Thioketal Nanoparticles Targeting Inflammation

The article Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines published in Nature Materials by Wilson, Dalmasso, Wang, Sitaraman, Merlin, and Murthy was one of the most dense articles we discussed this week, but arguably, one of the most well-thought out articles too in its processes. I'm going to do my best to summarize the logic that each experiment in the article was founded on. The researchers aimed to design a novel way to prevent the excess inflammation in inflammatory bowel disease and target the macrophages in the mucosal lining to downregulate pro-inflammatory cytokines that worsen symptoms. They developed a novel method where siRNAs, or small interrupting RNAs, are specifically delivered to areas of inflammation and reduce pro-inflammatory cytokine production locally.

Challenge 1: How do you target only diseased intestinal tissue in the release of siRNAs/drug delivery?

Researchers identified high levels of reactive oxygen species (ROS) that are released from sites of inflammation through studying ROS concentrations in biopsies from ulcerative colitis, colon cancer, and Helicobacter pylori infection patients. They developed the ROS-sensitive polymer thioketal nanoparticles (TKN) to encapsulate the siRNAs until they are released in the intestine at sites with high ROS, or inflammation sites, but still stay resistant to pH changes in the digestional tract. 

Challenge 2: Wait, how do we really know TKN degrades due to ROS and not due to pH changes? And how exactly do we know it can carry a drug/siRNAs?

Researchers took the polymer material and exposed it to three different solutions in an experiment: one solution was acidic, one was basic, and the final was concentrated with ROS. The polymer's weight reduced to less than a tenth of its original size in the superoxide solution, but stayed the same in the basic and acidic environments. They also tested delivery of an agent by making TKNs carrying a fluorescent dye and releasing them around activated macrophages. As such, the activated macrophages had higher count of fluorescent markers than inactive macrophages, indicating a release by TKN.

Challenge 3: So, now that we know the delivery method "works" releasing the material, how do we make sure the siRNA/drug gets into the cell and where we want it to go (ie. the bowels)?

Researchers added DOTAP, a positively charged lipid, to make sure the TNF-alpha siRNA particles in the TKNs (hereby known as "TNF-a-TKN"s). They also made the size roughly around ~600 nm that altered which cells would take them and where they would bind in the mucosa. 

Challenge 4: Remind me again, how do we know this "TNF-a-TKN" actually works to silence the TNF-alpha (pro-inflammatory) production in these immune cells?

Macrophages were 'activated' and then scientists studied how these cells reacted to TNF-a-TKN and controls (random siRNA fragments or placebo). TNF-a-TKN treated cells exhibited a significant reduction in TNF-alpha production.

Challenge 5: Can we get these siRNAs into the gut successfully of an IBD patient where it is supposed to work?

Mice models finally come into play here. To model an IBD patients, researchers induced an IBD-like reaction in the GI tract by tainting the experimental mice's water with DSS. These DSS mice and control mice were both given TKNs with fluorescence-tagged, random siRNAs in them to prove that the TKNs "can localize orally delivered siRNA to sites of intestinal inflammation." Researchers found three times the fluorescent siRNAs at inflammation sites compared to non-inflammatory sites.

Challenge 6: So, the entire capsule of TKNs can get into the body, through the digestive tract, and release its siRNAs directly to sites of inflammation instead of random sites in the GI tract. Does this actually work as we want now in mouse model to reduce the production of pro-inflammatory factors locally?
Once again, researchers induced IBD in mice by tainting their water. This time they administered TNF-a-TNKs (remember, TNF-a is a proinflammatory agent) to mice along with appropriate control groups. They calculated the levels of individual proinflammatory factors (the targeted TNF-alpha along with IL-6, IL-1, and IFN-gamma) in each of the groups of mice. Mice with TNF-a-TNKs exhibited a 10-fold decrease in colonic TNF-a mRNA (which produces this factor); these same mice also had inhibited levels of IL-6, IL-1, and IFN-gamma. Other experiments included in the paper came to similar conclusions, supporting the idea that TNF-a-TKNs did allow the delivery of this agent (siRNAs against TNF-alpha) to help treat local inflammation involved with IBD.


Researchers did continue to test for other factors during the study including the relative toxicity in comparison to the existing FDA-approved PGLA for drug delivery and a few tests that were aimed to discover the correct dosage level. However, this highlighted was the general logical pathway I imagined the researchers following, as conveyed by the logical progression of the article. As always there are some lose ends left by the researchers, but I believe they conveyed their main aim of the project (the development of the novel method for drug delivery specifically with IBD) and achieved that goal, despite occasionally missing some more medicinal or pharmacological areas of focus. As always with novel developments, the topic needs to be repeated and studied more thoroughly. However, I am very excited to hear of such an invention.

