi-layered control of the press. Like the writers of the Great Barrington Declaration and other Covid-19 dissidents, scientists who question HIV/AIDS dogma have been brutally punished for their heresy, no matter how prestigious their prior standing in the field and no matter how much evidence they have for their own claims. In 1987, the year the FDA's approval of AZT made AIDS the most profitable epidemic yet (a dubious designation Covid-19 has since surpassed), Fauci made it clearer than ever that scientific inquiry and debate - the basis of the scientific method - would no longer be welcome in the American public health sector, eliminating retrovirologist Peter Duesberg, then one of the most prominent opponents of the HIV=AIDS hypothesis, from the scientific conversation with a professional disemboweling that would make a cartel hitman blush. Duesberg had just eviscerated Gallo's 1984 HIV paper with an article of his own in the journal Cancer Research, pointing out that retroviruses had never before been found to cause a single disease in humans - let alone 30 AIDS-defining diseases. Rather than allow Gallo or any of the other scientists in his camp to respond to the challenge, Fauci waged a scorched-earth campaign against Duesberg, who had until then been one of the most highly regarded researchers in his field. Every research grant he requested was denied; every media appearance was canceled or preempted. The University of California at Berkeley, unable to fully fire him due to tenure, took away his lab, his graduate students, and the rest of his funding. The few colleagues who dared speak up for him in public were also attacked, while enemies and opportunists were encouraged to slander Duesberg at the conferences he was barred from attending and in the journals that would no longer publish his replies. When Duesberg was summoned to the White House later that year by then-President Ronald Reagan to debate Fauci on the origins of AIDS, Fauci convinced the president to cancel, allegedly pulling rank on the Commander-in-Chief with an accusation that the “White House was interfering in scientific matters that belonged to the NIH and the Office of Science and Technology Assessment.” After seven years of this treatment, Duesberg was contacted by NIH official Stephen O'Brien and offered an escape from professional purgatory. He could have “everything back,” he was told, and shown a manuscript of a scientific paper - apparently commissioned by the editor of the journal Nature - “HIV Causes AIDS: Koch's Postulates Fulfilled” with his own name listed alongside O'Brien's as an author.18 His refusal to take the bribe effectively guaranteed the epithet “AIDS denier” will appear on his tombstone. The character assassination of Duesberg became a template that would be deployed to great effectiveness wherever Fauci encountered dissent - never debate, only demonize, deplatform and destroy.    Even Luc Montagnier, the real discoverer of HIV, soon found himself on the wrong side of the Fauci machine. With his 1990 declaration that “the HIV virus [by itself] is harmless and passive, a benign virus,” Montagnier began distancing himself from Gallo's fraud, effectively placing a target on his own back. In a 1995 interview, he elaborated: “four factors that have come together to account for the sudden epidemic [of AIDS]: HIV presence, immune hyper-activation, increased sexually transmitted disease incidence, sexual behavior changes and other behavioral changes” such as drug use, poor nutrition and stress - all of which he said had to occur “essentially simultaneously” for HIV to be transmitted, creating the modern epidemic. Like the professionals at the Tri-State Healing Center, Montagnier advocated for the use of antioxidants like vitamin C and N-acetyl cysteine, naming oxidative stress as a critical factor in the progression from HIV to AIDS.19 When Montagnier died in 2022, Fauci's media mouthpieces sneered that the scientist (who was awarded the Nobel Prize in 2008 for his discovery of HIV, despite his flagging faith in that discovery's significance) “started espousing views devoid of a scientific basis” in the late 2000s, leading him to be “shunned by the scientific community.”20 In a particularly egregious jab, the Washington Post's obit sings the praises of Robert Gallo, implying it was the American scientist who really should have won the Nobel for HIV, while dismissing as “

In this episode, we review the high-yield topic of⁠ ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠Western Blot⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠from the Immunology section. Follow ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠Medbullets⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ on social media: Facebook: www.facebook.com/medbullets Instagram: www.instagram.com/medbulletsofficial Twitter: www.twitter.com/medbullets

Chiropractor Kim Bruno furthered her training with the Institute of Functional Medicine, ILADS, Horowitz Lyme Master Classes and holds a board-certification as a Certified Clinical Nutritionist. She owned a private practice for 17 years and was the functional medicine medical director for the largest immunology clinic in Colorado. She comes to us today as the Senior Medical Science liaison for Vibrant Wellness Labs. Today we discuss their panel of 48 neurologically-oriented antibodies: the Neural Zoomer Plus. We start by discussing the lab technology itself, which is somewhat unique in the testing world- it's an 'Immunochip', also called a protein-peptide microarray as viewed through chemiluminescence which can be more sensitive than historical Eliza testing. The sensitivity (the ability to find the needle in the haystack) ranges from 95-98% and the specificity (it's definitely a needle, and not a pin or nail or something similar that is not actually a needle) is 96-100%. The range is because each analyte has its own metrics. Here, we take a tangent into describing the limitations of Eliza & Western Blot testing, especially in light of tick-borne testing for Lyme disease & co-infections. Then we touch on PCR- polymerase chain reaction testing and the use of glass beads to break up biofilms in test samples for even more accurate results.   Our next chapter (around 14:30) focuses on the immune system itself. Listen in for some helpful analogies for the immunoglobulins-IgM for ‘marines vs IgG for ‘ground troops', IgA with affiliation with mucus membranes like the gut or respiratory linings, and IgE for anaphylactic allergic reaction. This gives the total pool from which the Neural Zoomer Plus antibodies are pulling from as a sort of clinical calibration to weigh the presence of the specific antibodies.   At 22:20, we dive into the Neural Zoomer Plus test itself. Dr Bruno shares her brilliant ‘hierarchy of consideration' for putting these antibodies into a context. While she states outright ‘this is not a diagnostic test', the larger truth is that this test cannot be used for diagnosis by clinicians who don't have the scope to make diagnostic conclusions, for example dieticians or health coaches. For our purposes at Neuroveda Health, we absolutely use this test for clinical decision making and diagnosis. Dr Bruno calls out molecular mimicry against pathogens or even foods or toxins that can confuse the immune system. We consider the Cunningham Panel (recently renamed the Autoimmune Brain Panel), which has been used longer for PANDAS evaluation. And we walk through each category of antibody included on this test.   We finish with a discussion about treatment approaches based on results from this test, including the Neuroveda Health approach to evaluating and addressing neuroimmune disease. FOR MORE INFORMATION: To look at a sample report of this test: https://hello.vibrant-wellness.com/hubfs/Sample%20Reports/MK-0072-01NeuralZoomerPlusSampleReport.pdf To find out more about the Neural Zoomer Plus test: https://www.vibrant-wellness.com/test/NeuralZoomerPlus To get testing, contact us to schedule an appointment with a clinician at Neuroveda Health: - Phone: 206-379-1213 - Email Reception@neurovedahealth.com To find out more about our clinic (and request a call back): https://www.neurovedahealth.com/

For more information, visit https://thecirsgroup.com   CIRS, or Chronic Inflammatory Response Syndrome, can sometimes be “followed” or accompanied by co-infections. Today, we are diving into one of the most convoluted and confusing potential avenues CIRS patients might need to address: Tick-borne illnesses, of which there are many and not just Lyme disease! We are honored to have the leading expert in tick-borne illnesses with us today to help break it down: Dr. Peg DeTulio, who has decades of experience with 1000s of patients. Did you know it's possible 20-50% of people with CIRS are also struggling with a tick-borne illness? If your healing journey has stalled, this may be a rabbit hole worth investigating. For more information, support, and resources in your own CIRS healing journey, visit TheCIRSGroup.com   TIME STAMPS: 0:00 Intro and disclaimer 3:00 Tick-Borne illnesses vs Lyme 4:55 Symptoms of tick-borne illness 6:28 For CIRS patients: treat mold before you address tick-borne 9:38 Red flag: Improvements but some symptoms remain 11:20 Western Blot test after CIRS is treated 12:42 C3a and tick borne illness 14:36 Testing process for tick-borne illnesses 19:05 Night Sweats that do not resolve 20:15 Air hunger or sighing at rest 21:08 Using an herb before testing to wake up the illness 22:28 Endemic areas and other risk factors 27:36 How common is tick-borne illness within the CIRS community 28:33 Can GENIE or NeuroQuant rule out Lyme or tick-borne illness 31:58 How treatable are tick-borne illnesses? 35:42 The benefits of treating slowly and carefully 40:30 How to spread awareness of tick-borne illness HELPFUL LINKS: Dr. Peg Detulio's website: https://regenixhealing.com/ Dr. Peg's interview with Judy Cho: https://youtu.be/bjc2twvHGFs?si=9aJyIVGHmUp8Zj2r   The CIRS Group: Support Community: https://thecirsgroup.com Instagram: https://www.instagram.com/thecirsgroup/   Find Jacie for carnivore, lifestyle and limbic resources: Instagram: https://www.instagram.com/ladycarnivory YouTube: https://www.youtube.com/@LadyCarnivory Blog: https://www.ladycarnivory.com/   Find Barbara for business/finance tips and coaching: Website: https://www.actlikebarbara.com/ Instagram: https://www.instagram.com/actlikebarbara/ YouTube: https://www.youtube.com/@actlikebarbara   Jacie is a 4 year carnivore, certified nutrition coach, and carnivore recipe developer determined to share the life changing information of carnivore and CIRS to anyone who will listen. Barbara is a coach, facilitator, speaker, 3 year carnivore, and a big fan of health and freedom. Together, they co-founded The CIRS Group, an online support community to help people that are struggling with their CIRS diagnosis and treatment.  

“Your immune system is the best tool to combat Lyme disease. Nourish, support, and cleanse it so it functions optimally.”Do you feel like your symptoms of persistent fatigue, brain fog, or unexplained joint pain are being ignored or dismissed? You're not alone. Many Lyme warriors fight the same battle, and it can be frustrating and isolating. But there is hope.Dr. Jeff Wright, a Lyme-literate doctor and chronic illness expert, joins us today to shed light on this complex condition. Together, we explore what causes these persistent symptoms, clarify testing methods, and dispel the myth that Lyme is incurable. If you or a loved one are disheartened by chronic symptoms, this conversation provides validation and answers. Tune in to Episode 3 of Season 3 today! Show Highlights: 00:00 - Episode Start06:26 - What is this mysterious illness, and how did it get its name?09:34 - Discover the key to conquering Lyme disease: a healthy immune system11:36 - Explore the different ways Lyme disease can manifest itself13:59 - Learn about the various strains and complexities of Lyme16:09 - Are long-term antibiotics actually making it worse? Weigh the pros and cons21:12 - What factors create the ideal environment for Lyme disease to thrive?32:12 - Dive into the world of conventional and non-conventional testing for Lyme disease43:34 - Explore additional tests to identify hidden infections that could be impacting your health47:36 - Discover Dr. Wright's favorite therapies and treatment options for Lyme disease 52:25 - Find out why Juanique raves about Ultraviolet Blood Irradiation Therapy (UVBI)53:38 - What makes the HOCATT infrared ozone sauna so effective for detoxification?54:16 - Learn about additional helpful therapies such as high-dose vitamin C, IV vitamins, neurofeedback, dietary protocols56:33 -  Discover the importance of sleep, emotional well-being, environment, and detoxification as the foundations of healthImportant Links: Gutsy Health Podcast IG - https://www.instagram.com/gutsyhealthpodcast/Gutsy Health Academy - https://www.mygutsyhealth.com/Get access to more resources on mold - https://www.mygutsyhealth.com/moldHow to Heal from Mold Toxicity with Dr. Jeff Wright - https://pod.link/1474697743/episode/edaf80b960e9b38faf183f6ffc4a8406Feel free to reach out to ProvoHealth and schedule a consultation with Dr. Jeff Wright by calling 801-691-1765

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551674v1?rss=1 Authors: Omondi, C., Chou, A., Fond, K. A., Morioka, K., Joseph, N. R., Sacramento, J. A., Lorio, E., Torres-Espin, A., Radabaugh, H. L., Davis, J. A., Gumbel, J. H., Huie, J. R., Ferguson, A. R. Abstract: Western blot is a popular biomolecular analysis method for measuring the relative quantities of independent proteins in complex biological samples. However, variability in quantitative western blot data analysis poses a challenge in designing reproducible experiments. The lack of rigorous quantitative approaches in current western blot statistical methodology may result in irreproducible inferences. Here we describe best practices for the design and analysis of western blot experiments, with examples and demonstrations of how different analytical approaches can lead to widely varying outcomes. To facilitate best practices, we have developed the blotRig tool for designing and analyzing western blot experiments to improve their rigor and reproducibility. The blotRig application includes functions for counterbalancing experimental design by lane position, batch management across gels, and analytics with covariates and random effects. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Teste rápido, Sorologia, Carga Viral, Western Blot... são tantas as possibilidades de se realizar diagnóstico de HIV que a gente acaba ficando confuso! Nesse episódio, Mafê e Klinger irão discutir o diagnóstico da fase crônica do HIV e seus fluxogramas. Vem com a gente!

It's been 40 years since extracellular vesicles, or exosomes, were first connected to the way cells communicate and transfer information. Since then, researchers have studied what part they play in the normal physiology of a cell. There has also been an expanding amount of work in the role that exosomes can play in therapeutics and diagnostics of a wide range of diseases.  Jim West, CEO of Clara Biotech, joins us to share his perspective on research and innovation using exosomes as well as life in a biotech startup. He provides great insights into where this technology is today, the challenges being experienced in the field, and the potential and opportunity that the field of applied exosome research represents in the future. Tune in today. Subscribe to get future episodes. Share with a friend or colleague that might enjoy too! Visit https://thermofisher.com/molbioschool to access molecular biology resources and educational content. Experience the Speaking of Mol Bio podcast in its extended video format for a more immersive journey, while also ensuring accessibility with downloadable transcripts for each episode.Watch now at thermofisher.com/podcast-video

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536625v1?rss=1 Authors: Huckleberry, K. A., Calitri, R., Li, A. J., Mejdell, M., Singh, A., Bhutani, V., Laine, M. A., Nastase, A. S., Morena, M., Hill, M., Shansky, R. M. Abstract: Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, and here we asked specifically whether the endocannabinoid (eCB) system could modulate Pavlovian fear conditioning in a sex-dependent manner. Systemic (i.p.) injection of CB1R antagonist AM251 in adult male and female Sprague Dawley rats prior to auditory cued fear conditioning produced a female-specific increase in freezing that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. Notably, AM251 also produced robust freezing in a novel context prior to auditory cue presentation the day following drug administration, but not the day of, suggesting that CB1R blockade elicited contextual fear generalization in females. To identify a potential synaptic mechanism for these sex differences, we next used liquid chromatography/tandem mass spectrometry, Western Blot, and confocal-assisted immunofluorescence techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, respectively, focusing on the ventral hippocampus (vHip). Fear conditioning elicited increased vHip AEA levels in females only, and in both sexes, CB1R expression around vHip efferents targeting the basolateral amygdala (BLA) was twice that at neighboring vHip neurons. Finally, quantification of the vHip-BLA projections themselves revealed that females have over twice the number of neurons in this pathway that males do. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Acompaña a Ricardo Cartas en una emisión más de la revista cultural De eso se trata, espacio de ciencia, de cultura, de gastronomía, de libros y más, de lunes a viernes de 08:30 a 10:00 horas. En El cambiante mundo de las infecciones, la Dra. Indiana Torres Escobar, académica de la Facultad de Medicina, reflexiona sobre el Día mundial de la lucha contra el SIDA, enfermedad causada por el VIH y que cuenta ya con 38 millones de pacientes reportados a nivel mundial, aunque se cree que son muchos más, por lo que existe un tratado internacional que busca detenerlo antes del año 2030. En México el número de infectados por transmisión perinatal aumenta, por lo que te invitamos a protegerte durante la actividad sexual, a usar condón, y a realizarte una prueba de diagnóstico, como ELISA o Western Blot.

Lauren Murphree is a 29-year-old certified IV technician from Tennessee. She's also the author of Silent Suffering: Finding God's Faithfulness in Chronic Lyme Disease. Ms. Murphree was flourishing and working full time. She was an athlete and was very social. Ms. Murphree was first diagnosed with Lyme disease when she was 9 years old, but it didn't “wreak havoc” in her life until she was around 20 years old. Some of Ms. Murphree's symptoms have included neuropathy, neck and back pain, brain inflammation, loss of energy, jaw pain, tremors, sharp shooting pains in her head and body, heart palpitations, body weakness and numbness, cognitive dysfunction, arthritis, heat intolerance, memory loss, insomnia, chest pain, panic attacks, Lyme rage, depression, anxiety, loss of appetite, nausea, light and sound sensitivity, POTS, heart and lung weakness, muscle spasms, tinnitus, exercise intolerance, dizziness, and more. Ms. Murphree's Lyme relapse wasn't diagnosed until her early twenties after two years of suffering. She was finally diagnosed with a Lyme relapse through a Western Blot blood test and an IGeneX specialty test. Ms. Murphree was treated with Low Dose Naltrexone (LDN), Disulfiram, Low Dose Immunotherapy (LDI), Bee Venom Therapy (BVT), Cowden Protocol (Nutramedix), various antibiotics, rife, ozone, and more. If you'd like to learn how a young woman has used an arsenal of treatments to treat chronic Lyme disease and wrote a book about her experiences to help the millions of people suffering with this horrible disease, then tune in now! PS Michelle McKeon, the Lyme Specialist, special guest co-hosted this interview with Matt from Tick Boot Camp!

Jessica Smith is a 30-year-old local director with Child Evangelism Fellowship, a Bible and Theology student at Shasta Bible College, and a student at Trinity School of Natural Health. In 2014, Ms. Smith moved from Oregon to Missouri to attend ministry vocational school. Despite growing up on a farm with a general awareness of ticks and Lyme disease, she was not taught to take steps to avoid or check for ticks. While taking a hike on the 600-acre school campus, she discovered a small tick on her arm. Shortly thereafter, she exhibited acute Lyme disease symptoms such as fatigue, flu symptoms and brain fog. Her acute symptoms dissipated in a few weeks and she “did not notice any decline in her health” until 3 years later when she again suffered flu symptoms, lost an “alarming amount of weight,” followed by severe joint pain, mental struggles, and heart palpitations. The debilitating symptoms sent her to a Naturopathic doctor who diagnosed her with Lyme disease after she tested CDC positive on a Western Blot blood test. For the next several years, she battled through a grief cycle and both sides of the pride and imposter syndrome coin before learning to become a good steward of her life and health. Today, she is “105%” healthier than before she suffered the tick bite. If you would like to learn more about how chronic Lyme disease sent a minister on a “New Path” for healing and a school to study natural health, then tune in now!

Ally Lopes is a 24-year-old National Board-Certified Health and Wellness Coach (NBC-HWC) and kindergarten teacher from the New Jersey Lyme belt. Ms. Lopes' Lyme disease journey began with severe vertigo and flu-like symptoms the day before she flew to Walt Disney World for her high school senior class trip. She was initially diagnosed with a stomach virus and a sinus infection. During the following 2 years, her symptoms progressed forcing her to seek treatment from so many doctors she finally lost count. She was finally diagnosed with Lyme disease using a Western Blot blood test by the doctor that diagnosed her mother with Lyme disease one year earlier. Ms. Lopes treated with her mother's practitioner for 1 year and then decided to take responsibility for building her own treatment team. Blazing her own trail introduced her to eastern and western treatment tools and practitioners resulting in Ms. Lopes making sufficient health gains to allow her to complete her undergraduate degree in elementary education. Inspired by her health journey and a desire to assist others overcome chronic Lyme disease, Ms. Lopes studied Wellness Coaching at the prestigious Mayo Clinic School of Continuous Professional Development. If you would like to learn more about how Lyme disease inspired an elementary school teacher to diversify her professional skills to bring her lessons from her personal journey to other people battling Lyme disease, then tune in now!

Sam Lesch is a 20-year-old student originally from Auburn, New York currently residing in North Carolina's Outer Banks. Her Lyme disease journey began during her senior year of high school when she suffered migrating symptoms that included the loss of the use of her right leg, the use of her hand and fingers, brain fog, fatigue, and facial drops. Although suffering from classic migrating Lyme disease symptoms, the confirmation of her family's suspicion that she was suffering from Lyme was delayed due to the failure of the standard Western Blot testing. Ms. Lesch tested negative for Lyme on 4 separate LabCorp Western Blot tests. Finally, a family friend recommended a superior DNA ConneXions (Tick Boot Camp Podcast episode 168) urine test and her diagnosis of Lyme disease and co-infections was confirmed. After her diagnosis, Ms. Lesch's parents and siblings studied Lyme through course work and built a team of medical professionals to treat her disease. The treatment team included a kinesiologist and an out-of-state Lyme Literate Medical Doctor (LLMD). Ms. Lesch's age and illness forced her to rely on her family and medical team to establish a treatment plan that included 5 antibiotics, probiotics, hyperbaric oxygen therapy, IV Meyers' Cocktail, yoga, and glutathione. Her treatment protocol was so rigorous that she began to feel what she called “pill fatigue.” If you would like to learn more about how a young woman on the cusp of adulthood refused to allow Lyme disease to prevent her from transitioning to college and independence, then tune in now!

