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January 2011 | Issue 18 | www.meducator.org

From Policy to the Periphery A look at India's National Rural Health Mission and its presence in Northern India's Primary Healthcare System

Fighting Cystic Fibrosis on the Molecular Level

Small Molecule Inhibitors of Biofilm Formation

Breathless for a Breakthrough How Electrophysiology is Broadening our Understanding of Asthma

Cholesteryl Ester Transfer Protein: The Key to Longevity?

Using Genetic Variants to Combat Arterial and Cognitive Diseases


issue 18 | JANUARY 2011

MEDUCATOR STAFF

LETTER FROM THE EDITOR

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MEDWIRES

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MEDBULLETINS

FEATURE

FROM POLICY TO THE PERIPHERY:

A Look at India’s National Rural Health Mission

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by SHEIRY DHILLON

articles SMALL MOLECULE INHIBITORS OF Pseudomonas Aeruginosa BIOFILM DEVELOPMENT

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ELECTRICAL PROPERTIES OF PULMONARY LYMPHATIC SMOOTH MUSCLE CELLS

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by MATTHEW A. CHONG

by MUSTAFA AHMADZAI

President Ahmad AlKhatib Vice-President Hiten Naik Editorial Board Khaled Ramadan Keon Maleki-Yazdi Matthew Macdonald Shelly Ramsaroop Sohail Mulla Mohsin Ali Matthew Chong Mustafa Ahmadzai Charles Yin Shelly Chopra Vaibhev Mokashi Graphics & Design Brian Chin Daniel Lee Lebei Pi Xena Li Ellen Liang Web Directors Ijlal Syed Aashish Kalani Public Relations Alyssa Cantarutti Louis Winston ADDRESS

CHOLESTERYL ESTER TRANSFER PROTEIN: THE KEY TO LONGEVITY?

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by VITHOOSHAN VIJAYAKUMARAN & NEIL D. DATTANI

B.H.Sc. (Honours) Program The Meducator Michael G. DeGroote Centre for Learning and Discovery Room 3308 Faculty of Health Sciences 1200 Main Street West Hamilton, Ontario L8N 3Z5

EMAIL

ABOUT Us:

Established in April 2002 with the support of the Bachelor of Health Sciences (Honours) Program (B.H.Sc.), The Meducator is McMaster University’s undergraduate health sciences publication. Through biyearly publications, a web page, and Facebook, we aim to provide an opportunity for undergraduate students to publish their work and share information with their peers. Our protocol strives to maintain the highest standard of academic integrity by having each article edited by a postgraduate in the relevant field. We invite you to offer us your feedback by writing to our email: the.meducator@learnlink.mcmaster.ca

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LETTER FROM THE EDITOR Our New Vision This issue’s cover image is a photograph of Sheiry Dhillon, a third year undergraduate student, in the midst of a discussion with women of the Indian village Chola. We chose this photograph as the face of Issue 18 because it captured the immersive nature of her research experience: as Sheiry discusses in our feature article, she spent several months of her summer studying rural health institutions in the northern Indian state of Himachal Pradesh. This image reflects a new vision for The Meducator: a platform for research articles that are student centered. As students, we recognize that the opportunity to conduct research at the undergraduate level can be an exciting experience – the process of discovery is both rewarding and stimulating. We also realize that there are few opportunities for undergraduate students to express the nature and significance of their research in an open forum. We want The Meducator to provide you with that opportunity – to share with the student community the aspects of your research that excite you. In order to facilitate this vision, we have introduced a new article format that we call Research Insight. The Research Insight article allows students to discuss the nature of their work, while reflecting on its significance and implications in the broader context of their respective fields. In this issue, Matthew Chong, working in the lab of Dr. Lori Burrows, discusses his research on biofilm development by the opportunistic pathogen Pseudomonas aeruginosa, while Mustafa Ahmadzai, working in the lab of Dr. Luke Janssen,

discusses his research on the electrophysiological properties of lymphatic smooth muscle cells in the lungs. If you have a strong interest in a particular topic in the health sciences, we have introduced another article format that we call Critical Review. The Critical Review article allows students to take a critical look at a particular issue or topic. In this issue, Vithooshan Vijayakumaran and Neil D. Dattani discuss how variation in the gene for an important cholesterol transport protein may yield significant health benefits. The variety of topics in this issue reflects our goal of expanding the scope of The Meducator to include all subject matter within the broad, interconnected realm of health sciences: global health, medical science, epidemiology, health policy, health ethics, and everything in between. Thus, our new tagline: McMaster University’s undergraduate health sciences publication. The Meducator would like to stay in touch with you, our readers, throughout the year. In order to do so, we have created a Facebook page that is updated daily with MedWires, short updates (250 characters) on advances in the health sciences. I encourage you to subscribe. We have also launched a new website with a professional archive, so that your written content remains open to the public in the years to come. Visit the website at www.meducator.org or search for “The Meducator” on Facebook. I would like to thank Hiten Naik, Vice-President, and the remainder of The Meducator team for their tireless work in making the above changes possible.

AHMAD ALKHATIB

President & Editor-in-Chief Bachelor of Health Sciences (Honours) Program, Class of 2012

The Meducator | January 2011

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MEDWIRE 3

Optogenetics is a new branch of imaging that helps neuroscientists not only observe action potential firing between specific neurons, but also control electrical impulses traveling down an axon, thereby providing insight into neurological function.

Psychologists have started treating patients with katsaridaphobia—the fear of cockroaches— using augmented reality therapy, wherein phobics can confront digital renditions of their fears within a real-world environment.

McMaster researcher Dr. Mick Bhatia and his colleagues became the first to successfully transform human skin cells into blood cells. The applications of this kind of cellular alchemy seem endless.

The British Medical Journal found that a diet rich in green leafy vegetables may reduce the risk of developing type II diabetes by up to 14%.

Feeling homesick yet? Although the feeling is not new, there are new developments on what causes the feeling. A recent study has found evidence that homesickness is nothing more than a simple adjustment issue.

Researchers at Sunnybrook have developed a cellphone software that can potentially help teens with mood disorders. Instead of using ‘mood journals’ or other currently common psychiatric methods, teens would record their feelings on their cell phone.

Researchers have successfully engineered mice with human liver cells, thereby allowing them to study the processes of the human immune system during diseases such as hepatitis B and C.

There is new insight into which genes may cause breast cancer. Women with the “high risk” breast cancer gene will be able to find out more about their chances of developing the disease by determining which region of the DNA the gene is in.

Oxytocin, a hormone involved in feelings of security, has demonstrated its potential to improve social abilities in autistic adults. Researchers found autistic participants to prefer interaction with more co-operative opponents during a computerized game, after inhaling oxytocin.

Bacteria inside fruit flies have been found to alter their pheromone production in such a way as to favour mating between fruit flies that are on the same diet. Therefore, symbiotic organisms may indirectly drive evolution of distinct fly species by influencing mate choice.

Researchers at UCLA have mapped out how an autism gene alters connections between neurons, changing the physiology of the brain. This discovery provides a critical missing link, bringing together genetics, physiology and neuroscience together in order to better understand autism.

Scientists have successfully developed a robotic hand capable of performing intricate tasks such as drawing. Instead of mimicking the complexity of the joints in the human hand, this device is essentially a grain-filled sphere that moulds to grasp objects without losing grip.


Introspection has been linked to the amount of grey matter a person has in their prefrontal cortex. The study also found that introspective people were more confident in the decisions they have made.

Canadian Tobacco Use Monitoring Survey reports that there has been a 7% drop in teen smoking in teenagers 15 and older. However, the rate of decline has slowed.

While it is known that antibiotics can cause temporary stomach discomfort, new studies are now showing that repeat exposure to these drugs might be altering the ‘good’ bacteria that reside in the gut. The long term effects of this study remain unknown.

Variations in the amino acid sequence of HLA-B proteins allow the immune system to break down HIV peptides. This may be why some individuals infected with HIV do not progress into full-fledged AIDS, thus giving direction for future vaccine efforts countering HIV.

According to a study by the American College Health Association, severe mental illness is becoming more common on university campuses across the United States and Canada.

People concerned with healthy food choices in restaurants tend to underestimate calorie counts. Consequently, they reward themselves by overeating.

Neuroscientists have discovered that 20 minutes of electric stimulation, targeted at the right parietal lobe, significantly improves numerical processing. While ethically debated, it may prove to be an efficient strategy in enhancing cognition and elucidating the role of specific brain regions.

Scientists have sequenced the genome of musician Ozzy Osbourne. Osbourne wanted to know what made him the Prince of Darkness and his genes did not disappoint. Thousands of rare variants were found in Osbourne’s genome, including mutations affecting brain function and alcohol metabolism.

