Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Fighting Chaos with care, community trust, engagement must be cornerstones of pandemic response
Reporter: Amandeep Kaur, BSc, MSc (Exp. 6/2021)
According to the Global Health Security Index released by Johns Hopkins University in October 2019 in collaboration with Nuclear Threat Initiative (NTI) and The Economist Intelligence Unit (EIU), the United States was announced to be the best developed country in the world to tackle any pandemic or health emergency in future.
The table turned within in one year of outbreak of the novel coronavirus COVID-19. By the end of March 2021, the country with highest COVID-19 cases and deaths in the world was United States. According to the latest numbers provided by World Health Organization (WHO), there were more than 540,000 deaths and more than 30 million confirmed cases in the United States.
Joia Mukherjee, associate professor of global health and social medicine in the Blavatnik Institute at Harvard Medical School said,
“When we think about how to balance control of an epidemic over chaos, we have to double down on care and concern for the people and communities who are hardest hit”.
She also added that U.S. possess all the necessary building blocks required for a health system to work, but it lacks trust, leadership, engagement and care to assemble it into a working system.
Mukherjee mentioned about the issues with the Index that it undervalued the organized and integrated system which is necessary to help public meet their needs for clinical care. Another necessary element for real health safety which was underestimated was conveying clear message and social support to make effective and sustainable efforts for preventive public health measures.
Mukherjee is a chief medical officer at Partners In Health, an organization focused on strengthening community-based health care delivery. She is also a core member of HMS community members who play important role in constructing a more comprehensive response to the pandemic in all over the U.S. With years of experience, they are training global health care workers, analyzing the results and constructing an integrated health system to fight against the widespread health emergency caused by coronavirus all around the world.
Mukherjee encouraged to strengthen the consensus among the community to constrain this infectious disease epidemic. She suggested that validation of the following steps are crucial such as testing of the people with symptoms of infection with coronavirus, isolation of infected individuals by providing them with necessary resources and providing clinical treatment and care to those people who are in need. Mukherjee said, that community engagement and material support are not just idealistic goal rather these are essential components for functioning of health care system during an outburst of coronavirus.
Continued alertness such as social distancing and personal contact with infected individual is important because it is not possible to rapidly replace the old-school public health approaches with new advanced technologies like smart phone applications or biomedical improvements.
Public health specialists emphasized that the infection limitation is the only and most vital strategy for controlling the outbreak in near future, even if the population is getting vaccinated. It is crucial to slowdown the spread of disease for restricting the natural modification of more dangerous variants as that could potentially escape the immune protection mechanism developed by recently generated vaccines as well as natural immune defense systems.
Making Crucial connections
The treatment is more expensive and complicated in areas with less health facilities, said Paul Farmer, the Kolokotrones University Professor at Harvard and chair of the HMS Department of Global Health and Social Medicine. He called this situation as treatment nihilism. Due to shortage of resources, the maximum energy is focused in public health care and prevention efforts. U.S. has resources to cope up with the increasing demand of hospital space and is developing vaccines, but there is a form of containment nihilism- which means prevention and infection containment are unattainable- said by many experts.
Farmer said, integration of necessary elements such as clinical care, therapies, vaccines, preventive measures and social support into a single comprehensive plan is the best approach for a better response to COVID-19 disease. He understands the importance of community trust and integrated health care system for fighting against this pandemic, as being one of the founders of Partners In Health and have years of experience along with his colleagues from HMS and PIH in fighting epidemics of HIV, Ebola, cholera, tuberculosis, other infectious and non-infectious diseases.
PIH launched the Massachusetts Community Tracing Collaborative (CTC), which is an initiative of contact tracing statewide in partnership with several other state bodies, local boards of Health system and PIH. The CTC was setup in April 2020 in U.S. by Governor Charlie Baker, with leadership from HMS faculty, to build a unified response to COVID-19 and create a foundation for a long-term movement towards a more integrated community-based health care system.
The contact tracing involves reaching out to individuals who are COVID-19 positive, then further detect people who came in close contact with infected individuals and screen out people with coronavirus symptoms and encourage them to seek testing and take necessary precautions to break the chain of infection into the community.
In the initial phase of outbreak, the CTC group comprises of contact tracers and health care coordinators who spoke 23 different languages, including social workers, public health practitioners, nurses and staff members from local board health agencies with deep links to the communities they are helping. The CTC worked with 339 out of 351 state municipalities with local public health agencies relied completely on CTC whereas some cities and towns depend occasionally on CTC backup. According to a report, CTC members reached up to 80 percent of contact tracking in hard-hit and resource deprived communities such as New Bedford.
Putting COVID-19 in context
Based on generations of experience helping people surviving some of the deadliest epidemic and endemic outbreaks in places like Haiti, Mexico, Rwanda and Peru, the staff was alert that people with bad social and economic condition have less space to get quarantined and follow other public health safety measures and are most vulnerable people at high risk in the pandemic situation.
Infected individuals or individuals at risk of getting infected by SARS-CoV-2 had many questions regarding when to seek doctor’s help and where to get tested, reported by contact tracers. People were worried about being evicted from work for two weeks and some immigrants worried about basic supplies as they were away from their family and friends.