For more information about the tests undergone, the results recorded, and overall more information than the summary, please feel free to read the article for yourself.

Citation
Wilson, S., Dalmasso, G., Wang, L., Sitaraman, S.V., Merlin, D., and Murthy, N. (2010) Orally delivered thioketal nanoparticles loaded with TNF-a-siRNA target inflammation and inhibit gene expression in the intestines. Nature Materials.

Do parents really know what’s best when it comes to vaccinating their children?

According to a recent article in Pediatrics, more than 1 and 10 parents use an alternative vaccination schedule than what is recommended by the CDC Advisory Committee on Immunization Practices General Recommendation Work Group (GRWG). The most commonly delayed vaccines include H1N1, seasonal influenza, Varicella, Hepatitis B, and Measles-Mumps-Rubella (MMR). One would assume that a delay in vaccinations could be attributed to financial barriers to accessing health care; however, these beliefs were most common among parents with higher incomes.

Parents that subscribe to the alternative vaccination schedule believe that delaying vaccine doses will result in fewer side effects, is safer for their children, and that many of the recommended vaccinations are not necessary. There is diminutive scientific evidence to support these claims; on the odd occasion an individual experiences an adverse event they often demonstrate immunodeficiency. Moreover, 1 in 3,000-4,000 children vaccinated with MMR experience febrile seizers, scientific evidence does not support Hepatitis B vaccinations causing Multiple Sclerosis, and the Institute of Medicine issued a reporting stating that there isn’t a link between vaccines and Autism (signs of autism appear around the same time many vaccines are administered which has led many to believe that there is a correlation).

The GRWG recommendations are based on scientific evidence from the expertise of health-care providers, public health officials, and the Immunization Action Coalition Group. GWRG vaccination schedule recommendations are influenced by age-specific risks for disease, age-specific complications, age-specific responses to vaccinations, and potential interference with the immune response by passively transferred maternal IgG. GWRG recommends administration of vaccines as close to the recommended intervals as possible; delaying recommended vaccines are associated with a significantly increased risk of contracting and spreading vaccine preventable diseases. Additionally, fully vaccinated individuals are at risk of vaccine preventable diseases if they reside in a community with large proportions of under-immunized individuals.

References:

Stratton, K., Ford, A., Rusch, E., Wright, C. (2011) Adverse Effects of Vaccines: Evidence and Causality. Washington DC: Institute of Medicine National Academies Press

Centers for Disease Control and Prevention. Vaccine Safety . Retrieved October 19, 2011 from http://www.cdc.gov/vaccinesafety/Vaccines/MMR/MMR.html

Dempsey, A., Schaffer, S., Singer, D., Butchart, A., Davis, M., and Freed, G. (2011). Alternative Vaccination Schedule Preferences Among Parents of Young Children. Pediatrics.

Malaria Vaccine Shows Promise

The New York Times published an article today about the encouraging preliminary results of a clinical trial testing a malaria vaccine, known as RTS,S, made by GlaxoSmithKline.  The vaccine has been in development for more than 25 years, intitially for the American military and now with most of its support coming from the Bill and Melinda Gates Foundation.  The RTS,S vaccine has consistently shown protection against Plasmodium falciparum malaria in children and infants in phase 2 trials.  This ongoing phase 3 clinical trial is scheduled to continue through 2014 and will include tests on more than 15,000 children, from infancy on up.

Early results from the trial were released at a Seattle malaria conference today, which showed that three doses protected 47 percent of the 6,000 children ages 5 months to 17 months from severe malaria.  I found it interesting that the article mentioned that the age group was chosen because newborns have some protection from their mothers' antibodies, which must be IgG antibodies.  While 47 percent protection may not seem very effective with most vaccines not being released until they do better than 90 percent, 47 percent protection would save millions of lives over a decade with malaria estimated to kill about 780,000 people a year, most being African children.  

I also found it interesting that the article mentioned it is much more difficult to make a vaccine against a parasite like malaria than to make one against a virus because the malaria parasite changes shape as it moves from blood to liver and back to the blood, with each form having different surface proteins.  The actual paper from the New England Journal of Medicine also shows that the vaccine is not without severe adverse events, like convulsive seizures.  We may have to wait until 2014 at the conclusion of the trial to see whether the vaccine is effective enough to pay for and if the reduction in malaria outweighs the adverseside effects.

Original Article: The RTS,S Clinical Trials Partnership. (2011). First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. The New England Journal of Medicine, 1-13. http://www.nejm.org/doi/full/10.1056/NEJMoa1102287?query=featured_home

Link to The New York Times article: http://www.nytimes.com/2011/10/19/health/19malaria.html?_r=1&ref=health

The Hygiene Hypothesis and the Old Friends Hypothesis

The hygiene hypothesis was first developed in the 1980s as an attempt to explain the increase in allergic disorders in the developed world. It postulated that the advent of sanitation and hyper vigilant hygiene practices led to reduced exposure of childhood illnesses, microorganisms and parasites. As a result, the immune system was not able to develop properly leading to the development of allergies and autoimmune disorders. This idea explains why the incidence of allergies and autoimmune disorders are so much higher in developed countries which have access to better hygiene practices, clean water and public sanitation, among other things.