The American Academy of Sleep Medicine published a study recently reporting that people, especially men, still need their 8 hours of sleep. Men with chronic insomnia and people who slept less than 6 hours a day were four times more likely to die than ‘good sleepers’ (8 hours a day).

Ultrathin actin cables called membrane nanotubes allow communication between cells that are far apart. Electrical signals sent through nanotubes may explain synchronized cell behavior. This finding has broad implications in terms of cell communication in the brain.

Osteoarthritis is a debilitating joint disease involving cartilage degradation. A recent animal study showed that when ADAMTS5, an enzyme implicated in this process, was inhibited, no cartilage damage was observed. These findings suggest a potential therapeutic target for osteoarthritis treatments.

MEDWIRE

Geron Corp. treated a human spinal cord injury with embryonic stem cells. This is part of an early trial to determine the safety of this type of treatment in the future. While this is exciting news, years of testing are still required.

The Meducator | January 2011

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MedBulletin

Novel Tuberculosis Treatment: A Nano-Therapy for a Massive Disease by Vaibhav Mokashi

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uberculosis (TB) is caused by inhalation of airborne particles containing Mycobacterium tuberculosis, which subsequently infiltrate the immune system to give rise to a productive cough, fever and lethargy. Antibiotics against TB have become ineffective due to the emergence of multiple strains of drug resistant M. tuberculosis. Consequently, a novel therapy is being developed that involves oral or aerosol administration of antibiotics encapsulated in nanobeads.1 Nanobeads consist of a polymer membrane that protects commonly used anti-TB drugs such as isoniazid and rifampin. Once nanobeads enter the body, they are actively transported across the epithelial layer of the lungs and are taken up by macrophages, the cells principally infected with and harboring M. tuberculosis. Macrophages engulf nanobeads through phagocytosis. The nanobeads then enter a phagolysosome, a digestive organelle within macrophages. The acidic environment of the phagolysosome catalyzes the breakdown of the polymer coating the nanobeads, thus releasing active drug to combat invading bacteria.1 Interestingly, animal trials found that three oral doses of nanotherapy were as effective as 45 doses of traditionally administered antibiotic treatments.1 Based on these results, this new technology may greatly reduce the dosing frequency for patients, as well as the possibility of reoccurrence due to drug resistance. Furthermore, since the nanotherapy mainly targets infected cells, it enables administration of more toxic drugs without significant damage to healthy cells. Moreover, nanobead encapsulation does not decrease the shelf life or efficacy of antibiotics.1 Overall, this newfound nanotherapy has the potential to revolutionize treatment for TB, a disease that claims 1.8 million lives a year. Griffiths G, Nyström B, Sable SB, & Khuller GK. (2010). Nanobead-based interventions for the treatment and prevention of tuberculosis. Nature Reviews Microbiology, 8, 827-834. Available from: doi:10.1038/nrmicro2437 [Accessed 23rd October 2010]. 1

The Link between de novo Mutations and Sporadic Disease by Charles Yin

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hen one thinks of genetic disease, the image that most often comes to mind is a condition present in the parents and then passed on to the offspring. However, >97% of cases of genetic disease are sporadic: the mutations responsible are not present in the somatic cells of either parent, but arise during germ cell replication.1 Sporadic diseases are thought to be caused by either nondisjunction or de novo mutations in the sperm or egg cells. De novo mutations are novel mutations that arise naturally due to DNA replication errors. When these occur in the germ line, the mutation can be passed to offspring and a new mutation is created that can result in disease.1 Examples of the effects of de novo mutagenesis are numerous. Certain forms of Parkinson’s disease are sporadic and have recently been demonstrated to be the result of de novo mutations. 2 Some forms of childhood epilepsy are also the result of de novo mutations. A small number of disorders that constitute a subset of sporadic childhood epilepsy have been found to be the result of just a single mutation in the gamete of the parent.3

Now that the connection between de novo mutagenesis and sporadic disease has been clearly established, future research in this field will likely focus on the timing and location of the appearance of these mutations in the germ line. In a recent study published by Vadlamudi et al., the timing of de novo mutations turns out to be a critical factor in the occurrence of Dravet’s syndrome (a form of sporadic epilepsy) in twins.4 If a mutation occurs in a gamete, its chances of successfully undergoing fertilization will be dramatically decreased. However, if the mutation occurs in the germ line progenitor instead, every gamete produced will carry that mutation and every offspring produced will be affected. Lupski JR. (2007). Genomic arrangements and sporadic disease. Nature Genetics, 39, S43-7. Available at: http://www.nature.com/ng/journal/v39/n7s/full/ng2084.html [Accessed October 26, 2010] Berry AL, & Foltynie T. (2010). Gene therapy: A viable therapeutic technique for Parkinson’s disease? J Neurol. Available at: http://www.springerlink.com/content/484064jjwj283863/fulltext.pdf [Accessed October 26, 2010] 3 Stafstrom CE. (2009). Severe epilepsy syndromes of early childhood: The link between genetics and pathophysiology with a focus on SCN1A mutations. J Child Neuro, 24(8S), 15S-23S. Available at: http://jcn. sagepub.com/content/24/8_suppl/15S.long [Accessed October 26, 2010] 4 Vadlamudi L, et. al. (2010). Timing of de novo mutagenesis – A twin study of sodium-channel mutations. New Eng J of Med. Available at: http://www.nejm.org/doi/pdf/10.1056/NEJMoa0910752 [Accessed October 26, 2010] 1 2

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by Vaibhav Mokashi

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indings by Yale University’s Vanja Duric suggest that MPK-1, an enzyme of the MAPK signaling cascade, plays an important role in the deregulation of certain areas of the brain. Duric showed that MPK-1 expression increases during stressful situations and negatively regulates the MAPK pathway that is involved in neuronal plasticity, function and survival. Consequently, the negative regulation of MAPK decreases the growth and viability of neurons in the hippocampus – the region of the brain that plays a key role in depression. Further testing using rat models showed that chronic antidepressant treatments normalize stress-induced MPK-1 over expression. Dr. Duric’s team believes that developing drugs that reduce MPK-1 expression in depressives will be much easier now that MAPK mechanisms are understood thoroughly.1

MedBulletin

Developments in Depression Research

On a similar note, Dr. Alexander at Cornell successfully used gene therapy to reduce depression-like symptoms. In his animal model of depression, mice were missing the p11 gene, which regulates levels of serotonin, a neurotransmitter that is linked with mood, sleep and memory. Dr. Alexander’s team used a viral vector to deliver a functioning copy of the gene into the defective mice to restore p11 expression and reduce depressive behaviour.2 Overall, these exciting new developments in molecular neuroscience and pharmacogenomics have the potential to improve our understanding of depression and may lead to new treatment options. Alexander B, Warner-Schmidt J, Eriksson T, Tamminga C, Arango-Lievano M, Ghose S, Vernov M, Stavarache M, Musatov S, Flajolet M, Svenningsson P, Greengard P, & Kaplitt M. (2010). Reversal of Depressed Behaviors in Mice by p11 Gene Therapy in the Nucleus Accumbens. Science Translational Medicine, 2(54), 54-76. Available from: DOI: 10.1126/scitranslmed.3001079 [Accessed 1st November 2010]. 2 Duric V, Banasir M, Licznerski P, Schmidt HD, Stockmeier CA, Simen AA, Newton SS, & Duman RS. (2010). A negative regulator of MAP kinase causes depressive behavior. Nature Medicine. Available from: doi:10.1038/nm.2219 [Accessed 1st November 2010]. 1

Poke-Free Glucose Monitoring for Diabetic Patients

by Shelly Chopra

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iabetics prick their fingers multiple times per day to monitor their blood glucose level. While this method may provide accurate measurements, the data obtained are of a discontinuous nature and prevent physicians from discerning glucose level trends. Over the past few years, continuous glucose monitoring (CGM) devices have been developed in response to this limitation of traditional glucometers. Unfortunately, a common requirement of CGMs is the use of external devices to record data, a criterion giving rise to potential infection. In an attempt to overcome the shortfalls of current CGM and conventional monitoring devices, Shibata et al. designed a system that involves the implantation of biostable microbeads under the skin.1 Shibata et al. first synthesized a fluorescent monomer that not only binds to glucose reversibly but also maintains its properties when immobilized on the surface of a polymer, such as a bead. The monomer was also designed with a high surface area, enabling easy access to glucose-binding sites. After integrating the fluorescent monomer into a microbead, the researchers tested its efficacy in mice by injecting the beads beneath the epidermis of each mouse’s ear. As the fluorescent monomers proved highly responsive to glucose, Shibata et al. used fluorescent imaging to monitor continuous fluctuation in blood glucose levels. In regards to measurement accuracy, the glucose concentrations calculated by the fluorescence microscope were found to correlate closely with those obtained through blood tests.1 Being comparably less invasive, coupled with a high degree of sensitivity, glucose monitoring via fluorescent microbeads may preserve the index fingers of 171 million diabetics from ever being poked again.2