The CTC team received more than 7,000 requests for social support assistance in the initial three months. The staff members and contact tracers were actively connecting the resourceful individuals with the needy people and filling up the gap when there was shortage in their own resources.
Farmer said, “COVID is a misery-seeking missile that has targeted the most vulnerable.”
The reality that infected individuals concerned about lacking primary household items, food items and access to childcare, emphasizes the urgency of rudimentary social care and community support in fighting against the pandemic. Farmer said, to break the chain of infection and resume society it is mandatory to meet all the elementary needs of people.
“What kinds of help are people asking for?” Farmer said and added “it’s important to listen to what your patients are telling you.”
An outbreak of care
The launch of Massachusetts CTC with the support from PIH, started receiving requests from all around the country to assist initiating contact tracing procedures. In May, 2020 the organization announced the launch of a U.S. public health accompaniment to cope up with the asked need.
The unit has included team members in nearly 24 states and municipal health departments in the country and work in collaboration with local organizations. The technical support on things like choosing and implementing the tools and software for contact tracing was provided by PIH. To create awareness and provide new understanding more rapidly, a learning collaboration was established with more than 200 team members from more than 100 different organizations. The team worked to meet the needs of population at higher risk of infection by advocating them for a stronger and more reliable public health response.
The PIH public health team helped to train contact trackers in the Navajo nation and operate to strengthen the coordination between SARS-CoV-2 testing, efforts for precaution, clinical health care delivery and social support in vulnerable communities around the U.S.
“For us to reopen our schools, our churches, our workplaces,” Mukherjee said, “we have to know where the virus is spreading so that we don’t just continue on this path.”
The WHO team is expected to soon publish a 300-page final report on its investigation, after scrapping plans for an interim report on the origins of SARS-CoV-2 — the new coronavirus responsible for killing 2.7 million people globally
Recent Grim COVID-19 Statistics in U.S. and Explanation from Dr. John Campbell: Why We Need to be More Proactive
Reporter: Stephen J. Williams, Ph.D.
In case you have not been following the excellent daily YouTube sessions on COVID-19 by Dr. John Campbell I am posting his latest video on how grim the statistics have become and the importance of using proactive measures (like consistent use of facial masks, proper social distancing) instead of relying on reactive measures (e.g. lockdowns after infection spikes). In addition, below the video are some notes from his presentation and some links to sites discussed within the video.
Notes from the video:
approaching 5 million confirmed cases in US however is probably an underestimation
295,000 US COVID-19 related deaths estimated by December 1, 2020
however if 95% of people in US consistently and properly wear masks could save 66,000 lives
however this will mean a remaining 228,271 deaths which is a depressing statistic
Dr. John Campbell agrees with Dr. Christopher Murray, director of the Institute for Health Metrics that “people’s inconsistent use of these measures (face masks, social distancing) is a serious problem”
States with increasing transmission like Colorado, Idaho, Kansas, Kentucky, Mississippi, Missouri, Ohio, Oklahoma, Oregon, and Virginia are suggested to have a lockdown when death rate reaches 8 deaths per million population however it seems we should be also focusing on population densities rather than geographic states
Dr. Campbell and Dr. Murray stress more proactive measures than reactive ones like lockdowns
if mask usage were to increase to 95% usage reimposition to shutdown could be delayed 6 to 8 weeks
SEATTLE (August 6, 2020) – America’s COVID-19 death toll is expected to reach nearly 300,000 by December 1; however, consistent mask-wearing beginning today could save about 70,000 lives, according to new data from the Institute for Health Metrics and Evaluation (IHME) at the University of Washington’s School of Medicine.The US forecast totals 295,011 deaths by December. As of today, when, thus far, 158,000 have died, IHME is projecting approximately 137,000 more deaths. However, starting today, if 95% of the people in the US were to wear masks when leaving their homes, that total number would decrease to 228,271 deaths, a drop of 49%. And more than 66,000 lives would be saved.Masks and other protective measures against transmission of the virus are essential to staying COVID-free, but people’s inconsistent use of those measures is a serious problem, said IHME Director Dr. Christopher Murray.
“We’re seeing a rollercoaster in the United States,” Murray said. “It appears that people are wearing masks and socially distancing more frequently as infections increase, then after a while as infections drop, people let their guard down and stop taking these measures to protect themselves and others – which, of course, leads to more infections. And the potentially deadly cycle starts over again.”
Murray noted that there appear to be fewer transmissions of the virus in Arizona, California, Florida, and Texas, but deaths are rising and will continue to rise for the next week or two. The drop in infections appears to be driven by the combination of local mandates for mask use, bar and restaurant closures, and more responsible behavior by the public.
“The public’s behavior had a direct correlation to the transmission of the virus and, in turn, the numbers of deaths,” Murray said. “Such efforts to act more cautiously and responsibly will be an important aspect of COVID-19 forecasting and the up-and-down patterns in individual states throughout the coming months and into next year.”
Murray said that based on cases, hospitalizations, and deaths, several states are seeing increases in the transmission of COVID-19, including Colorado, Idaho, Kansas, Kentucky, Mississippi, Missouri, Ohio, Oklahoma, Oregon, and Virginia.
“These states may experience increasing cases for several weeks and then may see a response toward more responsible behavior,” Murray said.