Love it or hate it, western blotting forms the bedrock of countless studies across numerous disciplines. Explore its history, development and applications in this episode all about the marmite of life science techniques. Guiding me through the hand wringing, hair follicle destroying history and process of western blotting, and hopefully explaining the beauty and potential of the technique - is Kenneth Oh, Senior Project Manager at Bio-Rad Laboratories. Kenneth reveals some of the latest developments in western blotting. Revealing how researchers are now able to validate the success of each stage of a western blot and work with smaller samples, Kenneth provides key tips for ensuring each of your western blots is a triumph. Contents: Intro: 00:00-01:15 History of western blotting: 01:15-3:50 What makes western blotting so tricky? 03:50-05:30 Stepwise optimization: 05:30-06:50 Stain-free western blotting: 06:50-08:50 The different variants of western blotting: 08:50-10:20 The right blot for the right application: 10:20-11:30 The most exciting developments in western bloting:11:30-13:35 New horizons for western blotting: 13:35-15:25 The impact of multiplex-western blotting: 15:25-16:30 Tips for best practice: 16:30-18:10 The future of western blotting: 18:10-20:30 Yearning for automation: 20:30-21:30

Madeline Castellanos is a 9-year-old actor, student and author from Los Angeles, California. Her Lyme disease journey began 4 years ago when her mother, Alexandra, took her to an urgent care facility when she noticed the child had an “alarming rash." After being dismissed by the urgent care doctor, the formerly energetic Madeline began to nap frequently, fall asleep during car trips, suffer daily migraine headaches, and fall behind her classmates academically in school. The developing symptoms and a new “bullseye” rash caused her mother to schedule Madeline for an examination by her primary care physician. Madeline's primary care doctor diagnosed her with Lyme disease after she tested positive on a Western Blot test. Sadly, Madeline's doctor acknowledged that he did not have the experience or training to adequately treat her disease, so he urged Alexandra “find a good doctor” for her daughter. Alexandra's quest to find a “good doctor” required the pair to travel across the country from California to the birthplace of Lyme disease: Connecticut. There, Madeline treated with the famous Dr Jones of New Haven Connecticut for “10 months nonstop” before she achieved symptom relief. Today, Alexandra helps Madeline to prevent symptom flare ups through diet, limiting direct sun exposure, and avoiding stressful situations. The Castellanos family's Lyme journey taught the mother and daughter many powerful lessons that they wanted to share with other parents and children. Their first outreach project was to co-author a children's book about a young child's Lyme disease journey titled “Mylie's Lyme Story.” Mylie's Lyme Story is now available wherever books are sold including Amazon and ironically Target. If you would like to know more about how Lyme disease inspired a mother and child to share their journey through a child friendly medium, then tune in now!

AN EYEWITNESS ACCOUNT Of Gross Irregularities and Medical Incompetence in the Early Clinical Trials of AZT By Lynn Gannett 2000 Please keep in mind that these two write-ups represent only the "tip of the iceberg," and for everything that is recorded here, there are literally dozens of other examples that I could give. My information is NOT included in John Lauritsen's book on AZT, simply because neither of us had heard of each other at that time (although I wish we had!). His book documents information related to the Phase II studies of AZT, whereas I was involved with the Phase III studies (ACTG 019). But it was all the same kinds of shocking nonsense, incompetence and fraud. In addition to ACTG 019 (which was the major study at that time), I also worked on other studies of AZT and ddI (ACTG 002, 016, 020, 081, 116, 117 and 118). Some of these studies included other drugs, such as pentamidine. I was somewhat young (and naive) at the time, and didn't quite know what to make of what I was witnessing, or what to do with the information that I had documented, having NEVER witnessed anything even remotely similar. In fact, at that point in my life, I didn't even realize that human beings were even CAPABLE of this kind of corrupt, insane, self-motivated behavior. If I had known then what I know now, I would have been much more persistent in my attempts to report this information to the NIH. I don't even have a single letter – my communication with them was done entirely over the phone. Which means they could simply deny that these phone conversations ever took place. All I have to offer as evidence that I DID REPORT THIS TO THE NIH AND THEY DELIBERATELY IGNORED MY INFORMATION is my personal testimony, and some handwritten notes that I took at the time (this was in the Spring of 1990). I may be able to locate at least one witness who could corroborate my story (the NIH site monitor for Syracuse). But the way that I look at this is that it's never too late to report this to the NIH, AGAIN. And the important thing is that I still have ALL of my original documents (an entire box full of material) which I tried to share with them in 1990. I have precise names, patient numbers, dates, lab values, memos, etc. I KNOW that my information is accurate, because I was so thorough and meticulous (not to mention HONEST) in my record-keeping. That's why they hired me – I'm an extremely detail-oriented, "numbers" person. Thus, all of my information could, in theory, be verified by referring to the original research forms and NIH documents from that time period, which must still exist and (theoretically) could be obtained through the Freedom of Information Act. Ideally, my second attempts to report this could lead to a Congressional Investigation. That is certainly what I will be calling for. The seriousness of my allegations certainly warrants such an investigation. For the NIH to knowingly ignore serious and credible DOCUMENTED reports of gross scientific misconduct coming from someone working on the INSIDE of these trials – if that doesn't constitute scientific "fraud," I don't know what does. Especially when we look at the real-life gruesome outcome of this deliberate refusal to even INVESTIGATE my allegations to see if there was any merit to them (along with all of the other glaringly obvious, telltale signs and warnings coming from around the globe, which were also ignored at that time) – all of this intentional "blindness" on the part of the NIH, the FDA, Burroughs Wellcome, etc., led to the unnecessary and unimaginable suffering of countless individuals (of both "HIV+" people and their loved ones), and the unnecessary deaths of (in my view) tens of thousands of people. This dreadful holocaust continues to this day. Parks Mankahlana, couldn't have summed it up better when he said "...the profit takers who are benefiting from the scourge of HIV/AIDS will disappear to the affluent beaches of the world to enjoy wealth accumulated from humankind ravaged by a dreaded disease." Knowing what I know "first hand" about AZT, and knowing how it should NEVER have received FDA approval under ANY circumstances, this is one of many reasons why I am especially grateful to the members of the Perth group, Peter Duesberg, John Lauritsen, Celia Farber, Anthony Brink and many others for the outstanding work that they have done in documenting the case against AZT. *** I was an eyewitness (and later a whistle-blower) to gross negligence and fraud in the Phase III clinical trials of AZT (1987 to 1990). I've been saying to people for years that AZT was NEVER proven to be safe or effective. From the particular studies in which I was involved, it would have been impossible to prove anything. The data was such a mess! I now realize that AZT is a deadly poison. All "AIDS drug" trials since that time have been based on the same flawed model. The big difference is that now there is even LESS meaningful oversight, and even MORE of an economic incentive for physicians to enroll patients. And recent drug trials have also been characterized by an absurdly brief follow-up period (24 or 36 weeks, for example), and effectiveness is often said to be determined by "surrogate markers" which have never been proven to relate to actual clinical health and/or increased survival. But, the early AZT studies were like the big "granddaddy" of all of this ensuing insanity! I did not know John Lauritsen at the time that he wrote his book, AZT: Poison by Prescription (1990), but I later told him that, if I had known him at that time, I could have given him several additional chapters for his book! In this book he meticulously documents serious fraud which took place at other participating hospitals (I was in Syracuse, NY), particularly at a hospital in Boston. When I first read John's book, it was like reading my own autobiography! Talk about deja vu! In spite of what I witnessed, I was not aware, however, of the deeper problems within "AIDS science" until many years later. It was in 1997 that I first heard about the views of the "AIDS dissidents." After educating myself regarding the many unexplained and nonscientific paradoxes and absurdities of the orthodox "HIV/AIDS" model, and after studying the alternative views proposed by the various "AIDS dissidents," I started doing public speaking on this topic. I especially want to share my story with as many people as possible about the fraud which I witnessed firsthand in the early AZT trials. I always tell people that if the general public knew what I knew about AZT, this so-called "drug" would be banned immediately. *** My name is Lynn Gannett. As the Data Manager for the first two years and seven months of the NIH-sponsored AIDS clinical trials conducted at the Syracuse, New York, clinic (September 1987 to March 1990), my belief is that the data which came from the Syracuse site is ABSOLUTELY WORTHLESS! I would NEVER trust my health or my life to the results of this so-called "research" or in the hands of these so-called "medical professionals." I can only speculate that if these things occurred at this site, that similar things may have and in all likelihood did occur at the other participating sites. The things that I witnessed at this clinic would HORRIFY any reasonable observer. The level of medical incompetence, unprofessionalism, unethical, dishonest, corrupt, illegal and immoral behavior was shocking and inexcusable. The data was so inaccurate and so full of holes that I often compare it to Swiss cheese. I felt like I was trapped in the middle of an awful movie about "mad scientists." If there was a "rule" that could be broken - they broke it! The following examples outline some of the most egregious examples of what I witnessed: * The Principal Investigator, an MD, and the Study Coordinator, an RN, showed a huge interest in enrolling as many patients as possible on studies (which would entitle them to more money and perceived prestige) and showed little interest in the research itself - specifically in the integrity, accuracy and completeness of the data. * The Study Coordinator (and other medical staff responsible for study patients) often displayed a significant lack of understanding and unfamiliarity with the study protocols and important memos concerning their implementation - as though she had not even read them, or had totally misunderstood and misinterpreted them, even in instances pertaining to terminology and procedures FUNDAMENTAL to the protocol itself, often months after the study had been underway. e.g. In what I consider to have been the most serious, disturbing and grossly incompetent situation that I witnessed, which came dangerously close to resulting in death, and unquestionably resulted in extreme unnecessary suffering, PID #110434, a black, obese, diabetic, HIV+ female with a history of serious heart problems, experienced severe hematologic toxicity from the 081 study drug (AZT), which had progressed to a GRADE IV toxicity by week 24 and resulted in her coming into the emergency room with "severe shortness of breath, fatigue and weakness," and required her to be hospitalized for a total of five days. BECAUSE NO ONE, DOCTOR OR NURSE, SHOWED ANY RESPONSIBILITY FOR, KNOWLEDGE OF, INTEREST IN OR RECOGNITION OF THE IMPORTANCE OF THE EXPLICITLY-DEFINED TOXICITY (ADVERSE REACTION) AND DOSE MANAGEMENT STEPS AND PROCEDURES OUTLINED IN THE PROTOCOL, instead of being taken OFF the AZT due to the Grade IV toxicity, her dose was reduced from 1000 mg/day to 500 mg/day, in complete violation of protocol requirements which explicitly require DISCONTINUATION of study drug. Additionally, early Grade I and Grade II toxicities should have indicated the need for interim lab monitoring of Hemoglobin, especially with this patient's complicated medical history, but instead this patient had NO lab work performed between week 20 (13DEC89) and week 24 (10JAN90), even though she was in for a pentamidine treatment on 20DEC89 and could have easily had a blood sample drawn. At some point during this time interval, her original medical chart was "lost," never to be found again, requiring a new chart to be made up, which subsequently obviously lacked significant information concerning her medical history. I was shocked, outraged and horrified when this whole situation occurred, and documented this gross medical incompetence and blatant violation of protocol requirements as carefully as I could because I wanted everything to be ON RECORD so that no one could later deny it or cover it up. (See attached memo dated 01FEB90 and lab flow chart showing toxicity progression.) * Patients were ROUTINELY enrolled who failed to meet eligibility requirements, especially when it came to specific required lab values and test ranges (i.e., within the required number of days prior to enrollment). There are many, many examples. Below are a few (also see attached 05OCT89 list of 081 screening lab eligibility failures): e.g. In the most blatant example, PID #110264C, a female partner of an HIV+ male, was enrolled on study 019 and took study drug for THREE weeks before it was discovered that she was actually HIV NEGATIVE! (The Elisa came back as a false positive but the Western Blot came back negative.) The test results date predated her enrollment on study date. She was also on oral contraceptives at the time, another eligibility violation. E.g. You might think that this would be the last time a patient would be enrolled without an HIV+ test result "in hand" - not so. PID #110316's HIV+ test results are dated 06JAN89, ONE MONTH AFTER his enrollment date (05DEC88)! e.g. Other patients had been routinely enrolled without HIV+ test results documentation available, often based simply on referrals from other physicians. An example is PID #110153H, a referral patient from Binghamton who, to my knowledge, has no HIV+ test results in his chart TO THIS DAY! After a year or two of my repeated requests to the Study Coordinator for this information, she finally obtained a LETTER ONLY from the referring physician "verifying" his HIV+ status, who also apparently had no supporting documentation. * There were COUNTLESS unreported (meaning unreported on the research forms) diagnoses, opportunistic infections, symptoms, concomitant medications, and adverse reactions. Except for "symptoms" (which were asked for at every visit), these significant things (which each REQUIRE reporting) should have been reported on one of several "as needed" or optional case report forms used to track this information - an extra step which was rarely taken. I often observed, as did the RTI (Research Triangle Institute) site monitors, this unreported information recorded or mentioned in the patient's regular medical chart. E.g. From the 22JUN89 site monitor's report: "Of the six protocol 002 charts which were reviewed for the first critical event verification, four reported death as the first event even though at least one OI [opportunistic infection] has preceded the death. These OI's were not reported. In another instance, it was impossible to determine what had happened to the patient between the time of randomization and death because the records were missing." * Incorrect lab tests were ROUTINELY ordered (either required labs omitted or unrequired labs ordered by mistake), and the wrong prescriptions were ROUTINELY written (for example, 1200 mg/day instead of 1000 mg/day). When I questioned these and other similar mistakes, I would be chastised by the Principal Investigator and/or Study Coordinator for being too "nit picky" or for inappropriately questioning someone's medical "expertise" since I did not have (nor did my position require) a medical degree. * Medical lab results were ROUTINELY transcribed incorrectly onto the research forms by the Study Coordinator (who I suspect may have dyslexia - at the very least, she does not have the "detail-oriented" type of mind necessary for this type of research position). * Syracuse had an unusually high and excessive rate of "no shows" (often meaning "not even scheduled to begin with") and "not dones" compared to the other clinics. * On a regular basis, I would have to REPEATEDLY request data from the Study Coordinator, and we routinely missed deadlines. E.g. There was one group of approximately 90 (!) forms which were "missing" for over a year and a half. When these forms were eventually "found," mostly blank, the Study Coordinator filled in much of the information with, in most cases, no supporting documentation or progress notes in the charts. I have always believed that this data was just "fudged" or made up, because there would have been no other written record of these things (such as vital signs). * Informed consent forms were ROUTINELY backdated, sometimes weeks or even months after enrollment. E.g. In at least one instance, a patient was asked to sign (and did sign, along with the Principal Investigator and Study Coordinator) an informed consent form for the WRONG study (PID #110076A signed an informed consent for study 116 but was being enrolled on study 117). * The Principal Investigator and Study Coordinator displayed such open hostility and contempt toward the site monitors that there was a high turnover (4 different site monitors in a 3-year period). These site monitors could have easily uncovered this corruption if they had done their jobs carefully (which the first 3, at least, did NOT do) and over an extended period of time. * On March 21, 1990, after attempting unsuccessfully for over one year to address and resolve these SERIOUS issues with the Principal Investigator and Study Coordinator, and after watching in horror as the situation worsened severely with the implementation of the DDI studies (see attached memo dated 19MAR90), and after being retaliated against in many vicious and mean-spirited ways by both the Principal Investigator and Study Coordinator merely for repeatedly raising these issues and insisting on some type of corrective action, I felt compelled, in good conscience, to resign my position as Data Manager and immediately report this critical situation to the RTI site monitor. E.g. After reporting this to the site monitor, she checked with her boss, who in turn checked with her boss, and the decision was made to launch a "special audit and investigation" of the Syracuse site by the program office. I was asked to mail copies of all of my documentation supporting my claims to the site monitor. In a 27MAR90, 1:40 PM, phone call, the site monitor told me that her boss "never heard of anything of this magnitude," and referred to the situation as "uncharted waters." E.g. In a 27MAR90, 4:00 PM (later that same afternoon), phone call with Carolyn Fassi, who called me from NIH on behalf of Dr. Kantz, she thanked me for bringing these issues to their attention and said it would be unnecessary for me to forward copies of my documentation to the site monitor or to NIH. She also stated that they "can't act directly" based on my claims or supporting documentation, that they would "keep a close eye" on the Syracuse site, that they "won't use my information against the site or me," and ended by saying that he (Dr. Kantz) "may not even need to call me, except to clarify something." In other words, "don't call us, we'll call you." I never received a call from their office or anyone else associated with these studies again.

Love it or hate it, western blotting forms the bedrock of countless studies across numerous disciplines. Explore its history, development and applications in this episode all about the marmite of life science techniques.Guiding me through the hand wringing, hair follicle destroying history and process of western blotting, and hopefully explaining the beauty and potential of the technique - is Kenneth Oh, Senior Project Manager at Bio-Rad Laboratories. Kenneth reveals some of the latest developments in western blotting. Revealing how researchers are now able to validate the success of each stage of a western blot and work with smaller samples, Kenneth provides key tips for ensuring each of your western blots is a triumph. Contents:Intro: 00:00-01:15History of western blotting: 01:15-3:50What makes western blotting so tricky? 03:50-05:30Stepwise optimization: 05:30-06:50Stain-free western blotting: 06:50-08:50The different variants of western blotting: 08:50-10:20The right blot for the right application: 10:20-11:30The most exciting developments in western bloting:11:30-13:35New horizons for western blotting: 13:35-15:25The impact of multiplex-western blotting: 15:25-16:30Tips for best practice: 16:30-18:10The future of western blotting: 18:10-20:30Yearning for automation: 20:30-21:30 See acast.com/privacy for privacy and opt-out information.

Love it or hate it, western blotting forms the bedrock of countless studies across numerous disciplines. Explore its history, development and applications in this episode all about the marmite of life science techniques. Guiding me through the hand wringing, hair follicle destroying history and process of western blotting, and hopefully explaining the beauty and potential of the technique - is Kenneth Oh, Senior Project Manager at Bio-Rad Laboratories. Kenneth reveals some of the latest developments in western blotting. Revealing how researchers are now able to validate the success of each stage of a western blot and work with smaller samples, Kenneth provides key tips for ensuring each of your western blots is a triumph. Contents: Intro: 00:00-01:15 History of western blotting: 01:15-3:50 What makes western blotting so tricky? 03:50-05:30 Stepwise optimization: 05:30-06:50 Stain-free western blotting: 06:50-08:50 The different variants of western blotting: 08:50-10:20 The right blot for the right application: 10:20-11:30 The most exciting developments in western bloting:11:30-13:35 New horizons for western blotting: 13:35-15:25 The impact of multiplex-western blotting: 15:25-16:30 Tips for best practice: 16:30-18:10 The future of western blotting: 18:10-20:30 Yearning for automation: 20:30-21:30

Why are chronic, persistent infections like Lyme, Bartonella, Babesia and others like long-COVID and Epstein-Barr Virus so difficult to diagnose and treat? Infectious Disease specialist, Dr Bela Chheda, MD, walks us step by step through testing options including Elisa, Western Blot, PCR, Immunoblot (Igenix, Vibrant America), FISH testing for Babesia (Igenix), and some of the uncommonly tested parts of the immune system like the T-cells (InfectoLab). She also reviews how these bacteria can hide deep within tissue compartments and intracellularly, and that they can change themselves to hide from our immune system as well as changing our immune system itself. This flows right into the discussion about treatments with a focus on antibiotics: when to start them, when to stop them, how long they may be used for and what they can or can't do for recovering from chronic persistent infections. Dr Chheda explains why the CDC-criteria for Lyme disease set in the 1990's to target surveillance of population health lacks the sensitivity to find the cases of Lyme disease out there that can be responsive to treatment and discusses the critical importance of interpreting all test results in the clinical context of the individual patient. This one-hour show cracks the nut on how to think about testing and treating of these tricky vector-borne diseases that can trigger a chronic persistent infection that impedes optimal health.