Allergic disorders arise from an imbalance of the Th1/Th2 immune response with the Th2 response being dominant. It was thought that reduced contact with pathogens led to an underdevelopment of the Th1 response. However, scientists saw a problem with this theory, because there was also an increase in autoimmune disorders such as IBD, multiple sclerosis, and type 1 diabetes, which are Th1 mediated responses. This brought about the idea that the problem was not with the Th1/Th2 balance but rather with the Tregulatory cells.

The Old Friends hypothesis is a revision of the hygiene theory that takes the Treg response into account in its explanation of the problem. It states that rather than the immune system not developing properly, it has evolved in a way that requires commensal microorganisms to function. These microorganisms are treated as “friends” because they are relatively harmless or because having an immune response to them would cause more harm than benefit. Instead of attacking the microbe or parasite, the immune system goes into immunoregulatory mode increasing the maturation of both dendritic cells and Treg cells to suppress the immune response to the invader. The regulation of the “old friends” also leads to a suppression of the immune system that would cause damage to self-cells. Some studies have been performed to show that probiotics can induce Treg giving more weight to the idea that commensal bacteria are needed for proper immune function.

Citations:
Rook G. A., Brunet L. R. (2005). Microbes, immunoregulation, and the gut. Gut 54, 317–320. doi: 10.1136/gut.2004.053785. [PMC free article] [PubMed] [Cross Ref]
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1774411/

Sachs, Jessica Snyder. Good Germs, Bad Germs: Health and Survival in a Bacterial World. New York: Hill and Wang, 2008. Print.

Trichuris suis therapy in Crohn’s disease

As we have learned and discussed in class, Inflammatory Bowel Disease is considered by some researchers to be more prevalent in more developed countries in the Western World and less prevalent in developing countries that do not have access to adequate sanitation and clean water and food supplies. The article "Trichuris suis therapy in Crohn’s disease" is based on this idea. The researchers of the article believe that people living in more developed countries are not often exposed to bacteria and parasites so they do not carry parasites inside of them. Thus, they believe that people with IBD create an immune response to the natural flora normally present in the gut. The researchers believe that by feeding these people worms, specifically Trichuris suis, there will be a reduction in inflammation by down-regulating the host's immune response to natural gut flora.

The study was conducted at the University of Iowa over a time period of 24 weeks and involved 29 patients. There was a large diversity in ages of the patients, ranging from 18 to 72 years old. All of them were allowed to continue any medications they were taking at the time as long as they met the criteria for the clinical trial set by the researchers. The worms themselves were grown in vitro after they were isolated from the colon of a pathogen free pig the worm ova were fed to in the first place. Once the worms had matured, the patients were given a commercial drink filled with worms to drink every three weeks. Patients also kept diaries of the symptoms they experienced during the treatments.

Halfway through the clinical trial, 22 of the 29 patients enrolled had responded to the treatment and 19 of the 29 patients were considered to be in remission. By the end of the 24 weeks, 23 patients had responded to the therapy and 21 patients were considered to be in remission. It was also noted that patients that were using immunosuppressive drugs experienced greater improvement compared to patients that were not immunosuppressed.

Article Citation:

http://www.ncbi.nlm.nih.gov/pubmed/15591509

Natural Remedies and Inflammatory Bowl Disease

One of the lay articles we discussed in class, "What is Inflammatory Bowel Disease?" from the NativeRemedies website, highlighted basic information about inflammatory bowel disease (IBD) and later delved into treatments and IBD flare-up preventions. Natural and holistic treatments such as therapeutic homeopathic herb remedies and allopathic medical treatments were the clear focus of the article as it continued to describe and promote products sold by the same NativeRemedies website.

I remember many of us in the class were upset with the connection of the medical information to what developed into product placement. The information, while valid in its own rights, appeared to be packaged in a vehicle that could sway certain readers through language and selectivity of information provided. Environmental factors, for example, included people who had little physical activity, higher socioeconomic status, stress, and smoking which all could somehow to apply to the majority of the population in the Western world. They failed to specific areas or give information on how each directly factor affected IBD. Homeopathic medicines sold on the site were composed by a clinical psychologist and not a dietician, which hopefully should at mildly question the product.