In addition, since July 15, several states have added mask mandates. IHME’s statistical analysis suggests that mandates with no penalties increase mask wearing by 8 percentage points. But mandates with penalties increase mask wearing by 15 percentage points.
“These efforts, along with media coverage and public information efforts by state and local health agencies and others, have led to an increase in the US rate of mask wearing by about 5 percentage points since mid-July,” Murray said. Mask-wearing increases have been larger in states with larger epidemics, he said.
IHME’s model assumes that states will reimpose a series of mandates, including non-essential business closures and stay-at-home orders, when the daily death rate reaches 8 per million. This threshold is based on data regarding when states and/or communities imposed mandates in March and April, and implies that many states will have to reimpose mandates.
As a result, the model suggests which states will need to reimpose mandates and when:
August – Arizona, Florida, Mississippi, and South Carolina
September – Georgia and Texas
October – Colorado, Kansas, Louisiana, Missouri, Nevada, North Carolina, and Oregon.
November – Alabama, Arkansas, California, Iowa, New Mexico, Oklahoma, Utah, Washington, and Wisconsin.
However, if mask use is increased to 95%, the re-imposition of stricter mandates could be delayed 6 to 8 weeks on average.
There are several similarities between SARS-CoV-2 and SARS-CoV, because both viruses share a high degree of homology to SARS-like coronaviruses isolated from bats. The entire genome of SARS-CoV-2 has 86% similarity with SARS-CoV. COVID-19, SARS-CoV-2 has a higher transmissibility than SARS-CoV, where more patients with COVID-19 have mild symptoms that contribute to spread because the patients are usually missed and not isolated.
Even in terms of disease dynamics, the similarities include transmission route via respiratory droplets. The angiotensin-converting enzyme 2 (ACE2), found in the lower respiratory tract of humans, has been identified as the receptor used for cell entry for both SARS-CoV and SARS-CoV-2.
So even though the viruses seem similar, there are some strong differences as well. Patients reports from China, Europe and United states show that some patients have also cardiac issues. The scientist do not truly understand what is happening at this point, whether people are having heart attacks (myocardial infarction) or whether the virus is actually invading the heart tissue to cause inflammation (myocarditis)
The great concern is that many people are asymptomatic with this condition, have no symptoms. This is what makes the virus so complicated is because you can have a group of patients severely sick and in the intensive care unit and in some cases, there are older individuals and some with underlying diabetes and heart disease, hypertension, renal disease.
Even though, the US has a large number of cases of over one million and at least 84,000 deaths, but due to undertesting, the true numbers of cases are probably far higher. The big unknown is that there is no clear understanding what is going to happen in the next coming months or years with the virus. However, the investigation models indicate that the virus has a probably of returning seasonally in the coming years.
Yet, people have to be mindful and recognize that even if we begin relaxing social distancing and transmission diminishes, that it could come back in these periodic waves, as suggested by the model.
The term ‘antibiotic’ was introduced by Selman Waksman as any small molecule, produced by a microbe, with antagonistic properties on the growth of other microbes. An antibiotic interferes with bacterial survival via a specific mode of action but more importantly, at therapeutic concentrations, it is sufficiently potent to be effective against infection and simultaneously presents minimal toxicity. Infectious diseases have been a challenge throughout the ages. From 1347 to 1350, approximately one-third of Europe’s population perished to Bubonic plague. Advances in sanitary and hygienic conditions sufficed to control further plague outbreaks. However, these persisted as a recurrent public health issue. Likewise, infectious diseases in general remained the leading cause of death up to the early 1900s. The mortality rate shrunk after the commercialization of antibiotics, which given their impact on the fate of mankind, were regarded as a ‘medical miracle’. Moreover, the non-therapeutic application of antibiotics has also greatly affected humanity, for instance those used as livestock growth promoters to increase food production after World War II.
Currently, more than 2 million North Americans acquire infections associated with antibiotic resistance every year, resulting in 23,000 deaths. In Europe, nearly 700 thousand cases of antibiotic-resistant infections directly develop into over 33,000 deaths yearly, with an estimated cost over €1.5 billion. Despite a 36% increase in human use of antibiotics from 2000 to 2010, approximately 20% of deaths worldwide are related to infectious diseases today. Future perspectives are no brighter, for instance, a government commissioned study in the United Kingdom estimated 10 million deaths per year from antibiotic resistant infections by 2050.
The increase in antibiotic-resistant bacteria, alongside the alarmingly low rate of newly approved antibiotics for clinical usage, we are on the verge of not having effective treatments for many common infectious diseases. Historically, antibiotic discovery has been crucial in outpacing resistance and success is closely related to systematic procedures – platforms – that have catalyzed the antibiotic golden age, namely the Waksman platform, followed by the platforms of semi-synthesis and fully synthetic antibiotics. Said platforms resulted in the major antibiotic classes: aminoglycosides, amphenicols, ansamycins, beta-lactams, lipopeptides, diaminopyrimidines, fosfomycins, imidazoles, macrolides, oxazolidinones, streptogramins, polymyxins, sulphonamides, glycopeptides, quinolones and tetracyclines.