Quantum Nurse www.quantumnurse.life Freedom International Livestream Friday, May 14, 2021 @ 2:00 PM EST 7:00 PM UK 8:00 PM Germany Features: People's Nurse International: Natural Health, Body Autonomy & Divinity Guests: Kate Shemirani, Nurse Practitioner Qualifed Nurse, UK Kate Shemirani has been a nurse for 36 years. She is also a personal nutritionist advisor and nurse practitioner. Kate Shemirani's inspiring story about how she came to stand up against the tyranny of mandatory vaccines, and as a result, she was arrested and charged with six felonies, JUST for standing up For The People. Kate is also a Christian that puts God's Will in the center of her life. Be inspired! Kristen Nagle Registered Nurse, BScN, RHN Ontario, Canada www.canadianfrontlinenurses.ca/ Nurse Kristen has been a nurse for 14 years, primarily in the Neonatal Intensive Care Unit, with previous experience in Pediatrics. When Kristen's first son was born she realized the importance food would have in creating the foundation for his growth and development and enrolled at the Canadian School of Natural Nutrition. Rachel Celler Registered Nurse http://theforensicnurse.org “The forensic nurse, exposing medical crime one misdiagnosis at a time. A sanctified healer, Navy veteran hospital corpsman, Registered nurse, and global health educator. My covenant is established with God and I teach biblical healing and cure according to the Holy Bible.I have an online healing and vaccine Re-education ministry, Fisher of men ministry.org. My nine minute video titled cancer laced vaccines exposes the MRC5 aborted fetal tissue used in vaccines is a weaponized cancer cell line engineered to make you sick. There is no mystery to reversing diseases like cancer, auto-immune disease and autism. There are natural solutions to every health problem. I help people around the world take authority over their health and reverse dis-ease naturally at home.” Dr. Kevin Corbett, PhD Qualified Nurse https://kevinpcorbett.com/ Kevin P. Corbett has over thirty years experience in clinical and academic healthcare practice working in acute / primary care and higher education institutions. He completed both undergraduate and postgraduate training in Art at the University of Reading (1979) and the Slade School of Fine Art, University College of London (1981). Kevin qualified as a Registered Nurse in 1986 becoming part of the commissioned staff for Broderip Ward at the Middlesex Hospital, London, Britain's first purpose built HIV/AIDS unit, opened by Princess Diana in 1987. Postgraduate nursing research followed at King's College London (1987-1989) into improving metered dose inhalation through patient training in the physiology of the inhaled route. His doctoral research (1995-2001) focused on patients' indeterminate experiences of the tests used in HIV/AIDS, the ELISA, Western Blot and PCR tests.

Quantum Nurse www.quantumnurse.life Freedom International Livestream Thursday, April 29, 2021 @ 2:00 PM EST 7:00 PM UK 8:00 PM Germany Features: People's Nurse International: Natural Health, Body Autonomy & Divinity Guests: Kate Shemirani, Nurse Practitioner Qualifed Nurse, UK Kate Shemirani has been a nurse for 36 years. She is also a personal nutritionist advisor and nurse practitioner. Kate Shemirani's inspiring story about how she came to stand up against the tyranny of mandatory vaccines, and as a result, she was arrested and charged with six felonies, JUST for standing up For The People. Kate is also a Christian that puts God's Will in the center of her life. Be inspired! Kristen Nagle Registered Nurse, BScN, RHN Ontario, Canada www.canadianfrontlinenurses.ca/ Nurse Kristen has been a nurse for 14 years, primarily in the Neonatal Intensive Care Unit, with previous experience in Pediatrics. When Kristen's first son was born she realized the importance food would have in creating the foundation for his growth and development and enrolled at the Canadian School of Natural Nutrition. Rachel Celler Registered Nurse http://theforensicnurse.org “The forensic nurse, exposing medical crime one misdiagnosis at a time. A sanctified healer, Navy veteran hospital corpsman, Registered nurse, and global health educator. My covenant is established with God and I teach biblical healing and cure according to the Holy Bible.I have an online healing and vaccine Re-education ministry, Fisher of men ministry.org. My nine minute video titled cancer laced vaccines exposes the MRC5 aborted fetal tissue used in vaccines is a weaponized cancer cell line engineered to make you sick. There is no mystery to reversing diseases like cancer, auto-immune disease and autism. There are natural solutions to every health problem. I help people around the world take authority over their health and reverse dis-ease naturally at home.” Dr. Kevin Corbett, PhD Qualified Nurse https://kevinpcorbett.com/ Kevin P. Corbett has over thirty years experience in clinical and academic healthcare practice working in acute / primary care and higher education institutions. He completed both undergraduate and postgraduate training in Art at the University of Reading (1979) and the Slade School of Fine Art, University College of London (1981). Kevin qualified as a Registered Nurse in 1986 becoming part of the commissioned staff for Broderip Ward at the Middlesex Hospital, London, Britain's first purpose built HIV/AIDS unit, opened by Princess Diana in 1987. Postgraduate nursing research followed at King's College London (1987-1989) into improving metered dose inhalation through patient training in the physiology of the inhaled route. His doctoral research (1995-2001) focused on patients' indeterminate experiences of the tests used in HIV/AIDS, the ELISA, Western Blot and PCR tests.

Southern, Northern e Western Blot: as bússolas da Biologia Molecular são o tema do BeP desta semana. Descubra como os cientistas foram criativos em nomear os principais processos de identificação da presença de DNA, RNA e proteínas em amostras biológicas, como essas técnicas funcionam e claro, tem toda a irreverência do nosso time. O Fofoca em Pauta entra em cena com temas como o Instagram da Alessandra Negrini, o Messi Careca e a Pucca, e o debate esquenta sobre o desenho Corrida Maluca e os novos termos criados pela falta de dicção dos podcasters. Então se ajeite em suas atividades e dá o play na gente! Elenco: Anderson Freitas, Lívia Cardoso, Gabriel Nunes e Flávia Gan. Campanha apoia-se: https://apoia.se/biotecempauta2 (pontual - doação única); https://apoia.se/biotecempauta1 (contínua - seja assinante) Vinheta: Cold Funk by Kevin MacLeod Link: https://incompetech.filmmusic.io/song/3522-cold-funk License: https://filmmusic.io/standard-license Background: Wholesome by Kevin MacLeod Link: https://incompetech.filmmusic.io/song/5050-wholesome License: https://filmmusic.io/standard-license

Meet Lindsey Dockins a 28 year old mom of 3 girls. She's been married to her college sweetheart for 9 years. In 2018 Lindsey was diagnosed with mycotoxins, Lyme and 5 co-infections, parasites, Epstein Barr virus, MTHFR and other genetic issues, allergies, sensitivities, vinyl toxicity, gun cleaner toxicity, candida, Mysterious black necrotic legions in my stomach lining, multi-chemical sensitivity and mast cell disorder. She had to move out of her house and start all over. Hear her harrowing story of surviving mold toxicity, recovering from Lymes and how she is helping others do the same. Some of the recovery methods and tests mentioned in her talk include: Hyperbaric Treatments Far Infrared Sauna by SaunaRay http://www.saunaray.com/index.html IQ Air Purifier iqair.com Beau biologist and other healthy and sustainable products for the home https://buildingbiologyinstitute.org/find-an-expert/products-stores/ Air Quality Tests: ServePro and EnviroPro Lyme disease testing: Western Blot, sensitive enzyme immunoassay (EIA), IGeneX labs Parasite Testing- GI Map test OAT- Organic Acid Test Food Allergy Testing Underourskin.com (documentary on Lyme's Disease) Disclaimer: Lindsey and host Jolene Fisher are not claiming to cure, prevent or treat any disease. This is a case study and one person's story of what they did to help their body recover. Please seek attention and advice from your doctor before attempting the advice listed here.

Tick Boot Camp’s guest today is Allyssa LaScala. Ms. LaScala is a 29-year-old integrative health practitioner from Reading, Pennsylvania. In 2011, during her sophomore year of college, she started to experience extreme fatigue. She scheduled an appointment with her primary care physician, who ran a multitude of tests, including a Western Blot. Ms. LaScala tested positive for Lyme. She took Doxycycline for 3 weeks, but still felt the symptoms of a tick disease. After 6 months, she asked to be retested, and still came back positive for Lyme. While developing new symptoms like depression and brain fog, Ms. LaScala got advice from other Lymies. She decided to see a Lyme literate doctor. If you would like to learn more about how Ms. LaScala’s Lyme experience changed the course of her life and led her to start the Biohacking Bombshell, then tune in now!

Gregory Hartley Brewer, from Bath in the United Kingdom shares his long battle to get a diagnosis for his Lyme-like symptoms, and then trying to access proper treatment once diagnosed with Lyme many years later.   But he is now embroiled in a long battle with the health care system as they try to cover up their missed diagnosis mistake and protect the doctors that not only denied Gregory medical care, but may have behaved in a criminal manner in conspiring to cover up their medical error.   But he has not given up: hear how Gregory is taking on the Goliath that is the United Kingdom health care system and exposing their attempts to hide a medical error that has morphed into much more serious and potentially criminal behaviour.   SHOW NOTES - TIME STAMPED   :04:00 Gregory, early 50s, born in Birmingham, UK - moved to States as a kid for a few years - back to London, then Bath - happy childhood, middle class upbringing - more sporty then academic - found drinking, girls and smoking so didn't go to university - bar, nightclub work - still looking for niche - loved helping people in community job :06:30 2005 Gregory get bitten by tic in backyard, but he's unaware of the existence of Lyme - rash and flu like aches - April 2005 collapsed with stabbing pain in chest chest, 'like being stabbed with a knife", thought he was dying - within a week went to the doctor and was diagnosed with hyperlipidemia, aka high cholesterol and triglycerides - couple of weeks later to another physician in same clinic and recounts rash and flu like symptoms - doctor says that sounds like Lyme, but it can't be Lyme because you're not seriously ill :07:45 A few weeks later he returns to doctor to say he thinks he has Lyme and she get angry and said he couldn't have Lyme because he's not seriously ill - Gregory believes her - in retrospect she was protecting her misdiagnosis that was only 4 weeks old - this is how uttterly their reputations come before patient treatment and safety - Gregory asks for Lyme testing, she refuses :08:55 Realizes some of his doctor appointment notes are missing out of his file, thought it was strange but had faith that the doctors knew what they were doing and there can't be a nefarious reason for that     :09:20 He worsens with Lyme symptoms: Peripheral neuropathy, palpitations / pericarditis, chest pain, anxiety from encephalopathy, low grade meningitis, headaches, sore shoulders - by 2008 in bad shape, reticent to raise Lyme disease for fear of denigrating and angry reaction - symptoms cause big impact on his social life, went from very social to feeling too anxious and stopped going out, lost friends from isolation :11:40 Gregory feeling anxious but not depressed, but no external anxiety trigger - no rhyme or reason when anxiety came - now knows it was bacteria impacting his brain - describes peripheral neuropathy in his legs - pain, squeezing, crushed and wants to explode - due to nerves being attacked by bacteria - may last minutes or hours, no rhyme or reason, sometimes hurts to walk - puzzling as to what is happening in his body, vacillates seeing doctors because they looked at him like a bloody idiot - by 2008 the doctor must have known these were Lyme symptoms - only 3 GPs in this clinic :15:20 2009 bitten again by a tic in his field and sees rash on the inside of his arm - its a Sunday so Gregory went to health center, diagnosed Lyme immediately and given one week supply of doxycycline, but later finds out he should have been given 2 weeks according to the National Institute of Clincal Excellence (NICE) guidelines at the time - doctor sent note to his GP about Gregory's Lyme diagnosis - about 9 months later symptoms worsen again, returns to his GP and is again denigrated and dismissed :16:20 GP says just because you had Lyme disease and it wasn't properly treated, doesn't mean you have it now - Gregory asks why their multiple discussions about Lyme disease are not in his medical file and the GP says she decides what goes into his medical file - Gregory requests that she make a note about his Lyme diagnosis in his file, she threatens to fire him as a patient if he keeps saying he has Lyme - turns out there is no mention of Lyme in his medical file from 2005 to 2014 :19:20 GP didn't want Lyme in his notes because it would come back and bite her - willful lack of insight about his symptoms - negligent since 2009 for failure to test and treat - switches to different doctor, and this doctor also says Gregory's symptoms are not Lyme - even though Bath high risk is endemic area there are no signs any where - Public Health England is responsible for responding to Lyme disease stopped Bath public heath from putting up signs after secret study found high concentration of tics in Bath area - without public signs Gregory wasn't aware Lyme in his area, mixed with phsycians who are Lyme illiterate - many, many patients sick but not diagnosed - a national scandal     :22:15 Lots of known unknowns about Lyme - NICE just changed guidelines saying that Lyme relapse can happen, and can access 4 more weeks of treatment - but lots of patients misdiagnosed and not diagnosed - but found new GP in March 2016 who is treating him well - gets 2 or 3 courses of antibiotics a year to manage, but not cure, his Lyme :23:40 In 2018 Gregory gets Macular Erythema on his hand, a sign of systemic disease, but if not for that sign, he wouldn't be able to get a Lyme diagnosis - about 300 GPs in UK treating long term Lyme sufferers outside NICE guidelines - lots of Lyme patients don't get treatment     :25:35 Lots of UK people get tested positive for Lyme in Europe labs but testing in UK finds Lyme negative - UK relies heavily on testing and not on clinical symptoms - Gregory contends that since Lyme testing is unreliable and inconsistent, that clinical symptoms should be used to diagnose - also because a person can test positive for Lyme and not have any symptoms because they've produced antibodies to the Lyme - can also have no antibodies and still have Lyme because the body did not produce antibodies - ELISA and Western Blot tests - a scandal in the UK as thousands have Lyme symptoms but no treatment - central Europe is better at treating :28:15 Gregory thinks when a doctor doesn't know what to do when a patient tells them one thing and testing the opposite, they fall back onto science (testing) - and it is very poor in Lyme disease - NICE guidelines basically say we have no confidence in what we're proposing, but we need to propose something - because doctors can't see symptoms they will attribute to another illness or give the patient a psychological diagnosis - Rob Hackett Australian medic on twitter wants aviation standards in medicine - Gregory re-tweeted Scott's tweet about a black box in the operating room :30:20 Hackett studies patient safety - bystander effect, ego, reputation - in Gregory's case it was ego that prevented the physicians from admitting they were wrong, thereby denying treatment to him - instead they conspired and were cruel and degrading to leave him ill deliberately - if not for diagnosis by another GP, Gregory would be still be suffering greatly with symptoms and still being told he has psych problems :31:00 December 2014 these GPs decided that they couldn't give Gregory a diagnosis of Lyme - goes to locum to see doctor about symptoms but he was misdiagnosed with prostatitis and given medication for that and Gregory had a bad reaction - says to locum doctor that he has Lyme, doctor believes him, but only gives 2 weeks of antibiotics - Gregory goes back to GPs who say he doesn't have Lyme because he has negative serology - he feels depressed and distressed :32:20 Gregory researches and finds that the Public Health England's Doctor Pathway says that antibiotic treatment may produce false-negative results - presents to GPs and this compels them to have a meeting with PHE but they send their newest and most junior GP to the meeting to withhold multiple information from PHE and to deny Gregory treatment in August 2015 :34:00 Gregory finds Lyme Disease Action help people with Lyme get a diagnosis - they write to his GP, who informs the PHE, who realize right away that they were previously withheld information about Gregory - however PHE leaves it to GPs to decide treatment and they say there is no Lyme to treat in Gregory     :36:45 LDA decides not to report these 3 GPs for criminal negligence, but Gregory is able to access treatment from them because if they refuse, the LDA can still report them - Gregory gets 4 weeks of treatment but did not yet know they were withholding clinical history from PHE so continues at the clinic - but if he had not got 2nd opinion, he'd still be suffering :37:50 Gregory emails PHE directly and gets almost immediate response apologizing for discrepancy in the reporting of his symptoms in all the discussion - Gregory finds out later the PHE forwards email to Gregory's GP with angry note, so GP now knows Gregory knows that PHE was not given full medical file - this leads to more forgery down the line :38:45 Gregory realizes the GPs are being willful, but he does not know why they wouldn't give full medical file to PHE - GP avoids talking to LDA because LDA has full medical file and will ask GP why she only sent partial file to PHE :39:45 Gregory files complaint with NHS - GPs are employed by NHS - and recieves their 1 page report in March 2016 and it is "so corrupt" - ignores early diagnosis - Gregory files another complaint and this time NHS realizes he knows something is wrong - they produce 10 page report and dismisses Gregory's symptoms and blames him for poor medical care - so they are protecting GPs and covering up their harm :41:40 July 27, 2012 - Eureka moment - if he is diagnosed with Lyme in 2015 from 2009 onset and denied medical care, then that is criminal negligence - now Gregory understands their motive and explains all their behaviour and actions: to cover their asses from criminal negligence which now not just a mistake, but a conspiracy to deny a patient treatment - they must have sat down at some point to decide to deny he had Lyme     :43:15 NHS notorious for covering up mistakes and attacking whistleblowers, be they patients or medical staff - there is a culture of defend, deny, delay - Gregory investigates himself by getting access to emails and puts more pieces of the hidden puzzle together - more denial, lying, created fraudulent email but never sent, blaming PHE, to protect their position and prevent exposure of their crime :47:40 Gregory continues to research and submit evidence to Palriamentary and Service Ombudsman (PSO) and General Medical Council (GMC) in December 2016 - LDA says can't support Gregory, with subtext they will be punished as whistleblowers and closed down by National Health Services (NHS) - more coverup happens by GMC and dismiss his evidence - PSO even slower - Gregory appeals GMC decision, they admit Gregory's case meets their high threshold for investigation, but claim they see no evidence of doctor misconduct and dismiss the case - PSO also dismiss and ignore evidence :49:30 Gregory takes the GMC to court in September 2017 but loses - appeals again to be heard - PSO closes investigation because "you will be disappointed with the outcome" - PSO well known as dust bin for complaints - September 2018 launches Judicial Review against PSO and gets oral hearing - Judge agrees PSO behaved badly but says PSO can act at own discretion - PSO fails to tell Gregory they must agree on scope of investigation, this gives him leverage to appeal - can't get legal support because nobody wants to believe 3 GPs intentionally denied a patient treatment for years :53:30 Gregory has found case law that doctors must treat, otherwise a criminal act - PSO has broad powers, but refuse to interview 2 witnesses, who's careers would be in jeopardy for not reporting the GPs for non-treatment - this is all a lot of stress, not a normal life, wish for happy days - GPs just 500 yards away and other patients at risk - willfull lack of insight into patients Lyme symptoms - knows a woman who was dismissed by GPs, turns out she had cancer for 2 years, and died soon after proper diagnosis, and refused to apologize - they are a danger and Gregory will do everything in his power to expose them :57:30 Others who are whistleblowers suffer greatly from blowback from goverment institutions - at end of the day, CEO of NHS Trust do not want their dirty laundry aired in public and will crush any thing that threatens that, no matter how obvious - recent case Dr Day in England where they tried to exclude junior doctors from whistleblower protection - statutory duty to protect patients - misconduct in public office - UK police rarely charge people in power 1:00:40 End of day, patients and medics suffer - if try to stand up, you are beaten down - most patients just want recognition and apology - but instead wilfull denial of their mistake - yet so wilfully doing harm by denying treatment - Gregory will have to report to police - conspiracy because junior doctor had no reason to lie, except to protect her senior doctors     1:02:30 Gregory sent email to junior doctor asking her to testify and the police show up on his doorstep with a 'community protection warning' to protect community from harassment - senior partner made sure junior doctor not available to be interviewed by police - 2 senior partners are professional liars - police refuse to acknowledge crime against Gregory, but feels he has to compel them to investigate 1:04:30 Started twitter April 2018 to put his experience out there - realize many others with similar experiences - solace and support finding others - next thing is to form groups and attack these organizations that are covering up all these cases - produce blog, keep campaigning - show its all a scam saying to public we've got orgs to support you, but its all a lie - they are in own silos and don't want to kick up a fuss and lose their job, pay cheque - huge scandal in UK and other countries 1:07:15 Last few months have been very difficult - started smoking again, stopped exercising, depression, self doubt, anxiety - some days better if get a bit of good news, but most of time it is a crushing weight - drinking to get to sleep - irritable, angry 1:08:55 Can't remember feeling happy, part due to Lyme symptoms, but mostly due to the situation - affects all parts of life - bloody nightmare - constant worry - affecting relationships, compounds Lyme - borderline sociopaths in high positions shown by their actions 1:10:45 GPs don't want to discuss trauma he's experienced by health care system, so can't get help for trauma - don't give up, just keep going 1:13:45 - end   Follow Gregory on twitter.   About the podcast I’m Scott Simpson, a personal counsellor by day, a podcast host by night, and a sick and disabled patient advocate surviving medical error. My hope is that by sharing stories of medical error, we can bring awareness to this 3rd leading cause of death, and implement solutions for patient safety. Host Scott Simpson But the podcast is not just about medical error experiences, I also interview people who are trying to make health care systems safer for all of us. Turns out that a lot of people working on patient safety, have personal experiences with medical error. The airline industry is quite transparent about their safety incidents. The exact opposite is true about the medical industry. They work hard to ensure the public does not get easy access to data about medical errors. Medical Error Interviews brings transparency to medical harm and death, giving voice to survivors and change makers. You can support the podcast and help make health care safer by becoming a Patron or Premium Patron.   Support Medical Error Interviews on Patreon by becoming a Patron for $2 / month. Or $5 / month to be a Premium Patron and watch the video versions of Medical Error Interviews.    