Shibata H, Heo YJ, Okitsu T, Matsunaga Y, Kawanishi T, & Takeuchi S. (2010). Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring. Proceedings of the National Academy of Sciences of the United States of America, 107(42), 17894. Available from: http://www.pnas.org/content/early/2010/09/27/1006911107. abstract. [Accessed 29 October 2010]. 2 Rathmann W, & Giani G. (2004). Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care, 27(10), 2568-9. 1

The Meducator | January 2011

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RESEARCH INSIGHT

From Policy to the Periphery: A Look at India’s National Rural Health Mission Sheiry Dhillon Bachelor of Health Sciences (Honours) Program, Class of 2012 Global Health Specialization Sheiry is a third year student in the Global Health Specialization of the Bachelor of Health Sciences (Honours) Program. She spent this past summer in Northern India’s Himalayan region completing a research project for Jagori Grameen, a local non-governmental organization. The central focus of her research was to understand the various rural health standards set by the Indian government and to examine whether primary health centers in the district of Kangra, Himachal Pradesh, are meeting these standards.

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he World Health Organization refers to health policy as “decisions, plans, and actions that are undertaken to achieve specific health care goals within a society.”1 This description is deceivingly simple; health policy is complex and requires short and long term goals, prioritization, and the involvement of a wide array of stakeholders ranging from funding bodies and healthcare providers to, of course, the members of the community.1 Implementation of health policies, therefore, requires a multidimensional approach and when completed successfully, is a vital tool in the improvement of the overall health of a population.

In today’s world of policy, it is not uncommon to encounter the terms “bottom-up” and “top-down” in regards to policy implementation. The ‘top’ refers to governing bodies and policy-makers where schemes originate, whereas the “bottom” is in reference to the grassroots where we find the community and lower levels of governance.2 Many national policies and programs, specifically the ones that will be further discussed in this paper, are defined from the top, yet require administration and implementation from the bottom. This is an integrative approach, where communication and accountability between service delivery on the grassroots and the expectations from the “top,” must be reconciled.3,4 The National Rural Health Mission (NRHM) introduced by the Government of India (GOI), is an interesting case in point. Today, the GOI’s agenda places heavy emphasis on this integrative process in order to increase access and quality to healthcare for the country’s rural areas. However, with a large and extremely diverse population, integrating the multiple levels of governance to deliver improved healthcare can be difficult. This paper will investigate India’s NRHM to better understand the importance of an integrative approach to health policy.

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HEALTH POLICY IN INDIA India has a population of 1.2 billion people, the second largest in the world—70% of that number lives in a rural setting where healthcare is lacking in access and quality.5 The rural-urban gap across India has consistently plagued the country’s healthcare sector; the rural sector faces a constant lack of resources, infrastructure, and primary healthcare providers. Socio-cultural dynamics and harsh living conditions also contribute significantly to barriers for accessible healthcare.6 In an attempt to target this inadequacy in the rural health sector, the central government launched the NRHM, a seven-year program to be implemented by 2012.7 Amongst its many goals, the NRHM emphasizes the importance of increasing the standards at which peripheral health centers operate. As a tool to meet these goals, the GOI has developed the Indian Public Health Standards (IPHS) for health centers, which outline the bare minimum requirements for the delivery of adequate and quality healthcare.7,8,9

THE IMPORTANCE OF THE PRIMARY HEALTH CENTRE The rural healthcare system in India is three-tiered in nature. At the most fundamental level is the health sub-center, which triages and refers patients to the second pillar, the primary health center (PHC). At a PHC, a physician and other health workers provide primary care. The Community Health Centre, which is the highest pillar of the three-tier system, functions similarly to the PHC, but caters to a larger population with more specialized services. Coverage


Health Personnel

# Req’d as per IPHS

Medical Officer

1

Pharmacist

1

Nurse-midwife

1

Healthworkers (Female)

1

Health Educator*

1

Health Assistant*

2

TABLE 1: PHC staffing requirements specified by the IPHS. Depicts the minimal staffing positions and number of personnel required at each PHC under the NRHM. *Indicates male and female

of each of these centers can be seen in Table 2.9 The three health centers operate in unison to collaboratively achieve quality and accessible healthcare for all. The PHC is the first contact point between the community and the public health doctor, making it India’s ‘cornerstone of rural healthcare.’9 The PHC therefore acts as an essential FIGURE 1: Map of the Kangra district of Himachal Pradesh. hub, accepting patients from sub-centers lacking the clinical knowledge, and referring patients further when needed.9 THE REALITIES OF THE Though PHCs do not solely indicate the quality of the rural PRIMARY HEALTH CENTRE healthcare sector, their importance makes them a highly reflective indicator of the rural community’s access to public health facilities. As the NRHM aims to increase quality and To better understand the current resources and quality of access to healthcare for rural populations by 2012, it is crucial healthcare delivery for its constituency, Jagori Grameen, a that PHCs, critical actors in the implementation process of local non-governmental organization in the Kangra district, the program, are assessed to evaluate whether the mission has evaluated six Primary Health Centers against the standards had an effective presence on the grassroots level. The results set by the NRHM. Through consultation of the official are ultimately a reflection of how well the top down and the governmental IPHS document and public health officials, bottom up approaches to policy are integrated. Using the five integral areas of assessment were identified for the GOI based IPHS documents as a basis of assessment, the questionnaire: Treatment & Testing, Resources & Services, aforementioned integration will be assessed by evaluating Manpower, Educational Services, and Monitoring. the PHCs in the rural, hilly, and tribal regions of the Kangra The results were far from proclaiming the PHC as the district in Himachal Pradesh (see Figure 1). “cornerstone of rural healthcare.” Of the PHCs surveyed,

Centre

Population Norms Plain Area

Hill/Tribal/Difficult Area

Sub-Centre

5,000

2,000

Primary Health Centre

30,000

20,000

Community Health Centre

120,000

80,000

TABLE 2: Coverage of 3-tier healthcare system. Listed above are the three pillars of India’s healthcare system. The health centres covering larger areas (eg. community health centres) are typically the most adequately prepared in regards to resources and health personnel.

The Meducator | January 2011

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Primary management of wounds

Primary Primary management management of fractures of poisoning

Primary management of burns

Minor surgeries

Labour room and services

Cataract surgery

PHC1

yes

yes

yes

yes

yes

yes

no

PHC2

yes

no

yes

yes

yes

no

no

PHC3

yes

no

no

no

no

no

no

PHC4

yes

yes

no

yes

yes

no

no

PHC5

yes

yes

yes

yes

no

no

no

PHC6

yes

yes

yes

yes

yes

yes

no

Percentage Yes (%)

100%

67%

67%

83%

67%

33%

0%

Percentage No (%)

0%

33%

33%

17%

33%

67%

100%

TABLE 3: Availability of treatments across PHCs.

Moreover, all five areas of assessment in the questionnaire across all PHCs uncovered significant deficiencies. Simple standards set by the IPHS (i.e. physician availability at each PHC)9 were not met. Two PHCs were missing doctors entirely; the other four lacked at least one other important healthcare professional outlined in Table 1.9 Table 3 and Figure 2 further outline the list of vital treatment and testing facilities that should be available at each PHC and their actual prevalence across the centers. Overall, although well intentioned, the apparent failure to achieve many of the goals pursued under the NRHM leaves little room for optimism. All personnel interviewed had the same response: resources are scarce, quality of healthcare delivery is low, and therefore the community lacks confidence in the rural healthcare system. Consequently, the following questions naturally arise: are the standards set by the IPHS realistic or just appropriate on paper, and who exactly is accountable for ensuring that these standards created by the central government are met at the periphery?

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# PHCs Offering Laboratory Tests 0

1

2

3

Bloodsmear for Malaria

Pregnancy Test Routine Urine, Stool, Blood Tests

Laboratory Tests

a mere 50% were aware of the term IPHS or its Hindi equivalent; none of those aware of the term had read or received an official IPHS document from the central, state, or local governments. This again highlights the importance of the top and bottom integration in policy implementation. Without a sound communication strategy between the varying levels of governance, awareness at the grassroots will not exist, and goals on a national level will not be met. The IPHS documents have been created by the central government and are hundreds of detailed pages outlining the roles of staff, programs, and resources that should be available at the PHC.9 In this case, however, crucial information has failed to reach important stakeholders at the periphery due to a lack of integration. Without awareness of the IPHS and their importance in healthcare delivery at the periphery, the documents are ultimately useless in increasing the quality of rural healthcare as they are intended to.