The increase in drug-resistant pathogens is a consequence of multiple factors, including but not limited to high rates of antimicrobial prescriptions, antibiotic mismanagement in the form of self-medication or interruption of therapy, and large-scale antibiotic use as growth promotors in livestock farming. For example, 60% of the antibiotics sold to the USA food industry are also used as therapeutics in humans. To further complicate matters, it is estimated that $200 million is required for a molecule to reach commercialization, with the risk of antimicrobial resistance rapidly developing, crippling its clinical application, or on the opposing end, a new antibiotic might be so effective it is only used as a last resort therapeutic, thus not widely commercialized.
Besides a more efficient management of antibiotic use, there is a pressing need for new platforms capable of consistently and efficiently delivering new lead substances, which should attend their precursors impressively low rates of success, in today’s increasing drug resistance scenario. Antibiotic Discovery Platforms are aiming to screen large libraries, for instance the reservoir of untapped natural products, which is likely the next antibiotic ‘gold mine’. There is a void between phenotanypic screening (high-throughput) and omics-centered assays (high-information), where some mechanistic and molecular information complements antimicrobial activity, without the laborious and extensive application of various omics assays. The increasing need for antibiotics drives the relentless and continuous research on the foreground of antibiotic discovery. This is likely to expand our knowledge on the biological events underlying infectious diseases and, hopefully, result in better therapeutics that can swing the war on infectious diseases back in our favor.
During the genomics era came the target-based platform, mostly considered a failure due to limitations in translating drugs to the clinic. Therefore, cell-based platforms were re-instituted, and are still of the utmost importance in the fight against infectious diseases. Although the antibiotic pipeline is still lackluster, especially of new classes and novel mechanisms of action, in the post-genomic era, there is an increasingly large set of information available on microbial metabolism. The translation of such knowledge into novel platforms will hopefully result in the discovery of new and better therapeutics, which can sway the war on infectious diseases back in our favor.
Tuberculosis is one of the world’s deadliest infectious diseases, which requires six-month course of daily antibiotics. To help overcome that, a team of researchers led by MIT has devised a new way to deliver antibiotics, which they hope will make it easier to cure more patients and reduce health care costs. In their approach a coiled wire loaded with antibiotics is inserted into the patient’s stomach through a nasogastric tube. Once in the stomach, the device slowly releases antibiotics over one month, eliminating the need for patients to take pills every day.
The device is a thin, elastic wire made of nitinol that can change its shape based on temperature. The researchers can string up to 600 “pills” of various antibiotics along the wire, and the drugs are packaged in polymers whose composition can be adjusted to control the rate of drug release once the device go in the stomach. The wire is distributed to the patient’s stomach via a tube inserted through the nose, which is used regularly in hospitals for delivering medications and nutrients. When the wire reaches the higher temperatures of the stomach, it forms a coil, which stops it from passing further through the digestive system. The researchers then tested the device in pigs and found that this device could release different antibiotics at a constant rate for 28 days. Once all of the drugs are delivered, the device is recovered through the nasogastric tube using a magnet that can attract the coil.
Giovanni Traverso and Robert Langer have been working on a variety of pills and capsules that can remain in the stomach and slowly release medication after being swallowed. This type of drug delivery, can expand treatment to several chronic diseases that require daily doses of medication. One capsule that shows promise appears to be for delivering small amounts of drugs to treat HIV and malaria. After being swallowed, the capsule’s outer coating disintegrates, allowing six arms to expand, helping the device to lodge in the stomach. This device can carry about 300 milligrams of drugs which is enough for a week’s worth of HIV treatment but it falls short of the payload of 3 grams of antibiotics every day needed to treat tuberculosis.
The researchers in addition to David Collins, an economist analyzed the potential economic impact of this type of treatment. He determined that if the treatment is applied in India, costs could be reduced by about $8,000 per patient. I think that such an approach can be helpful for longer regimens required for the treatment of extensively drug-resistant TB and even hepatitis C and this approach can be an vital milestone toward addressing this problem.
The Vibrant Philly Biotech Scene: Focus on Vaccines and Philimmune, LLC
Curator: Stephen J. Williams, Ph.D
I am intending to do a series of posts highlighting interviews with Philadelphia area biotech startup CEO’s and show how a vibrant biotech startup scene is evolving in the city as well as the Delaware Valley area. Philadelphia has been home to some of the nation’s oldest biotechs including Cephalon, Centocor, hundreds of spinouts from a multitude of universities as well as home of the first cloned animal (a frog), the first transgenic mouse, and Nobel laureates in the field of molecular biology and genetics. Although some recent disheartening news about the fall in rankings of Philadelphia as a biotech hub and recent remarks by CEO’s of former area companies has dominated the news, biotech incubators like the University City Science Center and Bucks County Biotechnology Center as well as a reinvigorated investment community (like PCCI and MABA) are bringing Philadelphia back. And although much work is needed to bring the Philadelphia area back to its former glory days (including political will at the state level) there are many bright spots such as the innovative young companies as outlined in these posts.