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to The Journal and it's editors. We're your co-hosts. I'm Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr Greg Hundley:             And I'm Greg Hundley, also Associate Editor from the Pauley Heart Center in Richmond, Virginia, VCU Health Sciences. Dr Carolyn Lam:                How well are we doing with guideline-directed stroke prevention therapy in atrial fibrillation? Well, there are going to be very important results that you need to hear about from Get With the Guidelines Atrial Fibrillation. That's our feature paper coming right up in a future discussion. But first, you've got Greg and I discussing really important papers that we've spotted in The Journal. Greg. Dr Greg Hundley:             Absolutely, Carolyn. And my favorite kind of follows from that 'cause it's really about left atrial electromechanical remodeling following two years of high intensity exercise training in sedentary middle-aged adults, kind of like me. The studies from Ben Levine at University of Texas Southwestern Medical Center in Dallas. So, what he's driving at here are moderate-intensity exercises associated with a decrease in incidents of atrial fibrillation. However, extensive training in competitive athletes is associated with an increased atrial fibrillation risk.                                                 So, in this study, they're looking at the effects of 24 months of high-intensity exercise training on left atrial mechanical as well as electrical remodeling in sedentary, healthy, middle-aged adults. So, he had 61 individuals, their average age was 53.5 years, quite young, who were randomized to 10 months of exercise training followed by 14 months of maintenance exercise and some stretching or stretching and balance control. He also had another group of 14 master's athletes that were added for a comparison and he looked at three of the echocardiograms to assess left atrial and left ventricular volumes and also had signal average EKG's for filtered P-wave durations and atrial light potentials. He made assessments at baseline, so before everyone started, and 10 and 24 months. Dr Carolyn Lam:                Hold on, hold on. Let's really understand here how much exercise were these sedentary middle-aged adults subjected to. Dr Greg Hundley:             So, let's talk about that because that was very interesting because a lot of us are out there exercising. So briefly the way he started this, there was an initial phase that was comprised of six months of regressive training during which an increase in the frequency, the duration, and the intensity of exercise, including two high-intensity aerobic interval sessions per week that were prescribed to peak training load. The peak training load included five to six hours of exercise per week that included two interval sessions, at least one being an hour-long session, and then two 30-minute sessions.                                                 Once you got that peak training load, that was sustained for four months and then he made these 10-month measurements as part of his study design. Now following that phase, a 14-month sort of a continuation, all of the 24 months, a 14-month period of maintenance exercise was completed where the frequency of high-intensity intervals was reduced to once per week plus continuous training all the way to that 24-month time point. And during the maintenance phase, participants performed a total of about three hours a week of aerobic exercise. Dr Carolyn Lam:                Well, don't keep us in suspense now. What did the study show? Dr Greg Hundley:             So at the 24 month time point of high-intensity exercise, it led to a disproportionate dilation of the left atrium compared to the left ventricle. So, mechanical changes, but no electrical remodeling was seen. And interesting, and remember he had that comparison cohort with master's athletes. Those participants randomized exercise training demonstrated lower absolute left atrial and left ventricular volumes, but a similar left atrial to left ventricular ratio after 24 months of exercise training.                                                 So, what's going on here, if you're middle-aged or young, some of us like to think, and you start one of these aggressive training sessions, you do have some changes mechanically in the shaping of your left atrium and left ventricle, but they're concordant, but no electrical remodeling that was observed in this situation. So, how do those elite athletes develop atrial fibrillation in the electrical remodeling? Don't know. It may be they need a longer duration of exercise. Maybe they start at a different time point because these are relatively sedentary individuals, and maybe their training regimen is very different.                                                 So, more research is needed, but it was interesting that these middle-aged folks that start with this little bit more aggressive regimen really didn't develop the electrical remodeling. So, Carolyn, you've got a couple of papers that are sort of tied together. Dr Carolyn Lam:                Indeed. A couple of papers centered on lipoprotein little A. Now, we know that lipoprotein little A levels predict the risk of myocardial infarction and this has been shown in populations of European ancestry, however there's very little data available in other ethnic groups. And so, this was addressed by Dr Paré from McMaster University and the Interheart Investigators who looked at more than 6000 cases of first myocardial infarction and more than 6800 controls, all from the Interheart study, and were stratified by ethnicity and included African, American, Chinese, European, Latin American, South Asian, and Southeast Asian ancestries.                                                 Lipoprotein little A concentration was measured in each participant, first using an SA that was insensitive to iso-form size and then iso-form size itself was also assessed by Western Blot in a subset of more than 4200 participants. So, what they found was that lipoprotein little A concentration and iso-form size varied markedly among the ethnic groups. Africans had the highest concentrations with the smallest iso-form size whereas Chinese had the lowest concentrations with the largest iso-form size.                                                 Furthermore, higher lipoprotein little A concentrations were associated with an increased risk of myocardial infarction and carried an especially high population burden in South Asians and Latin Americans. And a high concentration above 15 milligrams per deciliter was associated with significantly increased risk of myocardial infarction in all populations except Arabs and Africans. The iso-form size, on the other hand, was inversely associated with lipoprotein little A concentrations and did not significantly contribute to the risk. Dr Greg Hundley:             So, Carolyn, how do we use this clinically? I mean, do we measure this in folks? Dr Carolyn Lam:                Yeah. So, there are two take-home messages. I think one is about the monitoring or measuring and it supports a clinical use of the actual lipoprotein A concentration rather than iso-form size as a marker of myocardial infarction in this ethnically diverse population. But this is, other than Africans and Arabs where, remember that cut off did not seem to associate with a risk of MI's in these two ethnicities. The second take-home is that the effects of clinical interventions that reduce lipoprotein A should be investigated especially in South Asians and Latin Americans where the population attributable risk is really high. And that actually brings me to the second study.                                                 So, we've always been looking for intervention that can reduce lipoprotein A and this current paper is really interesting 'cause it talks about insights from the Fourier trial. So, we may finally have a therapy that can reduce it. Dr O'Donoghue from the TIMI study group and Brigham and Women's Hospital in Boston, Massachusetts and colleagues looked at the relationship between lipoprotein A levels, PCSK9 inhibition, and cardiovascular risk in the Fourier trial, which you remember is a randomized trial of Evolocumab versus placebo in patients with established atherosclerotic cardiovascular disease.                                                 So, they found that patients with a higher concentration of lipoprotein little A were at increased risk of coronary events independent of the LDL concentration. And individuals with a higher baseline LP little A concentration tended to have a greater relative and absolute coronary risk reduction with Evolocumab and therefore a lower number needed to treat. It was as low as four T for individuals with a lipoprotein A above the median versus 105 number needed to treat for those at or below a lipoprotein A level below the median. Dr Greg Hundley:             So should we start checking this in all our patients now, these lipoprotein little A levels? Dr Carolyn Lam:                Yeah. So, this issue was discussed beautifully in a company editorial by Dr Thanassoulis from McGill University Health Center. And here he mentions that there remains tremendous clinical inertia honestly for the measurement of lipoprotein A in North America and in fact, worldwide. For this to be successful, we really need to be proactively screening our patients with myocardial infarction and stroke and especially those with premature events or a family history. And particular attention will need to be made on screening individuals with recurrent events despite adequate lipid or LDL lowering who frequently may still have high lipoprotein little A. It's encouraging to know that the most recent version of the US Lipid Guidelines has newly recommended LP little A measurements in select individuals as a risk enhancer and so this should further raise awareness of lipoprotein little A as a risk marker.                                                 Finally, the editorialist mentioned that common misconception that we have a lack of therapeutic options to lower high LP little A. Still, we need to remember that these individuals may obtain significant benefit from more aggressive lifestyle modifications. And now we have these results of this trial that suggest that PCSK9 may be one of the few drugs that can lower lipoprotein little A. And so, the editorialist actually ended with targeting therapy for lipoprotein A is around the corner and a test of this hypothesis is really imminent, so we should watch this space. Dr Greg Hundley:             Yeah, so it sounds like another wonderment of PCSK9 inhibitors. Dr Carolyn Lam:                Yeah. Dr Greg Hundley:             Well Carolyn, let me jump in and finish our chat here talking about iron. This particular paper is from Dr Jean-Sébastien Silvestre from Paris, France, and he's looking at the iron regulator Hepcidin. So, we know that iron deficiency is frequent in patients with coronary artery disease and increases morbidity in those with high risk profiles such as those with diabetes and anemia and then conversely, excess iron is also detrimental to cardiac function. We see this with iron overload cardiomyopathies and as a major co-morbidity in patients with genetic hemochromatosis.                                                 So, among the multiple regulators of iron homeostasis is Hepcidin. It plays an instrumental role in fine-tuning systemic iron trafficking by modulating the transfer of dietary, recycled, and stored iron from intracellular compartments to extracellular fluids. Hepcidin is a catatonic peptide hormone. It's produced primarily by hepatocytes, but also, it's produced in macrophages. So, given the role of Hepcidin to locally regulate cardiac function and that inflammation guides cardiac remodeling after acute MI, the investigators hypothesized that inflammatory macrophages may control cardiac repair through a Hepcidin-dependent mechanism. And until now, the role of Hepcidin in some other cardiac diseases challenged by inflammation hasn't really been explored. Dr Carolyn Lam:                Huh, interesting. So, what did they find? Dr Greg Hundley:             Great question and let's lead to the main results of this study. The hormone Hepcidin, they found, was produced by a distinct sub-population of inflammatory cardiac macrophages residing in infarcted heart tissue and the deletion of Hepcidin in macrophages improved tissue remodeling and stimulated cardiomyocyte renewal in both, just as our wonderful basic science studies have, in both adult mice with myocardial infarction, neonatal animals with apical resection and also in human subjects. And so, this study provided novel insights into the complex roles of the immune response during cardiac repair following MI and suggests and deleterious role for the macrophage-derived Hepcidin in cardiac repair.                                                 Interesting, Carolyn. Another role for iron in acute MI and more research to come. Dr Carolyn Lam:                Indeed. Well, thanks Greg. Let's move on to our feature discussion, shall we?                                                 For our feature discussion today, we are talking about the first results from the Get With the Guidelines atrial fibrillation. That is huge, and I have none other than the first author, Dr Jonathan Piccini from Duke Clinical Research Institute, as well as Dr William Lewis from Case Western Reserve University here to discuss these really important results, so listen up. I think to start with it is such an honor to have you with us, Bill. I mean, as Chair of the Get With the Guidelines atrial fibrillation work group, could you give us a background on how did this start? How far has it come? Dr William Lewis:             The Get With the Guidelines program started in 2000. Greg Fonarow figured out that if we put in place mechanisms to improve adherence, that we could get people on appropriate therapies. In 2012, there was some focus on atrial fibrillation and I had been participating in the program since 2004 and I kept telling them that A-fib was a big, big problem. And in 2012, they said, "Let's do this," so we built this program to try to improve adherence in atrial fibrillation. Get With the Guidelines is a national, hospital-based, quality improvement program that improves adherence to guidelines over time and it has been very successful at doing that.                                                 So, by 2013 we were ready to start enrolling patients and we started getting patients in the database and we're now up to about 162 hospitals nationwide, in the United States, and we've enrolled about 75000 patients in the program. So, it's been very successful from that standpoint. Dr Carolyn Lam:                Congratulation. And today we're actually going to be talking about that very question you asked. Adherence. How well are we adhering to guideline-directed stroke prevention therapy for atrial fibrillation? Jonathan, wanna share the key results? Dr Jonathan Piccini:         I think you're getting exactly to the point of what was the rationale for this study and I think most individuals that are familiar with the field and atrial fibrillation and also clinicians across the world who are treating patients with atrial fibrillation know that most large reports, most nationwide studies have shown that adherence for oral anticoagulation to prevent stroke in patients with atrial fibrillation usually ranges in the 50, 60, 70 percent range at best. And there's been some notable publications in the past several years from nationwide registries that have shown rates as low as 50 percent or lower in high-risk patients. So, one of the main goals of the program, as Bill articulated, was to try and improve the use of oral anticoagulation in patients who had a guideline recommendation. So, patients who had a CHA2DS2-VASc score of two and higher with atrial fibrillation.                                                 And so, looking at over 30000 admissions between 2013 and 2017 and the guidelines A-fib program, we saw that just under 60 percent of patients who had known AF at the time of admission were on oral anticoagulation. And not surprisingly, the patients who were on oral anticoagulation had lower rates of stroke during their hospitalization. But the major finding from the program was that in this quality improvement program, the program was able to improve adherence to oral anticoagulation at discharge from 60 percent to admission all the way up to 93.5 percent in the overall cohort. And if you looked at results over time, adherence improved from 80 percent at discharge all the way to 96 percent and those improvements were sustained in follow up as well. Dr Carolyn Lam:                Could you tell us, what do you think are the key elements that help this improvement? Is it just because there's a program and people know they're being watched? Is it that there was a change? I mean, when you say oral anticoagulants I bet you mean both Warfarin and the newer oral anticoagulants, so how much did that help? What do you think is the key ingredient here? Dr Jonathan Piccini:         It was several things. Having visited several of these hospitals and spoken with them about the impact of the program, I think you can't emphasize enough that if you don't measure something, you can't really expect to improve it. So, just the fact that hospitals were having systematic data on their atrial fibrillation patients at discharge illustrating who was and who was not getting oral anticoagulation makes a big difference. Between the program itself and the conferences affiliated with the program and teaching sessions affiliated with the program, there's a heavy emphasis on education of the importance of guideline recommended treatments for atrial fibrillation, so that's a second component.                                                 And then there's an iterative relationship between the sites and the American Heart Association where improvements in the rates of oral anticoagulation are recognized and celebrated. And I think it's not any one thing, in my opinion. I think it's all of those things taken together. And again, Bill, who's been with the program since its inception probably has additional thoughts on that as well. Dr Carolyn Lam:                Bill, did you expect such remarkable results? Dr William Lewis:             No. I actually didn't expect 96, but in a previous study where we were looking at patients who had had a stroke in the stroke database, we were able to achieve 93 percent adherence. And so, 96 is remarkable and it's the highest number that's ever been seen in any A-fib program. I was going to mention about the idea of what makes the special sauce, if you will, and I think John put forth a number of items. I think, again, celebrating success, those kinds of things, but I think that docs, by their very nature, are very competitive and when you get a data report that says you're doing x percent and somebody else is doing y percent and their percentage is higher, you tend to get motivated to actually do better. And so, we provide these reports in the program to hospitals so that they can measure their success against other institutions. Dr Carolyn Lam:                That's such a good idea. And, you know, I practice here in Asia and there aren't these very massive programs that are accepted in many places. So, what do you think is the generalizability of something like this? Dr Jonathan Piccini:         That's such a critical question because a limitation is that these are hospitals that are saying voluntarily, "We want to commit to the program because we think quality care for atrial fibrillation patients is important." And so, you could argue that, well, these results really don't generalize to your run of the mill hospital in different parts of the world. And I think while that's a limitation, it's also a call for what the next steps are. So, having visited many of these hospitals, these are real hospitals of brick and mortar that face many of the same challenges other health systems and hospitals across the world do and I think the key message is that a hospital that implements these types of interventions is very likely to see the same improvement with their patients. And so, I think that's a very important message and a very positive message for patients all over the US and all over the world. Dr William Lewis:             I agree. I think it's, not turn-key, it's much more generalizable than we had ever expected. So, community hospitals do this. The American Heart Association is using other Get With the Guidelines programs in China. I think that there is a lot that has to do with the support that's provided by the program and the tools that are made available to them to be able to make it so that you can recreate it in a hospital. I agree, it is more difficult in some hospitals than others. Dr Carolyn Lam:                John, before we end, what are the take-home messages for clinicians listening out there? Dr Jonathan Piccini:         I'd have two messages. The first message is that this study shows that with some assistance any healthcare system or hospital can achieve optimal adherence to these medications for their patients and thus in so doing achieve a significant benefit for the public health. And the second message I would have, which isn't necessarily specifically related to the paper, but I think it's equally important, that this is just the beginning for the American Heart Association and the Heart Rhythm Society Get With the Guidelines A-fib registry. Though stroke prevention is obviously just one of many different aspects of quality care for atrial fibrillation and so keep an eye out 'cause you'll be seeing a lot of studies coming out about how Get With the Guidelines A-fib is better informing care and treatment for atrial fibrillation across many different therapy domains, including catheter ablation and rate control and other interventions for rhythm control. And again, on behalf of all the co-authors and the American Heart Association, the Heart Rhythm Society sponsors, we really appreciate to have the opportunity to talk about the program. Dr Carolyn Lam:                Thank you so much for sharing that with us.                                                 Audience, you heard it right here on Circulation on the Run. Don't forget to tune in again next week.                                                 This program is copyright American Heart Association 2019.  

Not all labs are created alike. I have a few negative LabCorp ELISA Lyme Tests. Shoot for a Western Blot from one of the following labs: www.IGENEX.com (California) www.mdlab.com (NJ) http://medicine.stonybrookmedicine.edu/pathology/tick (NY) If you have questions or concerns hit us up on Twitter, Instagram or Facebook @theguudcompany. Happy to help.

Why You Should Listen: In this episode, you will learn about the various testing options offered by IGeneX in the realm of vector-borne diseases. About My Guest: My guest for this episode is Dr. Jyotsna Shah. Dr. Jyotsna Shah PhD is the President and Laboratory Director of IGeneX Clinical Laboratory, Palo Alto, CA. Dr. Shah has over 40 years of research experience in immunology, molecular biology and microbiology, has been published, and holds more than 20 patents. After receiving her B.Sc. and M.Sc in Biological Sciences in the UK and her PhD. in diagnostic immunology from Nairobi University in Kenya, Dr. Shah joined the International Laboratory for Research on Animal Diseases (ILRAD) as a post-doctoral scientist and started the first DNA sequencing laboratory in East Africa. On completion of her fellowship, she went on to join Harvard University's Department of Tropical Medicine as a research fellow and continued work on development of molecular tools for diagnosis of parasitic diseases. Since then, Dr. Shah has worked at several Biotechnology companies focusing on the development of novel molecular technologies for diagnosis of infectious diseases and has become a world expert on use of Fluorescent in Situ Hybridization (FISH) technique for direct detection of pathogens in clinical samples. Dr. Shah joined IGeneX as the Director of Research and Development in 1997 and in 2003 became Laboratory Director. During her time with IGeneX, she introduced the first Fluorescent In Situ Hybridization (FISH) test for Babesia and also set up the PCR department for tick-borne diseases. Under Dr. Shah's direction, the laboratory has developed an excellent QA program and with her scientific guidance, IGeneX has become the world's leading reference laboratory for diagnosis of tick-borne diseases. Dr. Shah is now the owner of IGeneX and continues her mission to help those struggling with vector-borne illnesses. Key Takeaways: - What is the history of IGeneX? - What is the difference between antibody and antigen testing? - Why is IgM and IgG in Borrelia not the same as other infectious conditions? - Why are bands 31 and 34 included in IGeneX Western Blots but not other commercial blots? - What is the difference between the Western Blot and the ImmunoBlot? - What is the importance of the Relapsing Fever Borrelia test options? - What testing does IGeneX offer in the realm of coinfections? - What is the IGXSpot and when might it be useful to consider? - What microbes where found in her research on Morgellons? - How can tick testing be performed with IGeneX? Connect With My Guest: http://www.IGeneX.com Interview Date: April 23, 2018 Disclaimer: The content of this show is for informational purposes only and is not intended to diagnose, treat, or cure any illness or medical condition. Nothing in today's discussion is meant to serve as medical advice or as information to facilitate self-treatment. As always, please discuss any potential health-related decisions with your own personal medical authority.