Blood Grouping

Bleedingtime Clotting Test

Test for RTIs/STDs Sputum Testing for TB

Rapid HIV Test

FIGURE 2: Number of PHCs offering laboratory tests.

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POLICY AND THE PERIPHERY The Government of India has addressed the importance of “architectural correction” in the nation’s rural healthcare sector. It has also created tools such as the IPHS to improve the standards of rural healthcare delivery.10 Though the mission has indeed addressed the need for improvement in rural health, it has also been criticized for taking a “simplistic approach to a complex problem.”7 The development of the IPHS for PHCs is a case in point; creation of documents and standards is simply not enough. Policy, programs, and standards must have an avenue of reaching the periphery from a higher level of government. As seen in this study, PHCs in Kangra have no access or exposure to information considered vital to the success of the NRHM. Successful implementation of the NRHM and IPHS for PHCs requires a sound communication strategy to bridge national expectations from the “top” with the healthcare providers responsible for implementation at

the “bottom.” Moreover, once a communication strategy is in place and the peripheral PHCs are aware of the standards, they must be held accountable for meeting these standards. Lower levels of health governance, close to the periphery, such as district and block health officers, must be involved in ensuring that these standards are indeed met. The NRHM is headed in the right direction. It is addressing major needs in rural healthcare and creating tools to accomplish its goals. However, as it is approaching its 2012 deadline rather quickly, it is unacceptable for stakeholders at the periphery to be unaware of the policies. If the periphery is unaware of the policies and standards, accountability is essentially out of the question. Rather than just creating tools like the IPHS, the NRHM must focus on sound communication strategies so that health centers, like those in Kangra, have an awareness of these tools and policies and ensure accountability so the standards do not go unmet.

Reviewed by Kaelan Moat, Ph.D. Student

Kaelan is currently a second year PhD Student in the Health Policy program at the Centre for Health Economics and Policy Analysis (CHEPA), McMaster University, and is being supervised by Dr. John Lavis. His research interests include analyzing the influence of evidence-informed policy briefs as a mechanism to translate research evidence to health systems policy decisions, with an emphasis on low- and middle-income countries and a particular focus on Africa. He has also explored the impact of decentralization policy in India on its public health system and has a keen interest in comparative health systems studies in both low- and high-income countries. He is currently working within the Program in Policy Decision-Making as a research assistant on a project with the Canadian Cochrane Network and Centre to help develop a database of systematic reviews focusing solely on health systems research. Prior to arriving at McMaster, Kaelan earned a Master of Science degree at the London School of Economics and Political Science in International Health Policy and has worked for NGOs in both Canada and India.

REFERENCES The World Health Organization. (2010). Health Policy. Retrieved from http://www.who.int/topics/health_policy/ en/. 2 Baum F. (2007). Cracking the nut of health equity: top down and bottom up pressure for action on the social determinants of health. Promotion & Education, 14(2), 90-95. 3 Sheikh K, & Porter J. (2010). Discursive gaps in the implementation of public health policy guidelines in India: The case of HIV testing. Social Science & Medicine, 71(11), 2005-2013. 4 Nixon J. (1980). The importance of communication in the implementation of government policy at local level. Policy & Politics, 8(2), 127-144. 5 Government of India. (2001). Rural-urban distribution census of India: Census data 2001. Retrieved from http:// www.censusindia.gov.in/Census_Data_2001/India_at_ glance/rural.aspx. 1

Alcoba N. (2009). Hitting the road to alleviating India’s rural doctor shortage. Canadian Medical Association Journal, 180(10), 34-36. 7 Banerji D. (2005). Politics of rural health in India. International Journal of Health Services, 35(4), 783-796. 8 Zaman F, & Laskar N. (2010). An application of Indian public health standard for evaluation of primary health centers of an EAG and a Non-EAG state. Indian Journal of Public Health, 54(1), 36-39. 9 Directorate General of Health Services Ministry of Health & Family Welfare Government of India. (2006). Indian Public Health Standards (IPHS) for Primary Health Centres. New Delhi, India. 10 Hota P, & Dobe M. (2005). National Rural Health Mission. Indian Journal of Public Health, 49(3), 107-111. 6

The Meducator | January 2011

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RESEARCH INSIGHT

Small-molecule Inhibitors of Pseudomonas aeruginosa Biofilm Development Matthew A. Chong Bachelor of Health Sciences (Honours) Program, Class of 2010 The Laboratory of Dr. Lori Burrows, McMaster University Shinerama is one of the hallmark events of McMaster’s Welcome Week. Through this student-led initiative, Mac students raised $60,000 last year for cystic fibrosis research. Interestingly, and as some undergraduates would agree, that money is being put to great use right here at McMaster’s laboratories. Here we present the exciting new discoveries of one such group: the laboratory of Dr. Lori Burrows. Matthew A. Chong is a third year student in the Bachelor of Health Sciences (Honours) Program. Funded by a generous studentship from the Canadian Cystic Fibrosis Foundation, Matthew spent his summer researching biofilm development by opportunistic pathogen Pseudomonas aeruginosa. The central focus of his project was to characterize the structure activity relationship of potent biofilm inhibitor palmitoyl-DL-carnitine.

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ith a prevalence of 1 in 2500 live births, cystic fibrosis (CF) is the most common hereditary disease affecting Caucasians.1 CF normally manifests itself as a chronic, recurring lung infection that results in irreversible lung damage and ultimately respiratory failure. Pulmonary infections in patients with CF are notoriously difficult to treat because P. aeruginosa—the primary pathogen implicated in CF—exists as a surfaceassociated community known as a biofilm.1 In addition to being involved in the pathophysiology of CF, biofilms are associated with a long-list of diseases that include periodontitis, middle-ear infections, endocarditis, and infections of implanted medical devices such as catheters.2

Bacterial biofilms are defined as a surface-associated community of cells enclosed in a self-produced polymeric matrix.2 Biofilm formation, a ubiquitous characteristic of bacteria, allows them to better survive in the real-world where nutrients and environmental conditions are in a constant state of flux.3 Schematically, biofilm formation involves arrival at the surface, initial attachment, the formation of microcolonies (small aggregates of cells), biofilm maturation, and dispersal (leaving the biofilm to begin the cycle again).2,4 After microcolony formation begins, the developing biofilm initiates the synthesis of a slimy and highly-organized matrix composed primarily of DNA, protein and polysaccharides.2,3

(tagging of bacteria by antibodies).5,6 The biofilm matrix also binds antibiotics and contains antibiotic-degrading enzymes.3,7 Lastly, the matrix gives rise to metabolic heterogeneity (different rates of metabolism) of biofilm cells—a consequence of gradients in nutrient, waste product, and signaling molecule levels—that renders the cells themselves highly resistant to antibiotics, which generally depend on the disruption of active metabolism.2 To use an analogy, not only are biofilm cells hiding in a protective castle (the matrix) but they are also wearing armor (intrinsic antibiotic resistance). Indeed, biofilm cells are up to 1000 times more resistant to antibiotics compared to motile cells allowing them to persist—and subsequently recolonize the host—despite cycles of antibiotic therapy.2,8 Therefore, the development of inhibitors of biofilm formation represents a viable strategy to ameliorate biofilm-associated infections such as those experienced by CF patients.