First up I got to talk with Florian Schodel, M.D., Ph.D., CEO of Philimmune, which provides expertise in medicine, clinical and regulatory development and analytical sciences to support successful development and registration of vaccines and biologics. Before founding Philimmune, Dr. Schodel was VP in Vaccines Clinical Research of Merck Research Laboratories and has led EU vaccine clinical trials and the clinical development of rotavirus, measles, mumps, hepatitis B, and rubella vaccines. In addition Dr. Schodel and Philimmune consult on several vaccine development efforts at numerous biotech companies including:
\His specialties and services include: vaccines and biologics development strategy, clinical development, clinical operations, strategic planning and alliances, international collaborations, analytical and assay development, project and portfolio integration and leadership.
Successful development of vaccines and biologics poses some unique challenges: including sterile manufacturing and substantial early capital investment before initiated clinical trials, assay development for clinical trial support, and unique trail design. Therefore vaccines and biologics development is a highly collaborative process between several disciplines.
In his opinion, Dr. Schodel believes our biggest hurdle in vaccine development in a societal issue, not a preclinic development issue. Great advances have been made to speed the discovery process and enhance quality assurance of manufacture capabilities like
however there has not been a great history or support for developing vaccines for the plethora of infectious diseases seen in the developing world. As Dr. Schodel pointed out, there are relatively few players in the field, and tough to get those few players excited for investing in new targets.
However, some companies are rapidly expanding their vaccine portfolios including
The Louvre Museum has 8.5 million visitors per year. This blog was viewed about 220,000 times in 2013. If it were an exhibit at the Louvre Museum, it would take about 9 days for that many people to see it.
In 2013, there were 958 new posts, growing the total archive of this blog to 1,505 posts. There were 982 pictures uploaded, taking up a total of 253 MB. That’s about 3 pictures per day.
Leaders in Pharmaceutical Business Intelligence AND Bio-Tree Systems [pending Bio-Tree finding funding]
Leaders in Pharmaceutical Business Intelligence AND Lou Pharma [pending finding Licensees for drugs manufactured in Spain]
Leaders in Pharmaceutical Business Intelligence AND AlphaSzenszor Inc.
Leaders in Pharmaceutical Business Intelligence AND ValveCure, LLC
VENTURE #5:
Invented HERE!
1. Development of a NEW Nitric Oxide monitor to Alpha Szenszor Inc. sensor portfolio. A concept for a low cost POC e-nose, capable of real time ppb detection of Cancer The Cancer Team at Leaders in Pharmaceutical Business Intelligence under the leadership of Dr. Williams
2. Development of a NEW Nitric Oxide monitor to Alpha Szenszor Inc. sensor portfolio. A concept for Inhaled Nitric Oxide for the Adult HomeCare Market –
IP by Dr. Pearlman and Dr. A. Lev-Ari
a. iknow iNO is i-kNOw – Inhaled Nitric Oxide for the HomeCare Market
3. Cancer Genomics for NEW product development in diagnosis and treatment of Cancer Patients using sensory technology with applications for Radiation Therapy – The Cancer Team at Leaders in Pharmaceutical Business Intelligence under leadership of Dr. Sidney Kadish.
4. Developing Mitral Valve Disease: MRI Methods and Devices for Percutaneous Mitral Valve Replacement and Mitral Valve Repair
Augmentation of Patented Technology using RF – Dr. Pearlman’s IP Non-Hardware Mitral Annuloplasty – Dr. Justin D. Pearlman
CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC
Author: Larry H. Bernstein, MD, FCAP, Triplex Medical Science
Article 1.4 CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomics Analysis and Disease – Part IIC
Part I: The Initiation and Growth of Molecular Biology and Genomics – Part I From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?
Part IIB. “CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics” lays the manifold multivariate systems analytical tools that has moved the science forward to a groung that ensures clinical application.
Part IIC. “CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease “ will extend the discussion to advances in the management of patients as well as providing a roadmap for pharmaceutical drug targeting.
This final paper of Part II concludes a thorough review of the scientific events leading to the discovery of the human genome, the purification and identification of the components of the chromosome and the DNA structure and role in regulation of embryogenesis, and potential targets for cancer.
The first two articles, Part IIA, Part IIB, go into some depth to elucidate the problems and breakthoughs encountered in the Human Genome Project, and the construction of a 3-D model necessary to explain interactions at a distance.
Part IIC, the final article, is entirely concerned with clinical application of this treasure trove of knowledge to resolving diseases of epigenetic nature in the young and the old, chronic inflammatory diseases, autoimmune diseases, infectious disease, gastrointestinal disorders, neurological and neurodegenerative diseases, and cancer.
Recently, large studies have identified some of the genetic basis for important common diseases such as heart disease and diabetes, but most of the genetic contribution to them remains undiscovered. Now researchers at the University of Massachusetts Amherst led by biostatistician Andrea Foulkes have applied sophisticated statistical tools to existing large databases to reveal substantial new information about genes that cause such conditions as high cholesterol linked to heart disease.
Foulkes says, “This new approach to data analysis provides opportunities for developing new treatments.” It also advances approaches
to identifying people at greatest risk for heart disease. Another important point is that our method is straightforward to use with freely
available computer software and can be applied broadly to advance genetic knowledge of many diseases.
The new analytical approach she developed with cardiologist Dr. Muredach Reilly at the University of Pennsylvania and others is called “Mixed modeling of Meta-Analysis P-values” or MixMAP. Because it makes use of existing public databases, the powerful new method
represents a low-cost tool for investigators.