Robin is a Certified Integrative Nutrition Health Coach through the Institute for Integrative Nutrition and a member of the American Board of Drugless Practitioners. After coaching hundreds of patients with Chronic Lyme Disease, Autoimmune Disease, and Depression through her nutrition protocol, Robin launched Chronic Lyme Nutrition, a therapeutic nutrition protocol that teaches Chronic Lyme patients how to eat to reduce inflammation and depression, increase energy and strengthen the immune system. In today’s show, we talk about Lyme disease and the impact it can have on your health and fertility. This episode is brought to you by my 10 Week Fertility Awareness Mastery Program. The next session begins in April 2018. Click here to apply Don’t forget to sign up for my FREE FAM 101 video series. Click here for access.   Topics discussed in today's episode: What is Lyme disease, and what are the common symptoms? Can you ever completely eradicate Lyme disease from your body? What are the most common symptoms associated with Lyme disease? What are the co-infections that often accompany Lyme disease? How does Lyme disease affect your immune system? What are the most accurate ways to test for Lyme disease and the related co-infections? The Western Blot vs DNA Connexions tests to identify Lyme disease What should you ask your doctor if you want to know if you have Lyme disease? How is Lyme disease treated? How long does it typically take to treat Lyme disease? Why you want to find a Lyme literate doctor to support you if you think you may have Lyme How can Lyme impact your fertility? Connect with Robin: You can connect with Robin on her Website, on Facebook, and on Twitter. Resources mentioned: DNA Connexions Chronic Lyme Nutrition Better Health Guy Blogcasts - Effective Treatment of Lyme Disease with Dr. Lee Cowden, MD Take Back Your Health 10 Week Fertility Awareness Mastery Group Program Fertility Awareness 101 FREE Video Series Fertility Friday Facebook Group Related podcasts & blog posts: FFP 167 | SIBO and Gut Health | Dr. Allison Siebecker ND FFP 166 | SIBO, IBS, and Fertility | The Healthy Gut | Rebecca Coomes Fertility Awareness Podcast Episodes | Fertility Friday Join the community! Find us in the Fertility Friday Facebook Group Subscribe to the Fertility Friday Podcast in Apple Podcasts! Music Credit: Intro/Outro music Produced by J-Gantic A Special Thank You to Our Show Sponsor: Fertility Friday | 10 Week Fertility Awareness Mastery Group Program This episode is sponsored by my 10 Week Fertility Awareness Mastery Group Program! Master Fertility Awareness and take a deep dive into your cycles and how they relate to your overall health! Click here for more information!

Dos pruebas veraces y eficaces de deteccion del VIH y ETS, y quizas otra prueba mas, que de manera rapida y segura nos ayuda a saber si estamos infectados o no.

Dos pruebas veraces y eficaces de deteccion del VIH y ETS, y quizas otra prueba mas, que de manera rapida y segura nos ayuda a saber si estamos infectados o no.

Dos pruebas veraces y eficaces de deteccion del VIH y ETS, y quizas otra prueba mas, que de manera rapida y segura nos ayuda a saber si estamos infectados o no.

Originally broadcast Dec. 26, 2012 We talk with Al Shipley, musician, music writer and man of many projects, including Western Blot. Music by: Among Wolves, War on Women, Dave Fell, DDM & Rye Rye, Jumpcuts, and Western Blot. Listen now https://ia801509.us.archive.org/29/items/corridorcast_episode10/episode10.mp3

Originally broadcast Dec. 26, 2012 We talk with Al Shipley, musician, music writer and man of many projects, including Western Blot. Music by: Among Wolves, War on Women, Dave Fell, DDM & Rye Rye, Jumpcuts, and Western Blot. Listen now https://ia801509.us.archive.org/29/items/corridorcast_episode10/episode10.mp3

Hintergrund: Hyaluronan (HA) ist ein wichtiger Bestandteil von vielen Geweben und Flüssigkeiten des Körpers. HA beeinflusst die Makro- und Mikroumgebung und kann direkt über Rezeptoren wie CD44 (cluster of differentiation 44) und RHAMM (receptor for HA mediated motility) mit den Zellen wechselwirken. Dadurch hat HA Einfluss auf die Aktivierung, Migration und Proliferation von Zellen sowie auf den Umbau der extrazellulären Matrix. HA kann das Verhalten der Osteoblasten, Osteozyten und Osteoklasten beeinflussen und ist somit ein wichtiger Faktor sowohl für die gesunde Knochenhomöostase als auch für die Frakturheilung. Hyaluronansynthasen (HAS) sind komplexe Membranproteine, die für die Synthese von HA verantwortlich sind. Bei Säugetieren sind drei Isoformen bekannt: HAS1, HAS2 und HAS3. Sie zeigen eine hohe Homologie in ihrer Sequenz und Struktur, unterscheiden sich aber in Stabilität, Syntheserate und Länge des HA. Der genaue Regulierungsmechanismus der HAS ist noch nicht bekannt. Bisher wurde über eine Regulation durch externe Signalmoleküle, Ubiquitinierung oder Phosphorylierung berichtet. In der vorliegenden Arbeit wurde ein Modellsystem zur Untersuchung der Regulation der Aktivität der HAS aufgebaut. Mit diesem sollte die Interaktion der HAS mit dem Aktinzytoskelett als möglicher Regulationsmechanismus untersucht werden. Methoden: Zu diesem Zweck wurden drei Zelllinien hergestellt. Zum einen hTERT immortalisierte hMSCs (human mesenchymal stem cells), die sogenannten SCP1, welche jeweils eine der HAS-Isoformen, fusioniert mit einem eGFP-Tag, stabil exprimieren. Des Weiteren SCP1, die Lifeact-mRFPruby exprimieren, welches F-Aktin fluoreszenzmarkiert. Schließlich doppeltransduzierte hMSCs, welche sowohl HAS-eGFP als auch Lifeact-mRFPruby exprimieren. Als Gentransfersystem wurden Lentiviren eingesetzt. Zuerst wurden die Zellen hinsichtlich der stabilen und funktionellen Expression ihres Transgens anhand verschiedener Methoden untersucht. Mittels Immunfluoreszenzmikroskopie wurde eine Kolokalisation von Aktin und HAS dargestellt. In fluoreszenzmikroskopischen Timelapse-Aufnahmen wurden die Bewegungsmuster der HAS beobachtet. Ergebnisse: Mittels RT-PCR, Western Blot und Fluoreszenzmikroskopie wurde nachgewiesen, dass die Zelllinien SCP1-HAS1-eGFP D6, SCP1-HAS2-eGFP und SCP1-HAS3-eGFP E6 alle ihr jeweiliges HAS-eGFP-Gen stabil exprimieren. Die Funktionalität der HAS-eGFP wurde mit einem HA-spezifischen ELISA und mit einem selbst etablierten Aktivitätsassay untersucht, welcher das HA durch den biotinylierten HA-Bindekomplex (bHABC) färbt. Im ELISA zeigten alle Zelllinien eine statistisch signifikant höhere Hyaluronanproduktion als die Negativkontrolle. Die HAS3-überexprimierende Zelllinie erzielte von allen die höchste HA-Konzentration. In der Färbung mit bHABC war deutlich zu erkennen, dass diejenigen Zelllinien, in denen eine der HAS-eGFP-Isoformen überexprimiert wurde, eine stärkere Braunfärbung zeigten als Zellen der Negativkontrolle. Für den Nachweis, dass die HAS-eGFP in der Membran lokalisiert sind, wurden Immunfluoreszenzfärbungen gegen den Oberflächenmarker CD44 durchgeführt. Die fluoreszenzmikroskopischen Aufnahmen zeigten an Stellen, die durch die CD44-Färbung eindeutig als Membran zu erkennen sind, ebenfalls ein Signal für die HAS-eGFP. Dies bedeutet, dass die drei Isoformen der HAS-eGFP dort in der Zellmembran integriert vorlagen. Um eine Kolokalisation der HAS-eGFP mit dem Aktinzytoskelett darstellen zu können, erfolgte außerdem eine Färbung des Aktins mit Phalloidin. Bei allen Zelllinien konnte an ausgewählten Stellen eine solche Kolokalisation gesehen werden. Die hMSC-Lifeact-mRFPruby-Zellen wurden lebendig und fixiert im Fluoreszenzmikroskop betrachtet. Sie lieferten eine gute Darstellung des Zytoskeletts mit Stressfasern im Zellkörper und Aktinfilamenten im Zellcortex. Auffallend war, dass in den lebenden Zellen kurze Aktinfilamente zu sehen waren, die sich bei den fixierten Zellen nicht beobachten ließen. Um eine Interaktion zwischen den HAS-eGFP und dem Aktinzytoskelett in lebenden Zellen untersuchen zu können, wurden von den doppeltransduzierten hMSCs Timelapse-Aufnahmen angefertigt. Darin stellten sich die grün fluoreszierenden HAS-eGFP als globuläre Strukturen dar, die entlang der Aktinfilamente angeordnet waren und sich auch entlang dieser bewegten. Schlussfolgerung: Mit diesen Zellen wurde ein Modellsystem geschaffen, mit welchem eine Regulation der HAS über die Interaktion mit dem Zytoskelett untersucht werden kann. Genaueres Wissen über diesen Mechanismus kann für zukünftige Therapieansätze bei Frakturen und bei Knochenerkrankungen, wie z.B. der Osteoporose, richtungsweisend werden.

Die Strahlenempfindlichkeit des Normalgewebes ist in der humanen Bevölkerung sehr heterogen und kann bislang nicht über prädiagnostische Biomarker charakterisiert werden. Im Rahmen der vorliegenden Arbeit wurde ein Verfahren entwickelt, um die Strahlenempfindlichkeit in lymphoblastoiden Zelllinien von jungen Lungenkrebspatienten in einem Hochdurchsatz Screening-Ansatz zu untersuchen. Fünf Zelllinien mit unterschiedlicher Strahlenempfindlichkeit wurden gewählt, um in einem ungerichteten Versuchsansatz (2D DIGE Methode = two-dimensional difference gel electrophoresis) strahlenspezifische Proteinregulation nach gamma-Bestrahlung (137Cs-Quelle) zu untersuchen. Dabei konnten sowohl neue Proteine, wie z.B. Mcm7und SerpinB9 identifiziert werden, als auch Proteine (Strukturproteine, Chaperone), die bereits in der Literatur in Verbindung mit der zellulären Stressantwort beschrieben wurden. Die 2D DIGE Ergebnisse konnten beispielhaft anhand von vier Kandidatenproteinen im Westernblot validiert werden. Die Untersuchungen zeigten, dass die intraindividuellen Expressionsunterschiede nach gamma-Bestrahlung auf Proteinebene sehr gering waren. Die geringen Expressionsunterschiede konnten jedoch validiert werden. Die Untersuchungen gaben Hinweise darauf, dass die interindividuelle Strahlenantwort sehr unterschiedlich ist. Dies konnte in weiterführenden Experimenten bestätigt werden. Da die Proteinexpression der Regulation durch mikroRNAs unterliegt, wurde in einem weiteren Ansatz eine miRNA Array Analyse durchgeführt. Hier bestätigte sich ebenfalls die Beobachtung aus der 2D Proteinanalyse, dass die Strahlenantwort interindividuell sehr heterogen ist. Die Ergebnisse dieser Arbeit zeigten, dass die Strahlenantwort auf verschiedenen zellulären Ebenen intraindividuell kaum variiert, die interindividuelle Varianz aber sehr groß ist. Diese beobachtete Heterogenität erklärt die Problematik einzelne Biomarker zur Prädiktion der Strahlenempfindlichkeit zu identifizieren.

Hereditäre Myopathien, insbesondere Gliedergürteldystrophien, sind häufig mit einer begleitenden Kardiomyopathie assoziiert. Bei 15 der 27 bisher bekannten und molekulargenetisch analysierten Gliedergürteldystrophien konnte eine begleitende kardiale Komponente in unterschiedlicher Ausprägung gefunden werden. Dagegen wurde bislang nur unzureichend untersucht, wie häufig Patienten, die primär an einer hereditären Kardiomyopathie leiden, auch eine Skelettmuskelbeteiligung aufweisen, beispielsweise wie oft Patienten mit einer hypertrophen bzw. obstruktiven Kardiomyopathie (HCM oder HOCM) auch eine Gliedergürtelschwäche zeigen. Bei einer Familie mit einer bekannten familiären Kardiomyopathie konnte eine autosomal-dominante Mutation im MYBPC3-Gen gesichert werden. Mutationen in diesem Gen gehören zu den häufigsten genetischen Ursachen einer HCM. Einige schwerer betroffene Familienmitglieder litten zusätzlich an einer Gliedergürteldystrophie. Eine Gesamtexom-Analyse mittels Next-Generation-Sequencing lieferte Hinweise auf Varianten in möglichen weiteren Kandidatengenen, die die Schwere des Phänotyps erklären könnte. Alternativ wurde die Hypothese überprüft, ob Mutationen im MYBPC3-Gen neben ihrer bekannten Rolle als Ursache von Kardiomyopathien auch an der Entstehung von Gliedergürtelmuskelschwäche beteiligt sein könnten. Ein repräsentatives Patientenkollektiv wurde sowohl von neurologischer als auch von kardiologischer Seite ausgewählt, um eine klinische Kombination der Erkrankungen teilweise bestätigt zu finden. In einem kleinen Kollektiv von sieben Patienten mit einer hereditären hypertrophen (obstruktiven) Kardiomyopathie und verschiedenen Mutation im MYBPC3-Gen wurde bei drei Patienten eine bisher nicht diagnostizierte leichtgradige Muskelschwäche bestätigt, was die Möglichkeit einer Assoziation eines Gliedergürtelphänotyps und einer hereditären hypertrophen (obstruktiven) Kardiomyopathie mit Mutationen im MYBPC3-Gen nahelegte. Um zu überprüfen, ob MYBPC3-Genvarianten eine bisher übersehene Ursache erblicher Skelettmuskelerkrankungen sind, wurde ein Kollektiv von 59 Patienten mit dem klinischen Phänotyp einer Gliedergürteldystrophie, einem dystrophen Bild in der Muskelhistologie und positiver Familienanamnese ausgewählt, mit bislang aber negativer genetischer Testung in einer Reihe von bekannten LGMD-Genen. Eine molekulargenetische Analyse aller kodierenden Exons des MYBPC3-Gens lieferte jedoch keine pathogene Sequenzvariante. Auch bei einer anderen erblichen Myopathie, die als GNE-Myopathie oder hereditäre Einschlusskörpermyopathie bezeichnet wird, da sie durch Mutationen im GNE-Gen bedingt ist und muskelbioptisch charakteristische Einschlusskörper aufweist, ist neben der Skelettmuskelerkrankung auch eine kardiale Beteiligung beschrieben worden, die zwar selten auftritt, aber zu infausten Prognosen führen kann. Die GNE-Myopathie wird autosomal-rezessiv vererbt und präsentiert sich klinisch mit einer distal betonten und im weiteren Verlauf nach proximal fortschreitender Muskelschwäche, die eine typische Aussparung des M. quadriceps femoris zeigt. In der vorliegenden Arbeit wurde das GNE-Gen bei 20 klinisch ausführlich charakterisierten Patienten molekulargenetisch analysiert, mit dem Ziel, das bisher beschriebene Spektrum an bekannten Mutationen zu erweitern. Bei zwei Patienten konnten bereits publizierte Mutationen gefunden werden, wovon eine auch mit einem kardialen Phänotyp assoziiert wird. Die zusätzlich durchgeführte Proteinexpressions- und Lokalisationsdiagnostik von GNE in der Zelle mittels Western-Blot und Immunfluoreszenzmikroskopie sollte Aufschluss über die genaue Lokalisation und Funktion von GNE erbringen, um die Pathogenesemechanismen genauer zu verstehen und dadurch zielgerichtet therapeutische Strategien entwickeln zu können. Eine Einschlusskörpermyopathie findet sich ebenfalls bei der sehr seltenen hereditären IBMPFD, bei der es sich um einen Symptomenkomplex aus Einschlusskörpermyopathie (inclusion body myopathy, IBM), frontotemporaler Demenz und M. Paget des Knochens handelt, der durch Mutationen im VCP-Gen bedingt ist. Bei der IBMPFD wurden bislang weltweit 19 verschiedene Mutationen in 38 Familien gefunden. Die Erkrankung kann sich häufig auch klinisch oligosymptomatisch manifestieren, d. h. nur mit einer Myopathie. In einem Patientenkollektiv mit passendem Phänotyp ohne Nachweis einer Mutation im VCP-Gen, wurde die Analyse eines neuen, vielversprechenden Kandidatengens etabliert. Das HDAC6-Gen kodiert für ein gleichnamiges Protein, das durch die Interaktion mit VCP beim Abbau fehlgefalteter Proteine identifiziert wurde und an der Aggresombildung beteiligt ist. Somit wurden Patienten mit einem IBM- oder IBMPFD-Phänotyp und negativer genetischer Testung im VCP-Gen einer HDAC6-Genanalyse unterzogen. Eine pathogene Sequenzvariante ließ sich dabei jedoch nicht detektieren. Künftig werden neue Technologien wie die Gesamtexom-Sequenzierung die aufwändige Kandidatengenanalyse ablösen, um bei neuromuskulären Erkrankungen Mutationen in neuen oder bekannten Genen zu identifizieren, die wesentliche Erkenntnisse zur Genotyp-Phänotyp-Korrelation liefern.

Das West-Nil-Virus (WNV) ist ein zur Familie der Flaviviren gehörendes Arbovirus, das weltweit zunehmende Verbreitung findet. Das natürliche Reservoir des Virus sind Vögel. Nach Übertragung durch Stechmücken kann es zu Infektionen von „Fehlwirten“, insbesondere Pferden und Menschen, kommen. Die meisten Infektionen verlaufen asymptomatisch oder mit der Entwicklung des West-Nil-Fiebers, einer relativ milden, Grippe-ähnlichen Erkrankung. In einigen Fällen, vor allem bei immungeschwächten und älteren Individuen, können aber auch lebensbedrohliche Infektionen mit schwerer neurologischer Symptomatik (z.B. Enzephalitiden) die Folge sein. WNV-Impfstoffe sind bisher nur für die Veterinärmedizin zugelassen und diese benötigen für einen effektiven Schutz häufige Auffrischungen. Außerdem gibt es keine effizienten Therapiemöglichkeiten. Aus diesem Grund ist die Entwicklung weiterer wirksamer WNV-Impstoffe wünschenswert. Ziel dieser Arbeit war es, verschiedene rekombinante Vakzinkandidaten auf Basis des Modifizierten Vacciniavirus Ankara (MVA) zu entwickeln, zu analysieren und bezüglich ihrer Eignung als Vektorvakzin zu bewerten. Das in seiner Replikationsfähigkeit extrem limitierte und hoch attenuierte MVA gehört bei der Entwicklung neuartiger rekombinanter Virusvakzine zu den viel versprechendsten Kandidaten. Potentielle WNV-Vektorvakzine beruhen überwiegend auf der Expression der beiden viralen Hüllproteine prM/M und E oder Teilen davon. Gerade das E-Protein stellt nach einer Infektion das Hauptzielantigen der adaptiven Immunantwort dar, indem es eine Vielzahl an immunogenen und protektiven Epitopen aufweist. Die fünf in dieser Arbeit hergestellten rekombinanten Viren exprimierten zum Teil das E-Protein in unterschiedlicher Ausführung oder prM/M und E simultan. Damit wurden verschiedene Ansätze zur Induktion einer Immunantwort generiert und untersucht. Alle rekombinanten MVA-Vektorviren waren bis auf die inserierten Zielsequenzen identisch und erwiesen sich als genetisch stabil. Die Replikationsdefizienz der Viren in den humanen und equinen Zielzellen konnte eindeutig nachgewiesen und somit ihre biologische Sicherheit belegt werden. Für die Erzeugung hochtitriger Virusstocks und zur Impfstoffproduktion in größerem Umfang war es notwendig zu zeigen, dass sich die ins MVA-Genom inserierten Sequenzen nicht negativ auf das Vermehrungspotential der Viren in permissiven Zellen auswirkten. Es konnte belegt werden, dass alle Konstrukte dem Wildtypvirus ähnliche, und somit zur Produktion ausreichende, Wachstumsfähigkeiten besaßen. Als weitere wichtige Voraussetzung für die potentielle Verwendung der rekombinanten Viren als Kandidaten-Vakzine galt eine effiziente rekombinante Proteinexpression. Durch die Analyse der Proteinsynthese mittels Westernblot konnte nachgewiesen werden, dass diese bei allen Konstrukten stabil und produktiv verlief. Auch die Lokalisierung der rekombinanten E-Proteine durch Immunfluoreszenzfärbung und nachfolgender Konfokalmikroskopie infizierter Zellen brachte das erwartete Ergebnis. Abschließend wurden zur ersten Einschätzung der Immunogenität der rekombinanten Viren WNV-spezifische Antikörper- und T-Zellantworten im Mausmodell untersucht. Alle Vektorviren waren in der Lage humorale und zelluläre Immunantworten zu induzieren. Hierbei erwies sich MVA-WNVESOL, was die, mittels Antigen-ELISA ermittelte, Antikörperantwort anbelangt als viel versprechendster Kandidat. Bezüglich der CD8+-T-Zellantwort konnte sich dies jedoch nicht bestätigen. Es ist anzumerken, dass weiterführende Untersuchungen der Testimpfstoffe in anderen präklinischen Modellen in Zukunft noch durchzuführen sein werden. Die in dieser Arbeit hergestellten rekombinanten Viren und gewonnenen Erkenntnisse belegen die Fähigkeit von MVA als viel versprechenden Vektorvakzin-Kandidaten gegen WNV. Die nachgewiesene Sicherheit und zugleich gute Vermehrungsfähigkeit in permissiven Zellen, die effiziente WNV-Antigen-Expression und die ersten positiven Daten zur Immunogenität aller Konstrukte sprechen für eine zukünftige, weitere Nutzung und Untersuchung dieser Vektorviren, um als langfristiges Ziel einen potenten WNV-Impfstoff zu erhalten.