SMALL-MOLECULES AS INHIBITORS OF P. aeruginosa BIOFILM FORMATION

Previous small-molecule screening by Master’s candidate Iwona Wenderska of the laboratory of Dr. Lori Burrows identified the eukaryotic kinase inhibitor palmitoyl-DLcarnitine (pDLc) as a potent disruptor of P. aeruginosa biofilm formation. The purpose of this project was to characterize the structure-activity relationship of pDLc by In addition to providing structural support for the biofilm, exploring whether related compounds possessed similar the matrix protects biofilm cells from the host immune inhibitory properties. By understanding how certain system by shielding free radicals employed by neutrophils, chemical moieties (groups) of pDLc contribute to the hindering phagocytosis, and interfering with opsonization overall inhibition, we can gain a better understanding

11


N+

N+ O

O

O

OH

O

Hexadecanoic Acid

O-

O

Carnitine

O-

Palmitoyl-DL-Carnitine

FIGURE 1: Palmitoyl-DL-Carnitine contains two chemical moieties—carnitine and hexadecanoic acid—joined by an ester linkage. Note that hexadecanoic acid is also called the non-IUPAC name palmitic acid, and that is where the palmitoyl portion of palmitoyl-DL-carnitine’s name originates.

of the biofilm formation process and possibly identify more potent inhibitors. The Burrows laboratory uses a highly optimized assay to quantify the biofilm formation by P. aeruginosa. The biofilms are grown overnight (19hs) on plastic pegs immersed in culture containing P. aeruginosa strain PAO1. After this incubation period, the pegs are washed to remove nonadherent bacteria and then stained using crystal violet dye (a dye also used in gram-staining) to quantify the amount of biofilm. The starting point of the project was to determine the biofilm inhibitory properties of palmitoyl-DL-carnitine’s (pDLc) chemical moieties: the polar carnitine head-group and hexadecanoic acid. Interestingly, only hexadecanoic acid (and not carnitine) was able to inhibit biofilm formation. Since these results suggested that the lipid moiety of pDLc was of particular importance, other saturated fatty acids of varying chain length were tested for their biofilm-inhibitory properties. Only a subset of fatty acids is able to inhibit P. aeruginosa biofilm formation at micromolar concentrations (Table 2). Interestingly, the naturally occurring 14- and 16-carbon fatty acids, but not the synthetic 15-carbon acid, exhibited the most potent biofilm inhibition properties; this suggests that even modest differences in fatty acid chain length may affect their signaling properties. Hexadecanoic acid is found in nature as an antimicrobial present in plant extracts.9 Fatty acids are similar to detergents in so far as both compounds are known to make surfaces slippery (act as surfactants), which may physically interfere with bacterial attachment and subsequent biofilm formation. The surfactant properties of fatty acids made it unclear as to whether their inhibitory action was due to this physical effect or specific signaling mechanisms. Therefore, to elucidate the nature of the observed inhibition, the ability of common laboratory

Compound Palmitoyl-DL-Carnitine

EC50 N+ O

4μM

O

O-

O

Myristoyl-DL-Carnitine

N+ O

15μM

O

O-

O

Carnitine

N+

Not efffective O-

O

Hexadecanoic Acid

O

OH

30μM

TABLE 1: Summary of the effects of pDLc and its derivatives on biofilm formation. With the exception of the carnitine, all compounds shown inhibited biofilm formation by PAO1 in a concentration-dependent manner. The EC50 is defined as the concentration of the test compound at which biofilm formation is reduced to 50% of the vehicle-treated control. EC50 values below 25μM are generally considered to be excellent.

Systemic Name

Chain Length

EC50

Nonanoic Acid

9

Not effective

Decanoic Acid

10

Not effective

Dodecanoic Acid

12

Not effective

Tridecanoic Acid

13

Not effective

Tetradecanoic Acid

14

25 μM

Pentadecanoic Acid

15

Not effective

Hexadecanoic Acid

16

30 μM

Heptadecanoic Acid

17

Not effective

Octadecanoic Acid

18

Not effective

TABLE 2: Fatty acids differ in their ability to inhibit PAO1 biofilm formation. The EC50 is defined as the concentration of the test compound at which biofilm formation is reduced to 50% of the vehicle-treated control.

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Planktonic Growth

% Control

Biofilm Growth

FIGURE 2: Detergent-induced biofilm formation. The response to SDS is similar to the other detergents screened, and none were as effective as the fatty acids or pDLc at inhibiting biofilm formation. Planktonic growth is defined as growth in the culture medium (motile, non-biofilm cells) and is measured to ensure that test compounds are not inhibiting biofilm formation by simply killing the bacteria.

[SDS] (μM)

detergents to inhibit biofilm formation was investigated (as an example, Figure 1 shows data for the common detergent sodium-dodecyl sulfate). The detergents had only modest effects on biofilm formation (30% inhibition), and at higher concentrations they counterintuitively induced biofilm formation (>200% increase).

in the deaths of 22 Canadians.12 On the bright side, biofilms can be harnessed to bioremediate polluted environments and purify sewage.13 Nonetheless, in a medical context at least, the development of biofilm inhibitors may improve the efficacy of current antibiotics and increase the life expectancy and quality of life of patients with biofilm-associated chronic infections. Further research is currently underway to Based on the results, certain fatty acids and pDLc can act determine whether pDLc and related compounds increase as potent inhibitors of P. aeruginosa biofilm formation. the susceptibility of P. aeruginosa biofilms to antibiotics. Furthermore, the observation that common lab detergents are unable to inhibit biofilm formation in the same range of concentrations as the fatty acids and pDLc raises the ACKNOWLEDGEMENTS possibility that the observed effect is not physical but may instead act through specific signaling mechanisms. I thank Masters candidate Iwona Wenderska and the members P. aeruginosa has several fatty acid responsive transcription of the Burrows Laboratory for their excellent training and factors that modulate its gene expression.10 The ability of guidance. I also want to express my great appreciation for Dr. fatty acids to interact with these transcription factors was Lori Burrows’ support and mentorship throughout this work and shown to be highly dependent on the chain-length of as I continue as a thesis student under her supervision. This work the fatty acid, consistent with our observation that only was supported by a Summer Studentship from the Canadian the 14- and 16- carbon fatty acids appreciably inhibit Cystic Fibrosis Foundation. I would like to express my deep biofilm formation.11 Odd-numbered chain length fatty acids, gratitude for the hard work of the Foundation’s volunteers and which do not occur naturally, were interestingly less effective staff, as well as the generosity of their donors, for making this at inhibiting biofilm formation than their even-numbered experience possible. counterparts. The fact that the two chemical moieties of pDLc—hexadecanoic acid and carnitine—were individually less potent than pDLc at inhibiting P. aeruginosa biofilm formation illustrates beautifully that the whole is greater than the sum of its parts. Implicated in the etiology of fatal diseases like CF and many nosocomial infections, biofilms are clearly a widespread healthcare problem.2 Biofilms also make headlines in industry; for example, the recent contamination of a Maple Leaf Foods plant with Listeria monocytogenes biofilms resulted

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Reviewed by Dr. Lori Burrows, Ph.D.

Dr. Lori Burrows is a Professor at McMaster University in the Department of Biochemistry and Biomedical Sciences. Her research interests include type IV pili, peptidoglycan dynamics, and bacterial biofilms. Her lab employs a wide-range of genetic and biochemical techniques with the goal of identifying vulnerabilities in these systems that could be exploited for drug development.

REFERENCES Hassett DJ, Korfhagen TR, Irvin RT, Schurr MJ, Sauer K, Lau GW, et al. (2010). Pseudomonas aeruginosa biofilm infections in cystic fibrosis: insights into pathogenic processes and treatment strategies. Expert Opin.Ther. Targets, 14(2), 117-130. 2 Costerton JW, Stewart PS, & Greenberg EP. (1999). Bacterial biofilms: a common cause of persistent infections. Science, 284(5418), 1318-1322. 3 Karatan E, & Watnick P. (2009). Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol.Mol.Biol.Rev., 73(2), 310-347. 4 Glick R, Gilmour C, Tremblay J, Satanower S, Avidan O, Deziel E, et al. (2010). Increase in rhamnolipid synthesis under iron-limiting conditions influences surface motility and biofilm formation in Pseudomonas aeruginosa. J.Bacteriol., 192(12), 2973-2980. 5 Mah TF, & O’Toole GA. (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol., 9(1), 34-39. 6 Ryder C, Byrd M, & Wozniak DJ. (2007). Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr.Opin.Microbiol., 10(6), 644-648. 7 Schooling SR, & Beveridge TJ. (2006). Membrane vesicles: an overlooked component of the matrices of biofilms. J.Bacteriol., 188(16), 5945-5957. 1

Hall-Stoodley L, Costerton JW, & Stoodley P. (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nat.Rev.Microbiol., 2(2), 95-108. 9 Yff BT, Lindsey KL, Taylor MB, Erasmus DG, & Jager AK. (2002). The pharmacological screening of Pentanisia prunelloides and the isolation of the antibacterial compound palmitic acid. J.Ethnopharmacol., 79(1), 101107. 10 Kang Y, Nguyen DT, Son MS, & Hoang TT. (2008). The Pseudomonas aeruginosa PsrA responds to long-chain fatty acid signals to regulate the fadBA5 beta-oxidation operon. Microbiology, 154(Pt 6), 1584-1598. 11 Kang Y, Lunin VV, Skarina T, Savchenko A, Schurr MJ, & Hoang TT. (2009). The long-chain fatty acid sensor, PsrA, modulates the expression of rpoS and the type III secretion exsCEBA operon in Pseudomonas aeruginosa. Mol.Microbiol., 73(1), 120-136. 12 Charlebois S, & Horan H. (2010). Institutional and relational determinants in high- and medium-extent food product crises: the inner perspective of a public health crisis. Int.J.Environ.Health Res., 20(4), 299-312. 13 Cao B, Nagarajan K, & Loh KC. (2009). Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches. Appl.Microbiol.Biotechnol., 85(2), 207-228. 8

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RESEARCH INSIGHT

Electrical Properties of Pulmonary Lymphatic Smooth Muscle Cells Mustafa Ahmadzai Honours Biology & Pharmacology, Class of 2012 The Laboratory of Dr. Luke Janssen, McMaster University Mustafa Ahmadzai is a fourth-year student in the Honours Biology and Pharmacology (Co-op) Program. Mustafa spent this previous summer working at the Firestone Institute for Respiratory Health at St. Joseph’s Hospital in downtown Hamilton. His research focused on the electrophysiological properties of lymphatic smooth muscle cells in the lungs and how they participate in the etiology of asthma and other related pulmonary diseases. The work involved using a technique known as voltage clamping, wherein electrical probes are used to stimulate isolated cells and measure the resulting currents. Like pieces to a puzzle, these electrical responses may give us an insight into how the tissue behaves under complex disease states like asthma and chronic obstructive pulmonary disorder (COPD).