MixMAP draws on a principled statistical modeling framework and the vast array of summary data now available from genetic association
studies to formally test at a new, locus-level, association.
While that traditional statistical method looks for one unusual “needle in a haystack” as a possible disease signal, Foulkes and colleagues’
new method uses knowledge of DNA regions in the genome that are likely to
contain several genetic signals for disease variation clumped together in one region.
Thus, it is able to detect groups of unusual variants rather than just single SNPs, offering a way to “call out” gene
regions that have a consistent signal above normal variation.
The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] —
a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.
LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with
levels of Lp(a) and, as a consequence, strongly associated with CAD.
However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity),
as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism).
Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.
To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous
participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for
2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1]
Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to
widen the generalizability of their results.
Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with
CAD,
peripheral arterial disease,
abdominal aortic aneurysm,
number of CAD vessels,
age at onset of CAD diagnosis, and
large-artery atherosclerosis-type stroke.
They did not find an association with
cardioembolic or small-vessel disease-type stroke;
intracranial aneurysm;
venous thrombosis;
carotid intima thickness; or,
in a small subset of individuals, myocardial infarction.
English: Structure of the LPA protein. Based on PyMOL rendering of PDB 1i71. (Photo credit: Wikipedia)
Micrograph of an artery that supplies the heart with significant atherosclerosis and marked luminal narrowing. Tissue has been stained using Masson’s trichrome. (Photo credit: Wikipedia)
Scientists at the Gladstone Institutes have revealed the precise order and timing of hundreds of genetic “switches” required to construct a fully
functional heart from embryonic heart cells — providing new clues into the genetic basis for some forms of congenital heart disease.
In a study being published online today in the journal Cell, researchers in the laboratory of Gladstone Senior Investigator Benoit Bruneau, PhD,
employed stem cell technology, next-generation DNA sequencing and computing tools to piece together the instruction manual, or “genomic
blueprint” for how a heart becomes a heart. These findings offer renewed hope for combating life-threatening heart defects such as arrhythmias (irregular heart beat) and ventricular septal defects (“holes in the heart”).
They approach heart formation with a wide-angle lens by
looking at the entirety of the genetic material that gives heart cells their unique identity.
The news comes at a time of emerging importance for the biological process called “epigenetics,” in which a non-genetic factor impacts a cell’s genetic
makeup early during development — but sometimes with longer-term consequences. All of the cells in an organism contain the same DNA, but the
epigenetic instructions encoded in specific DNA sequences give the cell its identity. Epigenetics is of particular interest in heart formation, as the
incorrect on-and-off switching of genes during fetal development can lead to congenital heart disease — some forms of which may not be apparent until adulthood.
the scientists took embryonic stem cells from mice and reprogrammed them into beating heart cells by mimicking embryonic development in a petri dish. Next, they extracted the DNA from developing and mature heart cells, using an advanced gene-sequencing technique called ChIP-seq that lets scientists “see” the epigenetic signatures written in the DNA.
Map of Heart Disease Death Rates in US White Males from 2000-2004 (Photo credit: Wikipedia)
Estimated propability of death or non-fatal myocardial-infarction over one year corresponding ti selectet values of the individual scores. Ordinate: individual score, abscissa: Propability of death or non-fatal myocardial infarction in 1 year (in %) (Photo credit: Wikipedia)
simply finding these signatures was only half the battle — we next had to decipher which aspects of heart formation they encoded
To do that, we harnessed the computing power of the Gladstone Bioinformatics Core. This allowed us to take the mountains of data collected from
gene sequencing and organize it into a readable, meaningful blueprint for how a heart becomes a heart.”
For each of the above datasets, an upstream analysis from the identified transcription factors correctly identified the stimulus. IPA’s tools were very
easy to use and the
analysis time for the above experiments was less than one minute.
The performance, speed, and ease of use can only be characterized as very good, perhaps leading to breakthroughs when extended and used creatively. Ingenuity’s new transcription factor analysis tool in IPA, coupled with Ingenuity’s established upstream grow tools, should be strongly considered for every lab analyzing differential expression data.
NF-E2-related factor 2 (Nrf2) is an important transcription factor that
activates the expression of cellular detoxifying enzymes.
Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which
results in cytoplasmic Nrf2 degradation.
Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-
based screening, we observed miR-200a silencing in breast cancer cells and
demonstrated that upon re-expression, miR-200a
targets the Keap1 3′-untranslated region (3′-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may
contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a
in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with
Nrf2 nuclear translocation
and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription.
Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates
that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR-200a
regulation of Keap1 expression,
reactivating the Nrf2-dependent antioxidant pathway in breast cancer.
Nuclear factor-like 2 (erythroid-derived 2, also known as NFE2L2 or Nrf2, is a transcription factor that in humans is encoded by the NFE2L2 gene.[1]) NFE2L2 induces the expression of various genes including those that encode for several antioxidant enzymes, and it may play a physiological role in the regulation of oxidative stress. Investigational drugs that target NFE2L2 are of interest as potential therapeutic interventions for
oxidative-stress related pathologies.