Mesenchymale Stammzellen (MSCs) besitzen eine Vielzahl einzigartiger Eigenschaften, hierunter ihr Differenzierungs- und immunregulatorisches Potenzial, die sie sehr interessant für die biomedizinische Forschung machen. Ziel der vorliegenden Arbeit war es zu untersuchen, welche Mechanismen der immunmodulierenden Wirkung der MSCs zugrunde liegen. Zu diesem Zweck wurden mesenchymale Stammzellen aus dem Knochenmark von 5-16 Wochen alten Balb/c-Mäusen isoliert und in der Zellkultur expandiert. Das Oberflächenmarkerprofil der Zellen wurde durch Durchflusszytometrie (FACS) charakterisiert und die Expression potenziell immunsuppressiver Faktoren durch ELISA, Reverse Transkriptase-PCR beziehungsweise Western-Blot bestimmt. Für die Untersuchung der MSC-vermittelten Immunmodulation wurde ein Proliferationsassay etabliert, in dem Lymphozyten aus C57Bl/6-Mäusen durch das Mitogen ConA stimuliert und mit den murinen MSCs kokultiviert wurden. Durch die Zugabe spezifischer Inhibitoren gegen TGFβ, die Prostaglandin E2-Synthese (Indomethacin), den HGF-Rezeptor und den Adenosinrezeptor (SCH58261) wurde die Vermittlung der immunmodulierenden Effekte durch die MSCs untersucht. In der Studie konnte eine reine Population muriner MSCs mit charakteristischen Oberflächenmarkern (CD29, CD73, CD90, CD105, CD140b, Sca-1) expandiert werden, die in vitro starke immunsuppressive Effekte im Proliferationsassay aufwies. Die Untersuchungen potenzieller Effektormoleküle zeigten, dass bei den murinen MSCs TGFβ, HGF und das Enzym IDO keine entscheidende Rolle spielten und von den bekannten Mechanismen vor allem die Produktion von PGE2 die Lymphozytenproliferation hemmte. Als wichtigstes Ergebnis kann der Nachweis, dass die murinen MSCs die Ektonukleotidasen CD39 und CD73 koexprimieren, die extrazelluläres ATP über 5’-AMP in das extrem stark immunsuppressiv wirkende Adenosin konvertieren können, angesehen werden. Zusammenfassend wurde in der vorliegenden Arbeit erstmals die Expression von CD39 auf murinen MSCs beschrieben. Die Koexpression von CD39/CD73 auf den MSCs und die daraus folgende Produktion von Adenosin stellt einen neuen Ansatz dar, um die Funktion der MSCs besser zu verstehen. Die CD39+/CD73+-MSCs sind demnach eine vielversprechende Zellpopulation für die Behandlung von Autoimmunerkrankungen und für die Entwicklung neuer lokaler oder systemischer Therapien zur Toleranzinduktion nach Transplantation.

Background: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. Methods: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. Results: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual gamma-H2AX foci after 24 h. Conclusion: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Die Alzheimer-Krankheit ist die häufigste Form der Demenz mit einer stetig steigenden Zahl Betroffener. Trotz intensiver Forschung sind die genauen Ursachen noch immer weitgehend ungeklärt, mit der Folge, dass bislang keine kausale Therapie zur Verfügung steht. Als einer der vermuteten Risikofaktoren gilt das Geschlecht – Frauen erkranken häufiger und mit insgesamt schwererem Verlauf an Alzheimer als Männer. Ziel der vorliegenden Studie ist es, das transgene Mausmodell Tg2576, das ein humanes Amyloid-Vorläuferprotein mit der schwedischen Doppelmutation überexprimiert, phänotypisch umfassend zu charakterisieren und auf geschlechtsspezifische Differenzen hin zu untersuchen. Dabei werden jeweils sechs männliche und sechs weibliche Träger des Transgens (Carrier) im Alter von 6, 8, 10, 12, 14 und 16 Monaten mit ihren gesunden Wurfgeschwistern (Wildtypen) verglichen. Die Tiere werden im modifizierten Hole-Board-Test über acht Tage auf kognitive, verhaltensbedingte und motorische Parameter untersucht. Ab einem Alter von 8 bis 10 Monaten weisen die Carrier im Gegensatz zu den Wildtypen progressive, signifikante Beeinträchtigungen des deklarativen Gedächtnisses und des Arbeitsgedächtnisses auf. Damit einhergehend können eine verstärkte Anwendung nicht-räumlicher Suchstrategien sowie disinhibitorische Verhaltenstendenzen beobachtet werden. Im Geschlechtervergleich sind die kognitiven Defizite der weiblichen Carrier signifikant stärker ausgeprägt. Weiterhin zeigen sich ab einem Alter von 10 Monaten geschlechtsunabhängige, progressiv fortschreitende feinmotorische Störungen beim Fressen der Belohnungsmandeln. Die Mortalität der männlichen Carrier (48,4 %) ist signifikant höher im Vergleich zu den weiblichen Carriern (25,4 %) (p = 0,005). Die weibliche Tiere weisen ein signifikant niedrigeres Gewicht als die männliche Tiere auf (p < 0,001), wobei die Carrier signifikant weniger wiegen als die Wildtypen (p < 0,001). Laboranalysen zeigen im Geschlechtervergleich einen höheren Testosterongehalt im Blutserum der männlichen Tiere und einen höheren Östradiolgehalt bei den weiblichen Tieren. Bei der Untersuchung auf mögliche zugrundeliegende Störungen der Neurotransmission können keine mit zunehmendem Alter progressiven Veränderungen der Expression spezifischer Rezeptoren (NMDAR-NR2B, mGluR5 und PBR) im Western-Blot gezeigt werden. Insgesamt erweist sich das Tg2576-Modell als ein vielversprechendes Modell der Alzheimer-Krankheit für die weitere Grundlagenforschung und präklinische Testung therapeutischer Strategien. Dabei ist es insbesondere auch für weiterführende geschlechtsspezifische Forschungsansätze geeignet.

Für das Überleben von Bacillus subtilis ist eine verlässliche Überwachung der Integrität der Zellhülle essentiell, um diese zu schützen und bei Schäden adäquat zu reagieren. Neben den ECF � Faktoren spielen Zwei-Komponenten-Systeme (2KS) in der Zellhüllstressantwort von B. subtilis eine zentrale Rolle. Eines dieser Systeme, das LiaRS- 2KS reagiert auf eine große Anzahl verschiedener Zellwand-Antibiotika sowie andere zellhüllstress-auslösende Substanzen. Die zelluläre Funktion und Rolle des Lia-Systems konnte bisher nicht genau definiert werden. In der hier vorliegenden Dissertation wurde das Lia-System erstmals hinsichtlich seiner funktionalen Rolle in B. subtilis untersucht. Im ersten Teil der Ergebnisse wurde eine detaillierte Analyse der LiaR-vermittelten Zellhüllstressantwort in B. subtilisvorgenommen. Transkriptom-Studien dienten zur Identifizierung des LiaR-Regulons. Hierbei wurde die Genexpression des Wildtyps mit zwei Mutanten, die den „ON“ (�liaF) und „OFF“ (�liaR) Zustand des Lia-Systems repräsentierten, verglichen. Von den dabei identifizierten drei potentiellen LiaR-Zielloci (liaIH, yhcYZ-ydhA, ydhE) konnten durch anschließende Folgeuntersuchungen nur die Gene liaI und liaH als in vivo relevante Zielgene für LiaR verifiziert werden. Umfangreiche phänotypische Analysen zeigten, dass �liaIH-Mutanten nur schwach sensitiv auf einige Antibiotika sowie oxidativen Stress reagierten. Ebenso vermittelt eine Überexpression von LiaH in einer �liaF-Mutante keine Resistenz gegenüber stressauslösenden Substanzen. LiaH gehört zur Familie der Phagenschock-Proteine. Weitere Mitglieder dieser Familie sind PspA aus Escherichia coli und Vipp1 aus Arabidopsis thaliana, die große oligomere Ringstrukturen bilden. Die strukturelle Untersuchung von LiaH ergab, dass auch dieses Protein große Ringe bildet (>1MDa). Der zweite Ergebnisteil befasst sich mit der Untersuchung der Stimuluswahrnehmung der Zellhüllstress-detektierenden Systeme in B. subtilis. Die Zellhüllstressantwort auf das Antibiotikum Bacitracin wurde hierbei mittels �-Galaktosidase-Assay sowie Western Blot- Analyse erforscht. Das Bce-System reagiert dabei am stärksten und spezifischsten auf Bacitracin-Stress. Es wurde ebenfalls festgestellt, dass der ABC-Transporter BceAB essentiell für die Stimuluswahrnehmung ist und dass das Bce-System an sich eine Resistenzdeterminante in B. subtilis darstellt. Das Lia-System hingegen wird erst bei höheren Bacitracin-Konzentrationen induziert. Zusammengefasst deuten diese Ergebnisse darauf hin, dass das Bce-System Bacitracin direkt wahrnimmt (drug sensing) und das LiaSystem in indirekter Weise auf Zellhüllstress ausgelöst durch Bacitracin reagiert (damage sensing). Im dritten Teil der Ergebnisse wurdendie zelluläre Lokalisation von LiaI, LiaH und LiaG sowie die Beziehung der Proteine untereinander mittels Fluoreszenz-Mikroskopie und biochemische Ansätze untersucht. Die Membranproteine LiaI und LiaG sind unter Stressbedingungen in der Zellmembran lokalisiert. LiaH, ein cytoplasmatisches Protein verändert unter Stressbedingungen seine Lokalisation vom Cytoplasma an die Membran. Die Funktion von LiaH scheint sich also an der Zellmembran zu vollziehen, wobei LiaI als Interaktionspartner identifiziert wurde. Da in einer �liaI-Mutante LiaH unter Stressbedingungenebenfallsnoch an die Zellmembran assoziert ist, wurde nach weiteren Interaktionspartnern von LiaH gesucht. Eine umfangreiche bacterial-two-hybrid-Analyse ergab, dass sowohl LiaH als auch LiaI und LiaG in ein Interaktionsnetzwerk eingebettet sind, in welchem das bisher uncharakterisierte Protein YvlB eine Schlüsselrolle spielt.Die ebenso in dieses Netzwerk involvierten Proteine YjoB, DnaK und HtpG üben als Proteasen/Chaperone Funktionen in der Faltung und Degradierung von Proteinen aus. Ein Zusammenspiel des Lia-Systems und des Schlüsselproteins YvlB mit den Proteasen/Chaperonen als Reaktion auf Zellhüllstress ist denkbar. Die Phagenschock-Homologe PspA in Streptomyces lividans und E. coli üben einen erheblichen Einfluss auf die Proteinsekretion sowie die elektronenmotorische Kraft der Zelle aus. Daher wurde im letzten Teil der Ergebnisse die Rolle von LiaH in der Proteinsekretion sowie im Energiestoffwechsel näher analysiert. Ein Einfluß des Lia- Systems in der Aufrechterhaltung der elektronenmotorischen Kraft der Zelle konnte nicht bestätigt werden. Durch die Analyse des Sekretoms in B. subtilis konnte gezeigt werden, dass das extrazelluläre Proteom einer �PliaI-liaIH-Mutante im Vergleich zum Wildtyp signifikante Veränderungen in der Komposition aufwies.So wurde im Sekretom der �PliaIliaIH- Mutante vor allem das Zellwand-assoziierte Protein WapAidentifiziert, welches im Wildtyp oder in einer �liaF-Mutante nicht auftrat. Das Lia-System beeinflußt somit auch die Proteinsekretion von B. subtilis, wobei die molekularen Mechanismen noch unbekannt sind.

Human peritoneal mesothelial cells (MC) play an important role in inflammatory processes of the peritoneal cavity by producing various cytokines and chemokines, such as monocyte chemoattractant protein-1 (MCP-1). The present study was designed to assess the effect of the peroxisome proliferator-activated receptor-gamma- (PPAR gamma-) activator rosiglitazone on the mesothelial MCP-1 expression and release. Primary cultures of MC were obtained from omental tissue. MCP-1 antigen concentrations were measured in the cell supernatant by ELISA and MCP-1 mRNA levels by real-time polymerase chain reaction. The presence of PPAR. on MC was assayed in a Western Blot analysis. MC constitutively express PPAR gamma. Activation of this receptor via rosiglitazone (0,1-10 mu mol/L) resulted in significantly reduced amounts of mesothelial MCP-1 release as well as MCP-1 mRNA. The use of the PPAR gamma inhibitor GW-9662 could completely prevent the rosiglitazone effects. Rosiglitazone was also effective in reducing TNF alpha-induced enhanced secretion of MCP-1. Our findings indicate that glitazones are effective in reducing constitutive and TNF alpha-stimulated mesothelial MCP-1 mRNA expression and release.

Die vorliegende Dissertation befasst sich mit den durch S-Lost-verursachten Symptomen in der Haut, die zunächst durch starke Blasenbildung und später durch eine verzögerte Wund-heilung charakterisiert sind. Bei S-Lost handelt es sich um einen chemischen Kampfstoff, der erstmals im ersten Weltkrieg zum Einsatz kam und bis heute in vielen internationalen Kon-flikten großen Schaden anrichtete, obwohl der Gebrauch schon 1925 durch die Genfer Konvention verboten wurde. Aktuell stellt S-Lost zudem eine Bedrohung durch terroristische Aktivitäten dar. Da für S-Lost-induzierte Verletzungen bislang keine spezifisch wirksamen Behandlungs-methoden verfügbar sind, besteht großes Interesse an der Aufklärung der dem Krankheitsbild zugrunde liegenden molekularen Pathomechanismen, um daraus Rückschlüsse auf besser ge-eignete therapeutische Maßnahmen ziehen zu können. In unseren ersten Experimenten wurden als mögliche Auslöser der Blasenbildung die Expression und Sekretion ausgewählter Matrix-Metalloproteinasen (MMPs) und deren endogenen Inhibitoren, den tissue inhibitors of matrix metalloproteinases (TIMPs) in einem 3D-Haut-modell und in verschiedenen Zelltypen der Haut (Keratinozyten, Fibroblasten, mikrovaskuläre Endothelzellen, mesenchymale Stammzellen, monozytäre Zellen, PMN-Granulozyten) sowohl in Mono- als auch in Mischkultur untersucht. Unter Verwendung von molekularbiologischen und proteinbiochemischen Methoden wie qRT-PCR, Zymographie und Western Blot gelang der Nachweis, dass MMPs - insbesondere MMP-9 - nach Exposition der Zellen (v.a. Fibroblasten und monozytäre Zellen) mit S-Lost deutlich hochreguliert wurden. Zu Erhärtung der Annahme, dass MMP-9 durch Degradation der Basalmembran zwischen Epidermis und Dermis zur Blasenbildung beiträgt, konnte als Pathomechanismus nun erstmals eine parakrine Stimulation von Fibroblasten durch S-Lost-behandelte Keratinozyten identifiziert werden, als deren Folge eine vermehrte MMP-9-Sekretion resultierte. Darüber hinaus zeigte sich in weiteren Versuchen unter Verwendung des sog. Scratch-Assays und eines Transwell-basierten Invasionsassays, dass das Migrations- und Invasionsverhalten der Fibroblasten in Gegenwart des konditionierten Mediums der S-Lost-behandelten Keratinozyten positiv beeinflusst wurde. Aus klinischer Sicht sprechen diese Erkenntnisse für neue therapeutische Ansätze, die darauf beruhen sollten, die S-Lost-induzierte, auf proteolytischer Aktivität basierende Blasenbildung der Haut durch Applikation spezifischer MMP-Inhibitoren zu behandeln. In einem weiteren Projekt wurde die verzögerte Wundheilung als spätes Symptom der S-Lost-Vergiftung auf zellulärer Ebene untersucht, bei der eine eingeschränkte Re-Epithelialisierung der betroffenen Hautstellen beobachtet wird. Sowohl für den Prozess der Wundheilung als auch für die stetige Erneuerung der Haut werden epidermale Stammzellen benötigt, die für die Bildung von Keratinozyten verantwortlich sind. Diese unipotenten Progenitorzellen befinden sich in der basalen Schicht der Epidermis und sind in der Lage zu proliferieren und anschließend terminal zu differenzieren. Um eine Beeinflussung dieser Prozesse durch S-Lost zu untersuchen, wurden primäre unreife Keratinozyten (NHEK) verwendet und hinsichtlich ihres Differenzierungspotenzials untersucht. Dabei erwies sich S-Lost als potenter Induktor der Differenzierung von NHEK, was durch Bestimmung der Expression typischer Markerproteine wie Keratin-1, Involucrin und Loricrin gezeigt wurde. Die Induktion des Reifungsprozesses war sowohl von einem Rückgang der Proliferation als auch von einer verminderten Migrationsrate der Zellen begleitet. Die eingehende Analyse von mitogen-activated protein kinase (MAPK)-Signaltransduktionswegen führte zu der Erkenntnis, dass die Aktivitäten von p38 und ERK1/2 gegenteilige Rollen im Differenzierungsprozess einnehmen. Studien mit spezifischen Inhibitoren der MAPK be¬legten, dass p38 für den Reifungsvorgang in NHEK essentiell ist, während ERK1/2 diesem entgegen wirkt. So konnte durch Blockade von p38 die von S-Lost ausgelöste Differenzierung der Zellen verhindert werden. Ebenso war es durch diese Behandlung möglich, die von S-Lost stark beeinträchtige Migrationsfähigkeit der Keratinozyten wiederherzustellen, welche mit einer erhöhten MMP-1-Expression einherging. Davon abgeleitet erscheint es therapeutisch sinnvoll, selektive p38-Inhibitoren für die Behandlung von Wundheilungsstörungen nach Exposition der Haut mit S-Lost einzusetzen. Zusammenfassend erbrachten unsere Studien also den Nachweis, dass der S-Lost-induzierten Blasenbildung (als frühes Symptom) die spezifische Induktion der MMP-9 zugrunde liegt. Darüber hinaus konnte erstmals eine verfrühte Differenzierung in unreifen Keratinozyten der Haut (als mögliche Ursache für die verzögerte Wundheilung) nachgewiesen werden, wobei die MAPK p38 bei der Initiierung des Prozesses von entscheidender Bedeutung ist. Aufgrund dieser Resultate empfiehlt sich eine kombinierte Applikation von Inhibitoren der Aktivitäten von MMP-9 und p38, wobei der Einsatz jedoch zeitlich abgestimmt erfolgen sollte, um pathologische Effekte (Blasenbildung bzw. Differenzierungsinduktion in Keratinozyten) zu blockieren, ohne die positiven Auswirkungen von MMP-9 und p38 auf die Heilung (Migration von Immunzellen und Keratinozyten bzw. Reepithelialisierung) zu hemmen.