P

athology arises when a physiological function is compromised; treatment entails restoring this dysfunction to within homeostatic ranges. When concerned with illnesses like asthma or chronic obstructive pulmonary disorder (COPD), treatment depends exclusively on pharmacological interventions that target precise signalling pathways that underlie the outward symptoms associated with the disease. To understand the scope of these interactions, the researcher is confined to the level of the cell along with its many complexities. It is through these pursuits that the most meaningful and far-impacting results are obtained: data extracted at this microscopic level facilitate the production of life-saving drugs that attempt to target the right proteins and pathways, at the right time.

airflow into the pulmonary alveoli, where gas-exchange is known to occur.2,3 With this serving as a starting point, two crucial questions then arise: which signalling molecules are of greatest relevance to the activation of this pathway and secondly, how are they important to the overall etiology of the disease?

ISOPROSTANES AND THEIR NOVEL ROLE IN ASTHMA

It is well known that environmental allergens, pollutants and other irritants are strongly linked to the incidence of asthma.4,6 Whether or not these factors are direct or indirect contributors to this process is critical to understanding asthma. Isoprostanes comprise one When we consider asthma, the difficulty in breathing such family of molecules that are seen in situations arises from the hyperresponsiveness of the smooth of oxidative stress, which are characterized by the muscle cells that circumscribe the upper airway tract uncontrolled production of free radicals. Consequently, (see Figure 1).1 Ultimately, their constriction is mediated these compounds are now being implicated in airway by calcium influx into the cytosol through selective ion hyperresponsiveness.7 As structural isomers to the broad channels embedded within the cell membrane. In light family of prostaglandin signalling molecules, their plasma of this, it is important to note that an ion channel is levels have been classically interpreted as indicators of a large, membrane-bound protein that facilitates the the extent of oxidative damage that has occurred in a influx or efflux of ions. Since proteins are often specific tissue.8,9 A key difference between the prostaglandins and in function, some are selective for only one type of ion. isoprostanes, however, derives from the source of their A channel that permits sodium influx is referred to as a synthesis. Prostaglandins are produced as downstream sodium channel; one that is selective for potassium is a products of tissue damage in a reaction catalyzed by the potassium channel, and so on. The main physiological enzyme phospholipase A2. Alternatively, isoprostanes are ions of concern are sodium, potassium, calcium and by-products of lipid peroxidation, a process mediated by chloride. As such, the overstimulation of smooth muscle free radicals (see Figure 2).9,10 cells, mediated by these integral channel proteins impedes

15


mucous membrane bronchial muscle in spasm submucosa

bronchial muscle

inflammatory oedema

1

2 4

3

5

lesion of mucosa and submucosa

mucous membrane lumen constriction plug of mucous

infiltration of eosinophils

infiltration and oedema

FIGURE 1: Cross-section of the bronchus. Airflow into the lungs is controlled by the degree of lumen constriction, a process regulated by the bronchial smooth muscle cell layer. In asthmatic patients, these cells exhibit heightened sensitivity and constrict prematurely in response to allergens and pollutants thereby occluding airflow into the lungs.1

The newfound bioactivity of isoprostanes is believed to arise from their molecular similarities to prostaglandins. When applied to segments of the canine trachea, for instance, the agonistic properties of isoprostanes induce contractions of the smooth muscle layer, thereby mimicking the symptoms of an asthma attack.11 In order to explain these results, it has been hypothesized that the prostaglandin receptor, which normally mediates this response, cannot detect the minute differences between the prostaglandin and isoprostane ligands.12 An additional explanation put forth is the potential existence of an isoprostane-selective receptor that only responds to this ligand.12 On a large scale, free-radicalmediated production of isoprostanes, like that seen from inhaling toxic pollutants, could exacerbate the symptoms of asthma and perhaps even trigger a full-scale asthma attack.11 Conversely, pharmacological antagonism of these receptors would suppress this cycle and presents one possible direction for asthma drug development.

SHIFTING THE FOCUS FOR GREATER INSIGHT As we seek to develop new treatment options for asthma, our understanding remains incomplete without a thorough knowledge of how other tissue systems of the lungs interact with isoprostanes. How these adjoined tissue layers contribute to the onset of asthma may ultimately be far more profound than what was previously known, which is very little considering the lack of research within these areas. Pulmonary edema, which is characterized by a build-up of fluid in the lungs, is an often-cited symptom of numerous cardiothoracic pathologies in addition to asthma. In light of

this, it is necessary to investigate the impact of isoprostanes on the lymphatic system, which returns excess fluid to the heart for recirculation. The structure of the lymphatic vessel system is very diverse, region-specific and not as extensively studied to date.13 As a result, this inherent heterogeneity provides strong rationale for investigating how isoprostanes and lymphatic smooth muscle interact and contribute to symptoms of asthma – if at all. Like in blood vessels, the smooth muscle cells of lymphatic vessels encompass a layer of endothelial cells, which line the vessel wall (Figure 3). In veins and arteries, the communication between these two distinct layers gives rise to the body’s blood pressure control mechanisms; this process is regulated by the balance between vasodilation (mediated by nitric

O OH

O

HO

OH

FIGURE 2a: Prostaglandin E2 molecule. C7 COOH

C10 C13 HO

HO COOH

HO

Arachidonic acid

Oxidative injury HO

HO

HO 5-F2-isoprostanes or Type VI (Most abundant)

8-F2-isoprostanes or Type IV

HO

HO

COOH

COOH HO

COOH

HO 12-F2-isoprostanes or Type V

HO

HO 15-F2-isoprostanes or Type VII (Most analysed)

FIGURE 2b: Isoprostane molecule. FIGURE 2: Structural properties of prostaglandin and isoprostane molecules. The prostaglandin (Figure 2a) and isoprostane (Figure 2b) families share similarities in their overall molecular structures. Prostaglandins undergo production via an enzymatic pathway (initiated by phospholipase A2). Conversely, the production of isoprostanes is mediated by free-radicals and is notably more prolific, giving rise to multiple bioactive agents.9

The Meducator | January 2011

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oxide) and vasoconstriction (mediated by endothelin). Consequently, if isoprostanes are found affecting lymphatic smooth muscle in the same manner that they affect tracheal smooth muscle, then this could represent a novel inlet by which to treat edema and related pulmonary disorders. Moreover, as seen in airway hyperresponsiveness, lymphatic smooth muscle contraction is calcium-mediated. As such, this contractile response can be readily manipulated and measured through various advanced in vitro experiments, like those outlined below.

THE “CHARGE” BEHIND DRUG DEVELOPMENT Electrophysiological experiments measure ionic fluxes through selective channels such as the calcium channels that trigger bronchoconstriction, which were discussed above.14 Ultimately, the movement of an ion depends upon three basic factors. The first of these is the local electric field of the cell. The second factor is the chemical concentration gradient in which the ions are distributed across the membrane.14 Overall, equilibrium is established between these two forces, thereby giving rise to the so-called electrochemical gradient. The final element that determines ionic movement is the gating of the ion channel; that is, whether or not the channel’s pore is open to permit the flux of ions.15

Fibroblast Vascular smooth muscle cell Endothelial cell

Vessel lumen

Vascular tissue

FIGURE 3: Structure and organization of vascular walls. Like in the bronchii (Figure 1), the degree of vessel constriction depends on the contractility of smooth muscle cells that encompass the lumen. This balance between constriction and relaxation is controlled by factors released by the inner layer of endothelial cells and ultimately governs the ease with which blood flows through the vessel.17

they contribute to the occurrence of edema, asthma and related lung pathologies.