Doing some research from the NativeRemedies website, they list the main ingredients of one product as chamomile, meadowsweet, slippery elm, and Sutherlandia frutescens. Ginger, peppermint, rose-scented geranium, sweet fennel are the ingredients of another while the third listed product focuses on using "tissue salts" like Kali phos, Calc phos, and Nat phos (respectively potassium, calcium, and sodium phosphates). The combination of each ingredient in the products is never listed, only disclosing that the formula is 100% homeopathic.

In response to this, I found a review article written by L. Langmead and D. S. Rampton, Review article: complementary and alternative therapies for inflammatory bowel disease, that tried to combine and evaluate scientific studies done on the different forms of complementary and alternative medicine (CAM) available for IBD treatment. While they concluded that further clinical trials of potential effects of CAM approaches need to be conducted for IBD patients, I drew a few main ideas from the synopsis. First, there are so many options besides Western medicine even beyond herbal supplements that we do not always have the best methods in clinical trials to compare them against each other or through inconclusive trials. Second, most commonly the people who approach CAM practices are patients with "poor quality of life" already and an overall high "systemic steriod intake [which suggests a] poorly controlled disease" state. This could mean that Western medicine has already 'failed' these patients or the draw is emotionally based; either is not conclusive, but stands to point out the target audience of these companies. Third, studies have confirmed scientifically some of the positive effects of certain CAM procedures, debunking the claim that all of these procedures are based purely on placebo effect; however, these studies must compare aim to compare in vivo effects to be effective in humans. Lastly, serious side effects can come from either the current lack of regulation in the CAM procedures, the mixing of herbs and chemical toxicity that can come from supplements taken without dietary supervision, and from completely forgoing conventional medicine for CAM alternative.  

While naturopathic medicine does have its merits and plays a useful role in relieving symptoms for different diseases including inflammatory bowel disease among many others, the sources cited, reliability, and company practices need to be assessed before starting a regimen. As always, consulting a certified professional like a doctor, dietician, or pharmacist is recommended before drastically altering your diet or "self-dosing" with herbal supplements sold in the supermarkets today.

Sources cited
http://www.nativeremedies.com/ailment/natural-treatments-for-inflammatory-bowel-disease.html#question4
http://www.nativeremedies.com/products/digestiontonic-stop-common-heartburn-indigestion.html#tabs
http://www.ncbi.nlm.nih.gov/pubmed/16422993

Smoking and Crohn's Disease

Crohn’s disease (CD), a type of inflammatory bowel disease (IBD), is currently defined as idiopathic due to its largely unknown etiology. A chronic disease which affects the gastrointestinal tract, it’s thought to be multifaceted, involving multiple genetic and environmental factors. Rates of IBD are different across geography, time, age, and smoking, lending credence to the idea that the environment plays a role in disease pathogenesis. Out of all these factors, however, smoking and appendectomy (surgical removal of the vermiform appendix) are the most significantly correlated, with smoking being especially correlated for CD.

Smokers, in addition to having a decreased antioxidant capacity, show an increased production of reactive oxygen species. Some evidence exists that the tobacco glycoprotein is capable of promoting a Th1 cell response, which may lead to further inflammatory problems in patients with CD. The extent that smoking worsens CD-related symptoms appears to be dose-dependent: 48% intestinal inflammatory activity was reported for heavy smokers (> 10 cigarettes/day), 46% for moderate smokers (< 10 cigarettes/day), and 37% among non-smokers. Patients who smoke tend to require more aggressive treatment (higher doses of immunosuppressants and CD-specific drugs) and patients who stop smoking show a significantly lower relapse risk.

Despite the availability of anti-inflammatory drugs and other biological tools, intestinal resection remains one of the primary forms of treatment for those with CD. Patients suffering from CD have a 40-60% chance of undergoing an intestinal resection within five-ten years of disease diagnosis. Unfortunately, return of the disease post-resection is still very common, requiring additional surgeries (referred to as surgical recurrence). Patients who undergo this procedure to treat their ileal disease are significantly more likely to require surgical recurrence if they smoke or chew tobacco. Another study retrospectively analyzed 182 CD patients who had undergone an intestinal resection and found that smoking was an independent risk factor for surgical recurrence as well as endoscopic lesions.

How smoking affects the therapeutic response of CD patients seems to be dependent on the type of treatment. For example, no relationship was found between smoking and a patient’s response to infliximab (monoclonal antibody against tumour necrosis factor alpha). Smoking adversely affects a patient’s response to thiopurine, however, decreasing the efficacy of the treatment and increasing the drug’s side effects [1].

In sum, I think it’s quite apparent that smoking worsens CD-related symptoms, creating a demand for more aggressive treatment and decreasing the chance of disease remission.

Citations:

1. Nos P, Domènech E. Management of Crohn’s disease in smokers: Is an alternative approach necessary? World Journal of Gastroenterology 2011; 17: 3567-3574.