Infektionen mit Pigeon-Circovirus gelten als Wegbereiter für ein verlustreiches und bei jungen Brieftauben weit verbreitetes multifaktorielles Krankheitsgeschehen, der Jungtaubenkrankheit. Neben der klinisch manifesten Form einer PiCV-Infektion kommt es auch zu subklinischen Infektionen, die am lebenden Tier durch die bislang entwickelten Nachweismethoden nicht sicher zu diagnostizieren sind. Im Rahmen dieser Arbeit wurde ein indirektes Virusnachweisverfahren entwickelt und zur Untersuchung von Taubenseren eingesetzt. Da PiCV nicht mit klassisch-kulturellen Verfahren vermehrt werden kann, wurde das Antigen für die Antikörperdetektion, basierend auf dem PiCV-Kapsidprotein, rekombinant in E. coli exprimiert. Um die Expression des Virusproteins im prokaryotischen System zu erleichtern, wurde die cap-Sequenz ohne die Arginin-Codon-reichen, 5’-terminalen 117 Nukleotide kloniert. Ein 5’-terminal eingefügter 6xHistidin-Tag ermöglichte die Aufreinigung mittels Metall-Affinitäts-Chromatographie und den Nachweis des rekombinanten Kapsidproteins rCapPiCV über anti-His-Antikörper. Durch Expression des Konstrukts und anschließende denaturierende Aufreinigung konnte rCapPiCV in großer Menge und hoher Reinheit gewonnen werden. Durch vergleichende Untersuchung der Reaktivität von rCapPiCV gegen Präimmun- und Immunseren von experimentell PiCV-infizierten Tauben und einem anti-His-Antikörper im Western Blot konnte die spezifische Bindung von anti-PiCV-Antikörpern durch das rekombinante Protein gezeigt werden. Damit gelang erstmals der Nachweis von anti-PiCV-Antikörpern. Auf der Basis des rCapPiCV wurde ein indirekter ELISA entwickelt und zur Untersuchung von Taubenseren eingesetzt. Mit Hilfe des rCapPiCV-ELISAs konnte die Serokonversion experimentell PiCV-infizierter Tauben ein bis drei Wochen p. i. gezeigt werden. Die Untersuchung im Western Blot bestätigte bei allen Seren das Ergebnis der serologischen Untersuchung im ELISA und damit die Spezifität der Antigenpräparation. Der rCapPiCV-ELISA wurde daraufhin zur Untersuchung eines natürlich, subklinisch infizierten Taubenbestands eingesetzt und detektierte in 30 (81 %) von 37 getesteten Taubenseren anti-PiCV-Antikörper. Dadurch wurde gezeigt, dass das entwickelte indirekte Virusnachweisverfahren geeignet ist, PiCV-Infektionsgeschehen auf Bestandsebene nachzuweisen. Aus dem Patientengut der Klinik für Vögel wurden Seren von Tauben mit unbekannten PiCV-Infektionsstatus aus den Jahren 1989, 1991 und 1994 ausgewählt und im rCapPiCV-ELISA getestet. Von 81 Seren waren 59 (73 %) PiCV-seropositiv, was darauf schließen lässt, dass PiCV schon vor seiner Erstbeschreibung 1997 in Deutschland in Taubenpopulationen zirkulierte. Die vorgestellten serologischen Methoden können in weiterführenden Studien zu Epidemiologie, Pathogenese und Verlauf der PiCV-Infektion eingesetzt werden. Diese Daten aus der Forschung sind nötig, um die Bedeutung der PiCV-Serologie auf Bestands- und Einzeltierebene und ihren Nutzen für die klinische Diagnostik zu bewerten. Neben dem Einsatz zur Bindung virusspezifischer Antikörper, ist rCapPiCV eine potenzielle Subunit-Vakzine für die PiCV-Impfstoffentwicklung.

Background: Knowledge of differences in the cellular physiology of malignant and non-malignant cells is a prerequisite for the development of cancer treatments that effectively kill cancer without damaging normal cells. Calcium is a ubiquitous signal molecule that is involved in the control of proliferation and apoptosis. We aimed to investigate if the endoplasmic reticulum (ER) Ca2+-homeostasis is different in lung cancer and normal human bronchial epithelial (NHBE) cells. Methods: The intracellular Ca2+-signaling was investigated using fluorescence microscopy and the expression of Ca2+-regulating proteins was assessed using Western Blot analysis. Results: In a Small Cell Lung Cancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC) cell line but not in a Squamous Cell Lung Cancer (EPLC) and a Large Cell Lung Cancer (LCLC) cell line the ER Ca2+-content was reduced compared to NHBE. The reduced Ca2+-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Lowering the ER Ca2+-content with CPA led to increased proliferation NHBE and lung cancer cells. Conclusion: The significant differences in Ca2+-homeostasis between lung cancer and NHBE cells could represent a new target for cancer treatments.

Eine große Gruppe genetisch vererbter Erblindungskrankheiten steht im Zusammenhang mit Mutation in Genen, die in Photorezeptoren exprimiert sind. Diese Mutationen führen nicht nur zu einer Beeinträchtigung des mutierten Proteins selbst, sondern auch zu einer Störung von funktionell nachgeschalteten Proteinnetzwerken. In der Folge ändern sich die Zusammensetzung von Multiproteinkomplexen sowie die Proteinlokalisation, was schwerwiegende physiologische Konsequenzen nach sich zieht. Alleine im lichtwahrnehmenden Molekül Rhodopsin sind mehr als hundert unterschiedliche Mutationen beschrieben worden, die vermutlich im Zusammenhang mit Retinitis pigmentosa, einer degenerativen Erkrankung der Retina, stehen (http://www.sph.uth.tmc.edu/RetNet/). In Saccharose-Dichte Gadienten Experimenten von Dr. Magdalena Swiatek-deLange, die dieser Studie vorangegangen sind, wurde Rhodopsin als Teil eines potentiellen Rhodopsin/Ras Homolog Gene Family, Member A (RhoA)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/RhoKinase II (Rock II)/ Collapsin response mediator protein 2 (CRMP2) Signal-Multiproteinkomplexes in Außensegmenten von Stäbchen Photorezeptoren (ROS) identifiziert, welcher im Zuge dieser Studie bestätigt und eingehender untersucht wurde. Ein Zusammenhang zwischen einer Rhodopsin-vermittelten Degeneration von Photorezeptoren und der Regulation des Cytoskeletts durch die kleine GTPase Rac1, wurde von Chang und Kollegen (Chang and Ready, 2000) hergestellt. Sie haben gezeigt, dass die Expression von dominant-aktivem Rac1 in Rhodopsin-Null Mutanten von Drosophila die Rhabdomer Morphogenese erhalten kann. In Zellen fungiert Rac1 durch den Wechsel zwischen einem inaktiven, vorwiegend cytosolischen und einem aktiven, überwiegend membranassoziierten Zustand, als molekularer Schalter in der Signaltransduktion und vermittelt Signale von Membranrezeptoren an das Cytoskelett. Obwohl die Rolle von Rac1 bereits in einer großen Zahl unteerschiedlicher Zellen untersucht worden ist, ist seine Funktion in Photorezeptoren noch immer weitgehend ungeklärt. Die wenigen vorhanden Studien, in denen beispielsweise gezeigt wurde, dass Rac1 an der Fusion von Rhodpsintransportcarriern in Rana barlandieri (Deretic et al., 2004) oder auch an der lichtinduzierten Degeneration von murinen Photorezeptoren beteiligt ist (Belmonte et al., 2006), machen aber deutlich, dass Rac1 ein für die Funktion und Regulation von Photorezeptoren wichtiges Molekül ist. In dieser Studie wurde daher die Rolle von Rac1 in Photorezeptoren eingehender untersucht und ein Rac1-Interaktom in ROS, bestehend aus 22 Interaktoren, identifiziert. Von diesen 22 identifizierten Interaktoren sind fünf bereits als Interaktoren von Rac1 beschrieben worden, darunter CRMP2, einer der Hauptregulatoren von Polarität in neuronalen Zellen, sowie die cytoskelettalen Proteine Aktin ( and  und Tubulin ( and Unter den 17 neuen potentiellen Rac1 Interaktoren befindet sich das Aryl Hydrocarbon Receptor-Interacting Protein Like 1 (AIPL1), das im Zusammenhang mit Leberscher kongenitaler Amaurose (LCA) sowie mit retinalem Proteintransport steht (Sohocki et al., 2000), sowie eine Reihe von Proteinen, die Teil der Phototransduktionskaskade sind, wie die Untereinheit der 3´, 5´-cyclic-GMP Phosphodiesterase 6, Recoverin, Arrestin sowie die ,  und  Untereinheiten von Transducin. Rac1 verbindet damit die Lichtwahrnehmung durch Rhodopsin mit einer Regulation des Cytoskeletts und legt damit eine Interdependenz von Lichtwahrnehmung mit einer korrekten zellulären und funktionalen Struktur von Photorezeptoren nahe. In dieser Studie wurde nicht nur die Existenz des potentiellen Rhodopsin/RhoA/Rac1/Rock II/CRMP2 Multiproteinkomplexes in ROS bestätigt, sonder auch eine lichtabhängige Dynamik und Interaktion der einzelnen Komplexbestandteile beschrieben. In Übereinstimmung mit Daten aus verschiedenen Organismen ((Wieland et al., 1990), (Petrov et al., 1994), (Balasubramanian and Slepak, 2003)) konnte eine lichtabhängige Aktivierung von Rac1 in ROS von Schweinen nachgewiesen werden. Während lichtaktiviertes, GTP-gebundenes Rac1 überwiegend membranassoziiert vorliegt, konnte in dunkeladaptierten ROS insgesamt nur eine sehr geringe Menge an aktivem Rac1 detektiert werden. Des Weiteren wurden in dieser Studie auch deutliche Hinweise geliefert, die auf eine CRMP2 vermittelte Verbindung von Rac1 und RhoA assoziierten Signalwegen hinweisen, wohingegen die Kinase Rock II nur Teil des RhoA assoziierten Signalkomplexes zu sein scheint. Als Funktion von CRMP2 liegt daher eine Rolle als physiologischer Schalter nahe, der die Balance zwischen Rac1 und RhoA vermittelter Signaltransduktion koordiniert. Eine solche Funktion für CRMP2 wurde von Ariumura und Kollegen bereits für die Signaltransduktion in Neuronen vorgeschlagen (Arimura et al., 2000). Um die Signaltransduktion von CRMP2 in ROS eingehender untersuchen zu können, sind CRMP2 Antikörper unabdingbar, welche aber zu Beginn dieser Arbeit kommerziell nicht erhältlich waren. Daher war die Produktion und Charakterisierung von monoklonlalen CRMP2 spezifischen Antikörpern ein wichtiger Teil dieser Studie. Von den vier erhaltenen stabilen Linien monoklonaler, CRMP2 spezifischer Antikörper waren alle für den Einsatz im Western Blot sowie in der Immunohistochemie geeignet, aber nur ein Antikörper erwies sich auch als geeignet für die Immunopräzipitation von nativem CRMP2 aus primärem retinalen Gewebe. Dieser Antikörper stellt damit ein exzellentes Werkzeug für die weitere Charakterisierung der Funktion von CRMP2 in ROS dar. Drei Klassen von Proteinen regulieren die Aktivität von Rac1. Sie alle haben einen Einfluss auf den GTP/GDP-Austausch. Einer dieser Regulatoren ist der Rho GDP Dissociation Inhibitor (RhoGDI). Er kontrolliert die Interaktion von Rac1 mit weiteren regulatorischen Proteinen und Effektoren, sowie durch Interaktion mit dem Prenylrest von Rac1 das Pendeln zwischen Cytosol und Membran. Da aber der RhoGDI nicht in ROS nachgewiesen werden konnte (Balasubramanian and Slepak, 2003), legt dies den Schluss nahe, dass ein anderes Protein diese Funktion in ROS übernimmt. Das 17-kDa große Protein PDEdas lange Zeit als Untereinheit der retinalen cGMP Phosphodiesterase 6 aus Stäbchen galt, weist starke strukturelle Homologien zu RhoGDI auf. Es interagiert mit einer ganzen Reihe von prenylierten und unprenylierten Proteinen. Seine Fähigkeit, prenylierte Proteine von Zellmembranen zu lösen, erinnert stark an die Funktion, welche RhoGDI auf GTPasen der Rho Familie hat. Es wurde daher im Zuge dieser Studie untersucht, ob PDE in ROS GDI Funktion auf Rac1 ausübt. In dieser Arbeit konnte eine lichtabhängige Interaktion von Rac1 mit PDE in ROS von Schweinen nachgewiesen werden. Des Weiteren wurde gezeigt, dass aufgereinigtes PDE Rac1 von isolierten ROS Membranen lösen kann, eine Eigenschaft, die deutlich auf eine GDI-Funktion von PDE für Rac1 hinweist. Zudem wurde gezeigt, dass die Interaktion von Rac1 mit PDE mit einer lichtabhängigen Carboxylmethylierung von Rac1 in ROS korreliert, was ein Hinweis darauf sein kann, dass die die GDI Funktion von PDE durch die Methylierung von Rac1 reguliert wird. Alles in Allem zeigen diese Daten, das PDE für Rac1 in ROS die Funktion eines GDIs ausübt. In dieser Studie geben die identifizierten und mit Rac1 assoziierten Multiproteinkomplexe sowie deren lichtregulierte Dynamik einen deutlichen Hinweis darauf, dass Rac1 die Lichtwahrnehmung durch Rhodopsin mit Signalnetzwerken verbindet, die eine Rolle bei der strukturellen Integrität und Polarität von Photorezeptoren spielen. Dies deutet auf eine Abhängigkeit von Lichtwahrnehmung und funktioneller zellulärer Struktur hin. Mit der Bereitstellung von qualitativ sehr hochwertigen CRMP2 spezifischen Antikörpern liefert diese Studie zudem eine gute Basis für weiterführende Studien in diesem Forschungsfeld. Neben Rhodopsin assoziierten Komplexen stehen auch eine ganze Reihe von ciliären Komplexen in Zusammenhang mit degenerativen Erkrankungen der Retina. Im kürzlich entdeckten ciliären Protein Lebercilin (den Hollander et al., 2007) wurden Mutationen mit Leberscher kongenitaler Amaurose (LCA) in Verbindung gebracht, einer sehr schweren Form einer erblichen retinalen Dystrophie ((Kaplan et al., 1990), (Perrault et al., 1999)). Mit Hilfe von SF-TAP und LC/MS/MS Analysen konnten 24 Lebercilin Interaktoren in HEK Zellen identifiziert werden (den Hollander et al., 2007). Hier in dieser Studie wurden schließlich diese potentiellen Lebercilin Interaktoren auch in Photorezeptoren von Schweinen bestätigt (veröffentlicht in (den Hollander et al., 2007). Die identifizierten Interaktoren stellen mögliche Kandidaten für Gene für LCA und andere Ciliopathien dar und weisen Lebercilin als ein ciliär und mikrotubulär assoziiertes Protein in der Retina aus. Dies betont den Stellenwert, welche gestörte ciliäre Prozesse in der molekularen Pathogenese von LCA besitzen.

Die Synucleinopathien sind eine Gruppe neurodegenerativer Erkrankungen, die durch pathologische Ablagerungen des Alpha Synucleins charakterisiert sind. Sie umfassen unter anderem den Morbus Parkinson, die Demenz mit Lewy-Körperchen, die multiple Systematrophie und die Lewy Körperchen Variante des auch durch Ablagerungen des Tau Proteins gekennzeichneten Morbus Alzheimer. Ziel der vorliegenden Arbeit war die Analyse von Aggregationsprozessen des Alpha Synucleins durch einzelmolekülspektroskopische Verfahren und die Entwicklung neuer Techniken zur Analyse von Koaggregationsprozessen verschiedener Proteine. Hierzu wurden Synuclein und Tau Proteine rekombinant in E. coli hergestellt, aufgereinigt, durch Gelelektrophorese und Western Blot charakterisiert und Aggregationsprozesse unter in vitro Bedingungen untersucht. Rekombinantes Alpha Synuclein kann unter bestimmten Bedingungen in vitro zu fibrillären, amyloiden Strukturen aggregieren. Diese Aggregate ließen sich durch eine spezifische Änderung der Thioflavin T Fluoreszenz spektroskopisch nachweisen, mittels Elektronenmikroskopie darstellen, und zeichneten sich durch partielle Resistenz gegen die Einwirkung der Serin Protease Proteinase K aus. Durch Einbau von homologen Sondenmolekülen, die zuvor mit einer fluoreszenten Gruppe kovalent konjugiert wurden, ließ sich die Fibrillenbildung auch mit einzelmolekülbasierten, konfokalen, fluoreszenzspektroskopischen Analyseverfahren wie der Kreuzkorrelationsanalyse und der SIFT-Methode (engl. Scanning for Intensely Fluorescent Targets) darstellen. Der Nachweis der Fibrillenbildung war durch die fluoreszenzspektroskopischen Verfahren 100 fach sensitiver möglich. Im Vergleich mit nicht amyloidogenen Proteinen wie Beta Synuclein und Serum Albumin konnte zudem die Spezifität der Aggregation gezeigt werden. Bei neurodegenerativen Erkrankungen findet sich zum Teil die Beteiligung mehrerer amyloidogener Proteine am pathologischen Prozess. Durch die Möglichkeit der simultanen Anregung verschiedener Fluoreszenzsonden bei verschiedenen Wellenlängen und der Differenzierung des emittierten Fluoreszenzsignals konnten Koaggregationsprozesse in heterogenen Systemen mit Hilfe der Einzelmolekülspektroskopie untersucht werden. Es zeigte sich, dass Alpha Synuclein sowohl mit seinem Maushomolog, als auch mit den beiden humanen Parkinson-assoziierten Mutanten A30P und A53T gemischte Fibrillen bildet, während Beta Synuclein einen hemmenden Einfluss auf die Alpha Synuclein Fibrillenbildung aufwies. Mit Hilfe des zweifarbigen Systems ließ sich auch der Anlagerungsprozeß von Alpha Synuclein Monomeren an vorbestehende, amyloide Alpha Synuclein Aggregate darstellen, was einen weiteren Einblick in die molekulare Pathophysiologie der Synucleinopathien bot. Es wird vermutet, dass bereits Oligomerformationen einen neurotoxischen Effekt besitzen. Die besondere Sensitivität der konfokalen Einzelmolekülspektroskopie erlaubte ebenfalls die Detektion von frühen oligomeren Aggregaten des Alpha Synucleins sowie des Tau Proteins. Es konnte gezeigt werden, dass Alpha Synuclein bereits auf Oligomerebene heterogene Aggregate mit dem Tau Protein bildet, was durch die Tatsache, dass es sich bei beiden um intrazelluläre Proteine handelt, eine potentielle pathophysiologische Relevanz besitzt. Im Rahmen von Versuchsreihen zum Einfluss von Liquor aus an verschiedenen neurodegenerativen Erkrankungen leidenden Patienten, ließen sich Hinweise auf eine mögliche neuroprotektive Funktion des Liquors gewinnen. Dieser zeigte nach Gelfiltration in bestimmten Fraktionen deutlich aggregationshemmende Eigenschaften. Diese Fraktionen besitzen einen hohen Gehalt an Albumin. Zusammen mit in vitro gewonnenen Daten, die einen hemmenden Einfluss von Albumin auf die Aggregation des Alpha Synucleins zeigen, sind dies Hinweise, dass die protektive Eigenschaft an den Albumingehalt des Liquors gekoppelt ist. Das diagnostische Potential der entwickelten Messmethode verdeutlichten parallel durchgeführte Analysen von Influenza-Impfstoffpräparationen, in denen gezeigt werden konnte, dass sich Partikel mit unterschiedlichen Oberflächenepitopen auch in Mischungen differenzieren lassen. Durch den Einsatz von spezifischen fluoreszenzmarkierten Antikörpern ließen sich eindeutig bestimmte Influenzastämme anhand der Oberflächenepitop-Konfiguration identifizieren. Zusammenfassend konnte gezeigt werden, dass die konfokale Einzelmolekülspektroskopie eine äußerst sensitive und hochdurchsatzfähige Nachweismethode zur Untersuchung der molekularen Vorgänge im Rahmen der Alpha Synucleinopathien darstellt, deren Potential sich nicht allein auf die Grundlagenforschung beschränkt, sondern auch diagnostische Möglichkeiten beinhaltet und eine neue Methode zur Suche von bisher noch nicht beschriebenen, pharmakologisch interessanten Strukturen bietet.

Wir haben in der vorliegenden Arbeit das Vorhandensein von Defensinen, einer Gruppe natürlich vorkommender antimikrobieller Peptide, in der Peritonealhöhle untersucht. Unser Augenmerk richteten wir dabei auf die Adipozyten des Omentum majus, da eine mögliche Expression von Defensinen in Fettzellen auf Grund deren ubiquitären Verteilung im menschlichen Körper wichtige Erkenntnisse und Implikationen für zukünftige klinische Anwendungen haben würde (siehe auch „Ziel der Studie“ im Abschnitt „Einleitung“). Wir benutzten Nachweismethoden auf RNA- (RT-PCR, quantitative TaqMan-PCR, Northern Blot) und Protein-Ebene (Western-Blot, Immunhistochemie). Tatsächlich ist es uns gelungen, das humane α-Defensin 1 in hochgereinigten Adipozyten des Omentum majus und des subkutanen Fettgewebes in der PCR und in der Immunhistochemie nachzuweisen. Die Durchfühhrung des Western Blots erwies sich als komplex (siehe „Diskussion“). Daher kann gesagt werden, dass humane peritoneale Adipozyten wahrscheinlich das humane α-Defensin 1 synthetisieren. Zur Expression von humanem α-Defensin 5 können wir aktuell keine abschliessende Aussage treffen. Einerseits gelang dessen Nachweis in der PCR; Western Blot und Immunhistochemie blieben jedoch negativ (siehe „Ergebnisse“ und „Diskussion“). Die α-Defensin-Expression durch humane Adipozyten des Omentums (und der Subkutis) stellt einen weiteren Hinweis bezüglich der Bedeutung von Adipozyten in der primären Abwehr gegen bakterielle Infektionen dar.