Emerging experiments indicate that certain isoprostanes do in fact interact with lymphatic smooth muscle cells. The Basic physics dictates that charge separation, which in this exact nature of this interaction, however, is unclear. case is the differential distribution of ions across the cell Accordingly, much of this evidence has been derived using membrane, confers a voltage (also known as potential) upon a technique known as patch clamping. This technique the cell. When voltage changes occur, they serve as intrinsic involves the use of microscopic electrodes that are carefully signals that further propagate this initial depolarization: one annealed to a segment (a “patch”) of the cell membrane, channel’s opening leads to the opening of many others.14,15 while a computer amplifier varies (by “clamping”) the voltage As this continues, opposing ionic fluxes serve to “reset” the imposed upon the patch. As this occurs, the resulting electric cell to the initial voltage that prevailed before the whole current undergoes measurable fluctuations. The change in process began.16 The cycle repeats itself in this manner and these fluctuations in the presence of a drug – in this case, is the basis of cell physiology. Where asthma is concerned, isoprostanes – confirms a drug-cell interaction. a novel frontier involves studying how, and if, isoprostanes trigger a “turning on” of the excitatory pathway – such that Gaining a more profound understanding of this interaction contraction of the lymphatic smooth muscle occurs – or would potentially amount to the development of new whether it inherently triggers the “reset” mechanism. In drugs. Intense research, however, would have to be pursued this latter circumstance, cell contraction and the tissue’s within the industry before human trials can be conducted propulsion of lymph would decrease drastically since the to these ends, a process that may take years. Since the data calcium influx would be inhibited. Due to the role of obtained from patch clamping studies are to some extent the lymphatic system in draining excess fluid back to the absolute, this approach affords the researcher a direct means bloodstream, this inhibition would amount to a build-up by which to assess the effect of a compound on a cell. Such of fluid, which would clinically manifest as edema. Whether electrophysiological studies were pivotal in characterizing the “on” or “off” signal is preferentially stimulated not only how airway smooth muscle and isoprostanes interacted. determines how isoprostanes manifest their influence on the Whether or not this is the case with lymphatic smooth lymphatic system in the thoracic region, but also whether muscle cells, however, is only a matter of time.

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Reviewed by Dr. Luke Janssen, Ph.D.

Dr. Luke Janssen is a Professor of Medicine (Division of Respirology) at McMaster University. He obtained his PhD in Physiology and Pharmacology from McMaster University. Currently, his research interests span various topics, including smooth muscle physiology, ion channels, and isoprotane biology.

REFERENCES ParĂŠ PD, McParland BE, & Seow CY. (2007). Structural basis for exaggerated airway narrowing. Can J Physiol Pharmacol., 85(7), 653-8. 2 Zhang WC, Peng YJ, Zhang GS, He WQ, Qiao YN, Dong YY, Gao YQ, Chen C, Zhang CH, Li W, Shen HH, Ning W, Kamm KE, Stull JT, Gao X, & Zhu MS. (2009). Myosin light chain kinase is necessary for tonic airway smooth muscle contraction. J Biol Chem., 285(8), 5522-31. 3 Sanderson MJ, Delmotte P, Bai Y, & Perez-Zogbhi JF. (2008). Regulation of airway smooth muscle cell contractility by Ca2+ signalling and sensitivity. Proc Am Thorac Soc., 5(1), 23-31. 4 Dozor AJ. (2010). The role of oxidative stress in the pathogenesis and treatment of asthma. Ann NY Acad Sci., 1203, 133-7. 5 Bowler RP. (2004). Oxidative stress in the pathogenesis of asthma. Curr Allergy Asthma Rep., 4(2), 116-22. 6 Li N, Hao M, Phalen RF, Hinds WC, & Nel AE. (2003). Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin Immunol., 109(3), 250-65. 7 Clarke DL, Dakshinamurti S, Larsson AK, Ward JE, & Yamasaki A. (2009). Lipid metabolites as regulators of airway smooth muscle function. Pulm Pharmacol Ther., 22(5), 426-35. 8 Dunstan JA, Breckler L, Hale J, Lehmann H, Franklin P, Lyons G, Ching SY, Mori TA, Barden A, & Prescott SL. (2006). Associations between antioxidant status, markers of oxidative stress and immune responses in allergic adults. Clin Exp Allergy., 36(8), 993-1000. 1

Halliwell B, & Lee CY. (2010). Using isoprostanes as biomarkers of oxidative stress: some rarely considered issues. Antioxid Redox Signal., 13(2), 145-56. 10 Huwiler A, & Pfeilschifter J. (2009). Lipids as targets for novel anti-inflammatory therapies. Pharmacol Ther., 124(1), 96-112. 11 Catalli A, Zhang D, & Janssen LJ. (2002). Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol., 283(5), L1151-9. 12 Fukunaga M, Makita N, Roberts LJ 2nd, Morrow JD, Takahashi K, & Badr KF. (1993). Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells. Am J Physiol., 264(6 Pt 1), C1619-24. 13 Broadley KJ. (2006). Beta-adrenoceptor responses of the airways: for better or worse? Eur J Pharmacol., 533(1-3), 15-27. 14 Kitamura K, Teramoto N, Oike M, Xiong ZL, Kajioka S, Inoue Y, Nilius B, & Kuriyama H. (1991). Characteristics of the voltage-dependent calcium channel in smooth muscle: patch-clamp studies. Adv Exp Med Biol., 304, 209-27. 15 Catterall WA. (2010). Ion channel voltage sensors: structure, function, and pathophysiology. Neuron, 67(6), 915-28. 16 Bursztyn L, Eytan O, Jaffa AJ, & Elad D. (2007). Mathematical model of excitation-contraction in a uterine smooth muscle cell. Am J Physiol Cell Physiol., 292(5), C1816-29. 17 Hopkins J , McLaughlin CW, Johnson S, Warner MQ, LaHart D, Wright JD(1993). Human Biology and Health. Englewood Cliffs, New Jersey: Prentice Hall. 9

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CRITICAL REVIEW

Cholesteryl Ester Transfer Protein: The Key to Longevity? Vithooshan Vijayakumaran Honours Biology: Microbiology & Biotechnology Specialization, Class of 2011 Neil D Dattani Bachelor of Health Sciences (Honours) Program, Class of 2010 M.D. Candidate, Class of 2014, University of Toronto The negative attributes of cholesterol receive much attention in the media and scientific literature, with good reason. However, there are aspects of cholesterol metabolism that actually reduce the risk of developing cardiovascular disease. Furthermore, variations in the gene for an important cholesterol transport protein may yield significant health benefits. This critical review outlines the basic physiology of cholesterol, discusses cholesteryl ester transfer protein and potential variations in its gene, and identifies the implications of current research in this field for the pharmaceutical industry.

D

uring the past 25 years, the field of genetic lipoproteins (VLDL). Once it is released into the blood research has advanced greatly. Whole genomes of stream, cholesterol is converted to low density lipoproteins organisms ranging from small viruses to complex (LDL) as it is deposited into various organs. However, human beings have been sequenced. Since the completion LDL-cholesterol can be retained in arteries, leading to the of the human genome project in 2003, breakthroughs in formation of atherosclerotic plaques, which are implicated in sequencing technology have allowed for genetic screening causing peripheral vascular disease, strokes, and myocardial of certain diseases. Genetic screens can predict how likely infarctions.4 an individual or their offspring are to develop certain diseases. Conversely, genetic screening can also determine In contrast, high density lipoprotein (HDL), or “good the presence of beneficial genetic traits that may confer cholesterol�, removes cholesterol from arteries and protection against developing certain diseases. This paper transports them back to the liver for excretion or reuse.4 will elucidate how mutations in the cholesteryl ester transfer Epidemiological studies have shown LDL-cholesterol levels protein (CETP) gene confers a reduced risk of arterial and correlate positively with incidence of vascular diseases, while cognitive diseases.1,2 HDL-cholesterol levels inversely correlate with incidence of vascular diseases.6

BACKGROUND PHYSIOLOGY

Cholesterol metabolism is controlled by enzymes, membrane receptors, and transfer proteins in the gastrointestinal system Cholesterol is a steroid metabolite, responsible for and blood plasma. One particular cholesterol-regulating maintaining animal cell membrane permeability and transfer protein that has been extensively studied is CETP.6 structure. It is vital in the production of steroid hormones, CETP is highly expressed in the liver, heart, placenta, and bile acids, and some lipid-soluble vitamins.3 While the right lymph nodes.7 The protein transports cholesteryl esters and amount of cholesterol is beneficial, research has shown that triglycerides between VLDL, LDL and HDL.8 The net effect high levels are pathogenic.4 of CETP is a reduction in the content and size of HDL particles.4 Consequently, inhibition of CETP raises HDLCholesterol is mainly produced in the liver, intestines, cholesterol levels and lowers LDL-cholesterol levels in the adrenal glands, and reproductive organs. Given its body. Since higher concentrations of HDL-cholesterol and hydrophobic property, lipoproteins assist in the transport lower concentrations of LDL-cholesterol are preventative of cholesterol in the blood plasma.5 In the liver, cholesterol against cardiovascular disease, drugs which inhibit CETP is combined with apolipoproteins to form very low density are being tested.4