4. Highly active zinc finger nucleases by extended modular assembly
Zinc finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups,
the lack of robust non-commercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from
publicly-available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences.
However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might
be inherently inefficient due to its insensitivity to context-dependent effects.
Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted
loci in human and mouse cells. A novel Drop-Out Linker scheme was used to rapidly assess three- to six-finger combinations,
demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of
MA ZFNs of any array composition that exceed an ab initio
B-score cut-off of 15 were active.
MA ZFNs are able to target more DNA sequences with higher success rates than other methods.
These insightful reviews are based on the strategic data and insights from Thomson Reuters Cortellis™ for Competitive Intelligence. (A Review of April-June 2012).
The majority of diseases are complex and multi-factorial, involving multiple genes interacting with environmental factors. At the genetic level,
information from genome-wide association studies that elucidate common patterns of genetic variation across various human populations,
in addition to profiling, technologies can be utilized in discovery research to provide snapshots of genes and expression profiles that are controlled
by the same regulatory mechanism and are altered between healthy and diseased states.
The characterization of genes that are abnormally expressed in disease tissues could further be employed as
diagnostic markers,
prognostic indicators of efficacy and/or toxicity, or as
targets for therapeutic intervention.
As the defining catalyst that exponentially paved the way for personalized medicine, information from the published genome sequence revealed that much of the genetic variations in humans are concentrated in about 0.1 percent of the over 3 billion base pairs in the haploid DNA. Most of these variations involve substitution of a single nucleotide for another at a given location in the genetic sequence, known as single nucleotide polymorphism (SNP).
Combinations of linked SNPs aggregate together to form haplotypes and
together these serve as markers for locating genetic variations in DNA sequences.
SNPs located within the protein-coding region of a gene or within the control regions of DNA that regulate a gene’s activity could
have a substantial effect on the encoded protein and thus influence phenotypic outcomes.
Analyzing SNPs between patient population cohorts could highlight specific genotypic variations which can be correlated with specific phenotypic variations in disease predisposition and drug responses.
Prior to the genomic revolution, many of the established therapies were directed against less than 500 drug targets, with many of the top selling drugs acting on well defined protein pathways. However, the sequencing of the human genome has massively expanded the pool of molecular targets that could be exploited in unmet medical needs and currently, of the approximately 22,300 protein-coding genes in the human code, it has been estimated that up to 3000 are druggable. Furthermore, genomic technologies such as
high-throughput sequencing
and transcription profiling,
can be used to identify and validate biologically relevant target molecules, or can be applied to cell-based and mice disease models or directly to in vivo human tissues,
helping to correlate gene targets with phenotypic traits of complex diseases.
This is particularly important, as
insufficient validation of target gene/proteins in complex diseases may be a contributing factor in the decline in R&D productivity.
Personalized medicine no doubt is already having a tremendous impact on drug development pipelines. According to a study conducted by the Tufts Center for the Study of Drug Development, more than 90 percent of biopharmaceutical companies now utilize at least some
genomics-derived targets in their drug discovery programs.
However, pipeline analysis from Cortellis for Competitive Intelligence suggests that there is still a scientific gap that has resulted in difficulty optimizing these novel genomic targets into the clinical R&D portfolios of major pharmaceutical companies, particularly outside the oncology field. Selected examples of personalized medicine product candidates in clinical development include (see TABLE 4).
Mutations in Melanomaare in regions that control genes, not in the genes themselves. The mutations are exactly the type caused by exposure to ultraviolet light. The findings are reported in two papers in http://Science.com/ScienceExpress/
The findings do not suggest new treatments, but they help explain how melanomas – and possibly – other cancers – develop and what drives their growth. This is a modification found in the “dark matter”, according to Dr. Levi A. Garraway, the 99 percent of DNA in a region that regulates genes. A small control region was mutated in 7 out of 10 of the tumors, commonly of one or two tiny changes. A German Team led by Rajiv Kumar (Heidelberg) and Dirk Schadendorf (Essen) looked at a family whose members tended to get melanomas. Their findings indicate that those inherited with the mutations might be born with cells that have taken the first step toward cancer.
The mutations spur cells to make telomerase, that keeps the cells immortal by preventing them from losing the ends of their chromosome, the telomere. Abundant telomerase occurs in 90 percent of cancers, according to Immaculata De Vivo at Harvard Medical School.
The importance of the findings is that the mechanism of telomerase involvement in cancer is now within view. But it is not clear how to block the telomerase production in cancer cells.
A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)
Comment
This discussion addresses the issues raised about the direction to follow in personalized medicine. Despite the amount of work necessary to bring the clarity that is sought after, the experiments and experimental design is most essential.
The arrest of ciliogenesis in ovarian cancer cell lines compared to wild type (WT) ovarian epithelial cells, and
The link to suppressing ciliogenesis by AURA protein and CHFR at the base of the cilium, which disappears at mitosis or with proliferation.
There is no accumulation by upregulation of PDGF under starvation by the cancer cells compared to the effect in WT OSE.
Here we have a systematic combination of signaling events tied to changes in putative biomarkers that occur synchronously in Ov cancer cell lines.
These changes are identified with changes in
proliferation,
loss of ciliary structure, and
proliferation.