Thu, 21 Feb 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8706/ https://edoc.ub.uni-muenchen.de/8706/1/Burget_Lukas.pdf Burget, Lukas ddc:600, ddc:610, Medi

Hintergrund Humane mesenchymale Stammzellen sind ein viel versprechendes Ziel für die ex vivo Gentherapie, und Lentiviren sind exzellente Vehikel für den Gentransfer in hMSCs, da sie hohe Transduktionsfrequenzen mit langfristiger Genexpression erreichen. Dennoch könnte die Seneszenz von hMSCs die therapeutische Anwendung, infolge von zeitaufwendiger Zellselektion und Virus Titration, limitieren. Diese Arbeit beschreibt optimierte Protokolle für hoch effizienten ex vivo lentiviralen Gentransfer in hMSCs und eine schnelle und verlässliche Methode, um den funktionellen lentiviralen Titer mittels quantitativer Polymerase-Kettenreaktion (qPCR) zu bestimmen. Methoden EGFP wurde als Markergen/-protein verwendet, um verschiedene lentivirale Expressionsvektoren herzustellen. Die Produktion von Lentiviren wurde mit verschiedenen Verpackungssystemen getestet. Der Prozentsatz transduzierter Zellen wurde durch Polybrene und Blasticidinselektion erhöht. hMSCs von verschiedenen Spendern wurden mittels PCR und Western Blot analysiert. Regulierte Genexpression wurde durch Herstellung eines Tet-On selbstregulierten Expressionsvektors erreicht. Mit einem p24 ELISA-Test wurden übrig gebliebene virale Partikel im Zellkulturüberstand detektiert. Die Effizienz des lentiviralen Gentransfers wurde mittels Fluoreszenz-Mikroskopie beobachtet und mittels qRT-PCR und FACS-Analyse quantifiziert. Die lentiviralen Titer wurden mit qRT-PCR der exprimierten Transgene bestimmt. Die hMSC Differenzierung wurde histologisch untersucht. Ergebnisse Selbstinaktivierende lentivirale Vektoren der dritten Generation zeigten hoch effizienten Gentransfer in hMSCs bei der Verwendung von Polybrene. Die Blasticidinselektion hat den Prozentsatz der transgenen Zellen weiter erhöht unter Selektion von Zellen die mehrere Transgenkopien tragen. Die positiven Effekte von Polybrene und der Blasticidinselektion sind nicht von hMSCs eines speziellen Spenders abhängig. Präzise Regulation der Genexpression wurde durch Herstellung eines selbstregulierten Tet-On-Expressionssystems erreicht. Keine viralen Antigene wurden im Zellkulturüberstand nach aufeinander folgenden Medienwechseln detektiert, was auf die Abwesenheit von infektiösen Partikeln nach einigen Tagen hindeutet. In dieser Arbeit wurde ein starker linearer Zusammenhang zwischen der Virusverdünnung und der Stärke der Transgenexpression mittels qPCR Analysen beobachtet, wodurch die Virustitration durch Quantifizierung der Transgenexpression ermöglicht wird. Abschließend wurde durch Differenzierung in die adipogene, osteogene und chondrogene Richtung gezeigt, dass transduzierte hMSCs ihren Stammzellcharakter beibehalten haben und dass die Transgenexpression durch die Differenzierung nicht beeinflusst wurde. Schlussfolgerungen Die Quantifizierung der Transgen-Kopienanzahl durch qRT-PCR ist eine schnelle und verlässliche Methode, um den funktionellen lentiviralen Titer nach dem ex vivo Gentransfer in hMSCs zu bestimmen. Die in dieser Arbeit optimierte und charakterisierte Methode für die effiziente lentivirale Transduktion von humanen mesenchymalen Stammzellen, in Verbindung mit regulierbarer Transgenexpression, ist ein sicheres, verlässliches und leistungsstarkes Verfahren und bildet eine aussichtsreiche Grundlage für zukünftige Gentherapie und Tissue Engineering Anwendungen in hMSCs.

Abstract Participation of Neospora caninum in abortions in cattle populations in Northern Bavaria Stated aim of this study was to investigate the incidence and the potential importance of Neospora caninum as a cause of abortion in cattle populations in Northern Bavaria. For this purpose the available diagnostic methods of the LGL in Erlangen were compared with each other relating to their efficiency. Aim was to find out which method or which combination of methods is most useful in the routine diagnosis. To serve this purpose, all abortion cases which were sent in for analysis to the LGL Erlangen (232 cases) within a year (2005-2006) were tested with different direct and indirect methods. Direct diagnosis of the infection was determined by histopathology, immunohistochemistry and PCR (polymerase chain reaction). ELISA (Enzyme Linked ImmunoSorbent Assay) and Western Blot were used for indirect analysis. 23 of the 232 cases were considered to be infected by at least one of the diagnostic techniques used. That implies that Neospora caninum is involved in 10% of abortions in Northern Bavaria. It’s impossible to prove beyond doubt if Neospora caninum is the main cause of abortions or an innovator for further infections. Suspicious cases could be identified by routine histopathology because of characteristic lesions – particularly in brain and heart. Typical histological lesions consisted overbalancing in multifocal nonsuppurative necrotizing encephalitis and non-purulent myocarditis. Most of these cases were confirmed by immunohistochemical staining with using a polyclonal antibody. Frequently low numbers of disease agents necessitate more (up to 4) immunohistochemical runs in some cases to verify the agent. Especially in cases of bold autolysis or mummified fetuses histology was applicable only with restrictons, Immunohistochemistry not at all. PCR is able to detect DNA of the parasit in these cases. Altogether the PCR got the most positive results. In cause of this three different techniques (conventionell PCR, Real Time SYBR Green, Real Time TaqMan) could adduce approximately same results. The Real Time TaqMan Technique gained slightly advantage over the two other methods. High costs and difficulties to take positive results are detriments of the PCR-method. There is no qualified statement about the importance of Neospora caninum as a cause of abortion, using the PCR method only. Therefore the PCR method should be combined with one additionally morphological method. Serological tests of Neospora caninum intrinsic antibodies in fetal fluids proved not to be very useful. Fetuses in early pregnancy develop partly no specific antibodies against Neospora caninum so that these cases were seronegative in spite of showing typical morphological lesions. Compared with this, 4 seropositive cases did not show any histopathological alterations. A final interpretation of such cases is impossible. Summarising this study confirms the importance of Neospora caninum as a cause of abortion in Northern Bavaria and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted fetuses. The best combination is histopathology with PCR. After a screening with histopathology, suspicious fetal tissues should be verified by PCR. Furthermore the PCR has got the highest sensitivity of all used methods. It is able to detect also cases without lesions although inspite of presenting the parasit. PCR was confirmed to be the best tool for the diagnosis of Neospora caninum in aborted fetuses.

Die vorliegende Arbeit untersuchte im ersten Teil tierart-vergleichend das Vorkommen von PrPC bei Hauswiederkäuern in Eutergewebe und in den verschiedenen Milchfraktionen. Dies erfolgte mittels EIA und Western-Blot sowie für das Eutergewebe auch mittels Immunhistochemie (IHC). Für die Aufarbeitung von Rahm wurde dabei ein neues Verfahren etabliert, durch das die Proteinfraktion des Rahms als Pellet gewonnen werden konnte. Es fand seinen Einsatz auch im zweiten Teil bei der Untersuchung von Milch und Kolostrum BSE- bzw. Scrapie-infizierter Schafe mittels PrionScreen® und Western-Blot. Auch das Eutergewebe und die Milchfraktionen von drei BSE-Kühen wurden untersucht. Im Eutergewebe vom Rind war PrPC-Expression mittels EIA und IHC mit individuellen Unterschieden nachweisbar, nicht jedoch mittels Western-Blot. Bei den kleinen Wiederkäuern hingegen ließ sich PrPC mit allen drei verwendeten Methoden darstellen. In der IHC zeigte sich, dass die Expression das gesamte Zytoplasma der Laktozyten umfasste und PrPC-haltige Vesikel ins Alveolarlumen abgeschnürt wurden. Beim Rind dagegen beschränkte sich die PrPC Expression auf basolaterale Abschnitte der Laktozyten. In Kuhmilch konnte - im Gegensatz zu kürzlich erschienenen Publikationen - kein PrPC detektiert werden. Die kleinen Wiederkäuer zeigten jedoch eine individuelle und - wie anhand des Laktationszyklus eines Schafes nachvollziehbar - laktationsphasen-abhängige PrPC-Expression in den Milchfraktionen Magermilch, Molke und somatische Zellen. Neben den vom Hirngewebe bekannten drei Isoformen (zweifach-, einfach- und unglykosyliert) waren im Western-Blot mit mAk P4 zwei trunkierte Formen auf Höhe von ca. 8 und 14 kDa darstellbar. Dabei handelt sich vermutlich um N-terminale Fragmente. In Kasein war nur bei einigen Schafen PrPC im EIA nachweisbar. Rahm konnte aufgrund seiner Aufarbeitung mittels Detergentien nur im Western-Blot untersucht werden. Es zeigten sich bei Schaf und Ziege dieselben PrPC- Banden wie in den bereits beschriebenen Milchfraktionen. Bei den Proben TSE-infizierter Schafe zeigte sich sowohl bei Rahm aus Milch als auch bei Rahm und Molke aus fettreichem Kolostrum, die eine ähnliche Aufarbeitung erfuhren, PK-resistente Banden. Diese Banden zeigten sich auf Höhe der drei Volllängen-Isoformen sowie der 8-kDa-Form. Eine Abklärung erfolgte im PrionScreen®-EIA durch Einsatz der aus Rahm gewonnenen Proteinpellets. Darin reagierten sieben von 13 Milchproben der Scrapie-Tiere und acht von 108 der BSE-Schafe positiv. Dabei handelte es sich durchweg um Kolostrumproben (etwa 26 % der untersuchten Kolostrumproben). Auch im anschließenden Western-Blot mit einem Aliquot aus der Digestionsplatte des PrionScreen® zeigten sich bei einem Teil der positiven EIA-Reagenten Banden auf PrPC-Höhe. Durch die Kontrolle mit einem unspezifischen Primärantikörper konnte nicht eindeutig gezeigt werden, ob die Banden PrP-spezifisch sind. Ein ähnliches Phänomen bei somatischen Zellen aus Schaf-Kolostrum ist von EVEREST et al. (2006) bekannt. Man muss daher davon ausgehen, dass es sich um eine unspezifische Reaktion des Kolostrums handelt. Rahm aus Milch reagierte im PrionScreen® eindeutig negativ, ebenso Molke aus Milch im Western-Blot. Die Eutergewebeproben der drei BSE-Kühe reagierten negativ in EIA, Western-Blot und IHC. Auch in ihrer Magermilch war kein PrPres detektierbar. Die Kaseinfraktion reagierte positiv im PrionScreen®, wobei eine unspezifische Reaktion jedoch nicht ausgeschlossen werden konnte. Obwohl die Untersuchungen an klinisch gesunden Tieren gezeigt haben, dass PrPC als Replikationsmatrix für die Bildung von PrPSc im Euter und der Milch vorhanden ist, brachten die Analysen der TSE-infizierten Tiere letztlich keinen Beweis für das Vorkommen von PrPres in Milch. Lediglich im Kolostrum von TSE-infizierten Schafen zeigten sich untypische PK-resistente Formen. Eine Gefährdung des Verbrauchers durch Milch und Milchprodukte ist daher mit größter Wahrscheinlichkeit nicht gegeben.

Das leichte Schädelhirntrauma stellt in der Altersgruppe zwischen dem 15. und dem 25. Lebensjahr mit einer Inzidenz von 600 Personen/100000 Gesamtbevölkerung in Deutschland eine der häufigsten neurologischen Erkrankungen dar. 10-20% der Betroffenen leiden an chronischen posttraumatischen Leistungseinbussen, ohne zerebral einen makroskopisch erkennbaren Schaden aufzuweisen. In dieser Dissertation konnte im experimentellen Tiermodell des leichten Schädelhirntraumas erstmals der Verlust von Basalmembranbestandteilen als ein mikroskopisches Korrelat im Sinne einer deutlichen Störung der für die normale Funktion des Gehirns notwendigen Blut-Hirn-Schranke gezeigt werden. Dieses mikroskopische Korrelat äusserte sich in der Zerstörung der Integrität der Basalmembran und ihrer Verankerung mittels Integrinen in der extrazellulären Matrix. 24 Stunden nach einem milden Flüssigkeits-Perkussions-Trauma zeigte der Kortex der Ratten einen Verlust von 31 ± 6% (p < 0,03) des Basalmembran-bestandteiles Kollagen Typ IV im Western Blot gegenüber der nicht-traumatischen Seite und bestätigte somit den bereits in Vorarbeiten in der Immunhistochemie festgestellten Verlust von 19 ± 4% (p < 0,009) der gefärbten Kollagenfläche, als auch mit 29 ± 6% (p < 0,02) der Reduktion der Gesamtanzahl der durch Kollagen identifizierten Mikrogefäße. Dieser Verlust war nach 12 Stunden Überlebenszeit erst als Trend zu sehen und erst nach 24 Stunden Überlebenszeit signifikant. Der beobachtete Verlust war auf den Kortex der Traumaseite beschränkt, das heißt die Basalganglien blieben unbeschädigt. Verglichen mit ischämischen Veränderungen nach MCAO/R (Verschluss der Arteria cerebri media und Reperfusion) war der Kollagen Typ IV Verlust im milden SHT weniger ausgeprägt, als auch von einem unterschiedlichen Verteilungsmuster, da im MCAO/R der mikrovaskuläre Basalmembranschaden hauptsächlich in den Basalganglien zu finden ist, im experimentellen SHT jedoch in kortikalen Arealen. Auch im Bereich der Verankerung der Basalmembran fand sich ein ausgeprägter Verlust der Zell-Adhäsions-Molekül Untergruppe genannt Integrine. Die untersuchten Integrinuntereinheiten α1, α6 und β1 finden sich entlang des Endothels der kleinen hirnversorgenden Gefäße. Als direkte Folge auf ein moderates Schädelhirntrauma in der Ratte treten hier deutliche Verluste auf. Die mit α1-Integrin Antikörper durchgeführte Immunhistochemie zeigte von allen drei untersuchten Integrinsubgruppen die stärkste Reduktion: Die angefärbte maximale Fläche nahm sowohl in der 12-Stunden-Überlebensgruppe, als auch in der 24-Stunden-Überlebensgruppe signifikant gegenüber der nicht-traumatischen Seite ab. Die 12-Stunden-Gruppe erfuhr eine Reduktion der maximalen α1 Integrinfläche um 8 ± 2% (p < 0,01; Abbildung 30), die 24-Stunden-Gruppe sogar eine Reduktion der maximalen α1-Integrinfläche um 13 ± 2% (p < 0,001). Die ebenfalls untersuchte α1 Integrinintensität nahm in einem vergleichbarem Maß signifikant ab, in der 12-Stunden-Überlebensgruppe um 8 ± 1% (p < 0,01), in der 24-Stunden-Überlebensgruppe um 14 ± 2% (p < 0,01). Etwas weniger stark ausgeprägt und erst nach 24 Stunden Überlebenszeit signifikant, folgten die beiden Integrinuntereinheiten β1 und α6 mit 12 ± 2% (p < 0,005) respektive 8 ± 2% (p < 0,05) Verlust der gefärbten Integrinfläche, sowie 10 ± 3% (p < 0,05) respektive 7 ± 1% (p < 0,005) Verlust der Integrin Färbeintensität. Dieser Effekt konnte nur in kortikalen Arealen des Gehirns entdeckt werden, wie bereits zuvor die Schäden der Basalmembran, und war auch im Zeitverlauf parallel zum Verlust von Kollagen Typ IV. Auch war, verglichen mit ischämischen Veränderungen nach MCAO/R, der Integrinverlust im milden SHT weniger ausgeprägt und von einem unterschiedlichen Verteilungsmuster. Die vorbeobachteten Verluste dieser Integrinuntereinheiten (entsprechend Kollagen Typ IV) werden nach MCAO/R hauptsächlich in den Basalganglien gefunden, im experimentellen SHT jedoch in kortikalen Arealen. Als ursächlich für die beobachteten und deutlichen Schäden der mikrovaskulären Basalmembran des Gehirns auch nach dem milden experimentellen Schädelhirntrauma wäre am ehesten die Aktivierung der Matrix-Metallo-Proteasen als einer der drei Hauptwege der Proteolyse (Plasminogen-Plasmin-System, Matrix-Metallo-Proteasen und Inflammation) zu vermuten. Zudem könnten auch die Basalmembranbestandteile selbst, die als Reaktion auf ein Trauma freigesetzt werden, insbesondere Untereinheiten von Kollagen Typ IV, verborgene Bindungsstellen zur Signalvermittlung präsentieren, welche eine Kaskade von intrazellulären Zerstörungsmechanismen anstossen könnten. Zukünftige Untersuchungen sollten sich daher auf die drei bisher bekannten proteolytischen Hauptwege, sowie die Basalmembranbestandteile selbst und ihre Auswirkungen auf die Regeneration der Mikrogefäße stützen, um ein besseres Verständnis für die in den Verlust von Basalmembran involvierten Prozesse während eines Schädelhirntrauma zu entwickeln. Zukünftige Therapien des leichten bis moderaten experimentellen Schädelhirntraumas sollten daher möglicherweise bereits während der akutmedizinischen Versorgung in Betracht gezogen werden. Man sollte anhand der Ergebnisse dieser Dissertation in Betracht ziehen, dass die hier vorgestellte Studie auch im milden experimentellen Schädelhirntraumamodell bereits zum frühen Zeitpunkt von 24 Stunden Überlebenszeit stabil signifikante Verluste von Basalmembranbestandteilen nachweisen konnte. Therapeutische Strategien sollten sich daher auch nach mildem Schädelhirntrauma auf eine Wiederherstellung der endothelialen Basalmembran konzentrieren, insbesondere um die oben beschriebenen Folgeschäden durch im Blut gelöste Bestandteile der Basalmembran, ihre Interaktion mit Integrinen und den nachfolgenden intrazellulären Signalkaskaden zu unterbinden. Optimalerweise sollten diese Strategien bereits für den akutmedizinischen Zeitpunkt geplant werden.

Apoptose glatter Gefäßmuskelzellen spielt eine wichtige Rolle sowohl im Rahmen der Pathogenese der Atherosklerose als auch der Restenose nach Ballonangioplastie. Zu Beginn der Arbeit war bekannt, dass glatte Gefäßmuskelzellen in Abhängigkeit von der Zelldichte auf bestimmte Substanzen wie z.B. PDTC oder Lovastatin mit Apoptose reagieren. Bei hoher Zelldichte konnte keine Apoptose induziert werden. Ziel der Arbeit war es, die apoptoseinduzierenden Substanzen PDTC und Lovastatin zu charakterisieren und verantwortliche Mechanismen der Apoptoseprotektion zu identifizieren. Dazu wurden glatte Gefäßmusklezellen in hoher Dichte kultiviert und konditioniertes Medium gewonnen. Dieses konditionierte Medium induzierte Proliferation in glatten Gefäßmuskelzellen und vermittelte einen Schutz vor Apoptose. Durch Immunpräzipitation und Western Blot konnte eindeutig gezeigt werden, dass die verminderte Apoptoseempfindlichkeit glatter Gefäßmuskelzellen in hoher Dichte durch eine autokrine Produktion von FGF-2 vermittlelt wird.

CpG-Oligonukleotide stellen synthetische Imitate mikrobieller DNA dar, die über Toll like-Rezeptor9 B-Zellen und plasmazytoide dendritische Zellen aktivieren. Es existieren verschiedene Klassen, die sich in ihrer Fähigkeit zur Induktion von Interferon-alpha und zur Aktivierung von Immunzellen unterscheiden. Aktuell werden CpG-ODN in klinischen Studien zur Therapie von Malignomen, Infektionen und allergischen Erkrankungen und als Impf-Adjuvans erprobt. Zielsetzung dieser Arbeit war die genauere Charakterisierung der Effekte der verschiedenen CpG-ODN auf die PDC und die weitere Aufklärung der Signaltransduktionswege, die diese Effekte vermitteln. Die verwendeten Methoden waren ELISA, Durchflusszytometrie, Western Blot und molekularbiologische Methoden inclusive PCR, Klonierung und Sequenzierung. Dabei konnte gezeigt werden, dass nur CpG-A in der Lage ist, eine starke und lang anhaltende IFN-alpha-Produktion in der PDC zu induzieren mit einem bestimmten Spektrum an IFN-alpha-Subtypen. Sowohl die CpG-A als auch die CpG-B induzierte IFN-alpha-Produktion erwiesen sich als abhängig von p38-Aktivierung aber unabhängig von JNK und ERK. Interessanterweise beeinflusste NF-kB die CpG-induzierte IFN-alpha-Produktion gegensätzlich, wobei die CpG-A induzierte IFN-alpha-Produktion gefördert und die CpG-B-abhängige IFN-alpha-Produktion gehemmt wurde. Diese Ergebnisse liefern einen Beitrag zur weiteren Aufklärung des molekularen Wirkmechanismus der CpG-Deoxyoligonukleotide.