19


heterozygous for the D442G genotype has been associated with decreased risk for Alzheimer’s disease.9 Although there was no statistical significance, further adjustment of the data for sex and age showed that the G allele might be protective against the development of Alzheimer’s disease (corrected for multiple testing).9 This conclusion is supported by observations that the variants have altered CETP structure and function leading not only to reduced CETP levels, but lowered CETP activity in comparison to the wild type.9 Studies on heterozygotes showed that plasma CETP activity was only 60%-85% and HDL levels were elevated by 10%80% in comparison to the wild type.9

LIVER Cholesterol + Apolipoproteins

VLDL LDL

HDL

BLOOD VESSEL FIGURE 1: A Physiology of CETP Related Molecules & Compartments. A diagram of CETP function showing the key physiological processes involved. Cholesterol in the liver is packaged with apolipoproteins into VLDL, which enters the bloodstream. CETP plays a role in mediating the conversion of VLDL to HDL and LDL. Some CETP variants produce higher levels of HDLs and lower levels of LDLs resulting in a reduced risk of heart and arterial diseases. Images adapted from: http://www.medical-art-and-illustration.com/ http://www.zdsolutions.it/flash/gallery_med.htm

The I405V variant is the second most common variant found in over 25% of studied populations. Studies indicate that it is three times greater in the centenarian population than in people with a median age of 70.2 It is believed that individuals with the heterozygous genotype tend to produce both versions of CETP, as the alleles are co-dominant to one another.10 While both homozygous I/I and the heterozygous individuals produce almost identical amounts of CETP, the V/V homozygotes tend to have a 9-23% CETP deficiency.10,1 A decrease in CETP function increases HDL (high density lipoproteins) levels in the body, and decreases LDL (low density lipoprotein). The result of this is that HDL-c levels are approximately equal in individuals with the I/I or I/V genotypes, while they are ten percent higher in V/V individuals.10

Not only do I405V V/V homozygotes tend to have higher HDL levels and low LDL levels, they produce larger HDL CETP ALLELES AND POLYMORPHIMS and LDL particles, which are less likely to get trapped in vessels. This results in a decreased risk of coronary artery The CETP gene is located on chromosome 16 and contains disease or atherosclerosis.1 Furthermore, it is believed a large variety of mutations that are highly polymorphic. that lipid metabolism partially mediates the conservation Many of the mutations in the gene can be separated into of cognitive function in the brain, and as a result I405V three major groups including missense, nonsense and silent variants tend to have reduced risk and onset of dementia mutations.1 Missense mutations result in the production of as they age.2 158 Ashkenazi Jews between 95 and 107 years single amino acid substitutions. These mutations are often of age were evaluated for cognitive function and tested for milder, but some variants can have a significant effect on the CETP I405V variants. It was found that those with the protein function. Nonsense mutations produce premature CETP I405V genotype were twice as likely to have good stop codons, causing the protein product to be prematurely cognitive function (a score greater than 25 on the Minitruncated. This leads to either partial or complete CETP Mental State Examination).2 deficiency. Silent mutations do not produce an amino acid substitution due to the redundant nature of the genetic code. As a result they are thought to play only a minor role PHARMACOLOGICAL IMPLICATIONS in CETP function.1 Recognizing the therapeutic potential of CETP inhibition, A common missense mutation that causes partial CETP pharmaceutical companies have engineered and tested CETP deficiency is the D442G variant, where aspartic acid at inhibitors in clinical trials. Two prominent CETP inhibitors position 442 is substituted with a glycine.9 Heterozygotes for developed over the past decade are torcetrapib (engineered this allele retain 60-85% of normal CETP function.1 Being by Pfizer) and anacetrapib (engineered by Merck).11 The Meducator | January 2011

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A 2004 study with a small sample size in the New England Journal of Medicine showed that Torcetrapib significantly increased HDL-cholesterol and decreased LDL-cholesterol levels. The effect was even greater when the drug was combined with statin therapy.11 However ILLUMINATE, a subsequent study including 15,000 patients, indicated that while torcetrapib plus statin therapy raised HDLcholesterol levels, compared to statin monotherapy this combination therapy caused an excess number of adverse events such as death and myocardial infarction.12 A further study, the ILLUSTRATE study, showed no difference in atherosclerotic plaque levels in the torcetrapib-statin combination therapy and the statin monotherapy groups.12 This indicates that torcetrapib causes adverse cardiovascular events by mechanisms other than atherosclerosis, such

as vasospasm or potential interaction with the reninangiotensin-aldosterone system. Due to its harmful effects, torcetrapib is no longer in development. Anacetrapib on the other hand is still under development. Recent studies in The Lancet indicate that this drug has great potential. In healthy patients and patients with dyslipidemia it was shown to increase HDL-cholesterol and decrease LDL-cholesterol levels. Additionally, unlike torcetrapib it did not exhibit any off-target effects such as hypertension.13 Importantly, it also did not increase the incidence of adverse events.13 However, these are small phase 1 trials, and longterm safety and efficacy data for anacetrapib have not yet been reported.

Reviewed by Dr. Geoff Werstuck, Ph.D.

Dr. Geoff Werstuck is an Associate Professor at McMaster University in the Department of Medicine. His work at Henderson involves researching the molecular mechanisms by which diabetes mellitus promotes the progression and development of cardiovascular disease. His laboratory employs a broad range of molecular and cellular techniques to examine cell and tissue-specific responses to hyperglycemia. This includes identifying changes in atherosclerotic gene expression induced by hyperglycemia and determining the mechanisms of accelerated atherosclerosis in diabetic mouse models and human blood samples. His laboratory is especially interested in identifying and testing potential new targets for therapeutic intervention in the treatment and prevention of atherothrombotic cardiovascular disease.

REFERENCES Boekholdt SM, Kuivenhoven JA, Hovingh GK, Jukema JW, Kastelein JJ, & van Tol A. (2004). CETP gene variation: relation to lipid parameters and cardiovascular risk. Current Opinion in Lipidology, 15, 393-398. 2 Barzilai N, Atzmon G, Derby CA, Bauman JM, & Lipton RB. (2006). A genotype of exceptional longevity is associated with preservation of cognitive function. Neurology, 67, 2170-2175. 3 Leah E. (2009). Cholesterol. Lipidomics Gateway, [doi:10.1038/lipidmaps.2009.3]. 4 Barter P. (2000). CETP and atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 20, 2029. 5 Thompson GR. (2004). Is good cholesterol always good? Genetic and pharmacological increases of HDL cholesterol need further evaluation. BMJ, 329, 471–2. 6 Tanne JH. (2005). Activating stem cells may treat Alzheimer’s. BMJ, 330, 622. 7 GNF SymAtlas. (2005). Genomics Institute of the Novartis Research Foundation Web site: http://symatlas.gnf.org/ SymAtlas/symquery?q=CETP. Retrieved December 28, 2009. 1

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Bauerfeind A, Knoblauch H, Schuster H, Luft FC, & Reich JG. (2003). Single Nucleotide Polymorphism Haplotypes in the Cholesteryl-Ester Transfer Protein (CETP) Gene and Lipid Phenotypes. Human Heredity, 54, 166-173. 9 Chen D, Yang J, Tang Z, Dong X, Feng X, Yu S, & Chan P. (2008). Cholesteryl ester transfer protein polymorphism D442G associated with a potential decreased risk for Alzheimer’s disease as a modifier for APOE 4 in Chinese. Brain Research, 1187, 52-57. 10 Bruce C, Sharp DS, & Tall AR. (1998). Relationship of HDL and coronary heart disease to a common amino acid polymorphism in the cholesteryl ester transfer protein in men with and without hypertriglyceridemia. The Journal of Lipid Research, 39, 1071-1078. 11 Brewer HB. (2004). Increasing HDL cholesterol levels. NEJM, 350(15), 1491-1494. 12 Tall AR. (2007). CETP Inhibitors to increase HDL cholesterol levels. NEJM, 356(13), 1364-1366. 13 Duriez P. (2007). CETP inhibition. The Lancet, 370, 18821883. 8


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