In this described scenario,
WT OSE cells would be arrested, and
it appears that they would take the path to apoptosis (under starvation).
Even without more information, this cluster is what one wants to have in a “syndromic classification”. The information used to form the classification entails the identification of strong‘signaling-related’ biomarkers. The Gli2 peptide has to be part of this.
In principle, a syndromic classification would be ideally expected to have no less than 64 classes. If the classification is “weak”, then the class frequencies would be close to what one would expect in the WT OSE. In this case, in reality,
several combinatorial classes would have low frequency, and
others would be quite high.
This obeys the classification rules established by feature identification, and the information gain described by Solomon Kullback and extended by Akaike.
Does this have to be the case for all different cancer types? I don’t think so. The cells are different in ontogenesis. In this case, even the WT OSE have mesenchymal features and so, are not fully directed to epithelial expression. This happens to be the case in actual anatomic expression of the ovary. On the other hand, one would expect shared features of the
ovary,
testes,
thyroid,
adrenals, and
pituitary.
There is biochemical expression in terms of their synthetic function – TPN organs. I would have to put the liver into that broad class. Other organs – skeletal muscle & heart – transform substrate into energy or work. (Where you might also put intestinal smooth muscle).
They have to have different biomarker expressions, even though they much less often don’t form neoplasms. (Bone is not just a bioenergetic force. It is maintained by muscle action. It forms sarcomas. But there has to be a balance between bone removal by osteoclasts and refill by osteoblasts.)
Viewpoint: What we have learned
The Watson-Crick model proposed in 1953 is limited for explaining fully genome effects
The Pauling triplex model may have been prescient because of a more full anticipation of molecular bonding variants
A more adequate triple-helix model has been proposed and is consistent with a compact genome in the nucleus
The structure of the genome is not as we assumed – based on the application of Fractal Geometry. Current body of evidence is building that can reveal a more complete view of genome function.
transcription
cell regulation
mutations
Summary
I have just completed a most comprehensive review of the Human Genome Project. There are key research collaborations, problems in deciphering the underlying structure of the genome, and there are also both obstacles and insights to elucidating the complexity of the final model.
This is because of frequent observations of molecular problems in folding and other interactions between nucleotides that challenge the sufficiency of the original DNA model proposed by Watson and Crick. This has come about because of breakthrough innovation in technology and in computational methods.
Radoslav Bozov •
Molecular biology and growth was primarily initiated on biochemical structural paradigms aiming to define functional spatial dynamics of molecules via assignation of various types of bondings – covalent and non-covalent – hydrogen, ionic , dipole-dipole, hydrophobic interactions.
Lab techniques based on z/m paradigm allowed separation, isolation and identification of bio substances with a general marker identity finding correlation between physiological/cellular states.
The development of electronic/x-ray technologies allowed zooming in nano space without capturing time.
NMR technology identified the existence of space topology of initial and final atomic states giving a highly limited light on time – energy axis of atomic interactions.
Sequence technology and genomic perturbations shed light on uncertainty of genomic dynamics and regulators of functional ever expanding networks.
Transition state theory coupled to structural complexity identification and enzymatic mechanisms ran up parallel to work on various phenomena of strings of nucleotides (oligomers and polymers) – illusion/observation of constructing models on the dynamics of protein-dna-rna interference.
The physical energetic constrains of biochemistry were inapplicable in open biological systems. Biologists have accepted observation as a sole driver towards re-evaluating models.
The separation of matter and time constrains emerged as deviation of energy and space constrains transforming into the full acceptance of code theory of life. One simple thing was left unnoticed over time –
the amount of information of quantum matter within a single codon is larger than that of a single amino acid. This violated all physical laws/principles known to work with a limited degree of certainty.
The limited amount of information analyzed by conventional sequence identity led to the notion of applicability of statistical measures of and PCR technology. Mutations were identified over larger scale of data.
Quantum chemistry itself is being limited due discrete space/energy constrains, thus it transformed into concepts/principles in biology that possess highly limited physical values whatsoever.
The central dogma is partially broken as a result of
regulatory constrains
epigenetic phenomena and
iRNA.
Large scale code computational data run into uncertainty of the processes of evolution and its consequence of signaling transformation. All drugs were ‘lucky based’ applicability and/or discovery with largely unpredictable side effect over time.
Other Related articles on this Open Access Online Sceintific Journal include the following:
This post is in continuation to Part 1 by the same title.
In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.
Regulation of the redox immunomodulators—NO/RNS and ROS
In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.
Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)
Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.
Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.
NO levels dictate the immune signaling pathway
NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.
Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.
Figure and legend credits: (Wink et al., 2011)
Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.
This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.
Species-specific NO production
The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.
The next post in this series will cover the following topics:
The impact of NO signaling on an innate immune response—classical activation
NO and proinflammatory genes
NO and regulation of anti-inflammatory pathways
NO impact on adaptive immunity—immunosuppression and tissue-restoration response
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)
Fig 1. Image credit: (Wink et al., 2011)
Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).
In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.
NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.
Redox reactions in immune responses
NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).
The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONO–intermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.
The Phagosome ‘cauldron’
The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.
Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)
This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.
Bacteria
There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples
E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.
Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.
Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.
Parasites
Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, Helicobacter, Mycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).
Virus
Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
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