The Crohnie

It appears that some Azathioprine and 6-MP, which are used for the treatment of Crohn’s Disease, and whose mechanism of action is currently unknown, have antibiotic activity against Mycobacterium avium subspecies paratuberculosis (MAP).

http://www.paratuberculosis.org/pubs/proc9/abst182f_o6.htm

As noted by the authors, “These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen.”
=======================================

The in vitro susceptibility of human and bovine-origin Mycobacterium paratuberculosis to the thiopurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) were established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn’s disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP even at high concentrations (>=64.0 microg/mL). Among rapidly growing mycobacteria, M. phlei was susceptible to 6-MP and AZA whereas M. smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-amino- salycilic acid (mesalamine)) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin (CPX) used as control drugs were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6- MP and CPX resulted in significantly higher CFUs as compared to use of CPX alone. These data may partially explain the paradoxical response of Crohn’s disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs i.e. they do not worsen with anti-inflammatory treatment as would be expected with a microbial etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn’s disease: azathioprine drugs may confound interpretation of data on therapeutic responses both antibiotic-treated and control groups.

Liverpool, UK – 10 December 2007: Scientists at the University of Liverpool have found how a bacterium, known to cause illness in cattle, may cause Crohn’s disease in humans.

http://www.liv.ac.uk/newsroom/press_releases/2007/12/crohns-disease.htm

Crohn’s is a condition that affects one in 800 people in the UK and causes chronic intestinal inflammation, leading to pain, bleeding and diarrhoea.

The team found that a bacterium called Mycobacterium paratuberculosis releases a molecule that prevents a type of white blood cell from killing E.coli bacteria found in the body. E.coli is known to be present within Crohn’s disease tissue in increased numbers.

It is thought that the Mycobacteria make their way into the body’s system via cows’ milk and other dairy products. In cattle it can cause an illness called Johne’s disease – a wasting, diarrhoeal condition. Until now, however, it has been unclear how this bacterium could trigger intestinal inflammation in humans.

Professor Jon Rhodes, from the University’s School of Clinical Sciences, explains: “Mycobacterium paratuberculosis has been found within Crohn’s disease tissue but there has been much controversy concerning its role in the disease. We have now shown that these Mycobacteria release a complex molecule containing a sugar, called mannose. This molecule prevents a type of white blood cells, called macrophages, from killing internalised E.Coli.”

Scientists have previously shown that people with Crohn’s disease have increased numbers of a ‘sticky’ type of E.coli and weakened ability to fight off intestinal bacteria. The suppressive effect of the Mycobacterial molecule on this type of white blood cell suggests it is a likely mechanism for weakening the body’s defence against the bacteria.

Professor Rhodes added: “We also found that this bacterium is a likely trigger for a circulating antibody protein (ASCA) that is found in about two thirds of patients with Crohn’s disease, suggesting that these people may have been infected by the Mycobacterium.”

The team is beginning clinical trials to assess whether an antibiotic combination can be used to target the bacteria contained in white blood cells as a possible treatment for Crohn’s disease.

The research was funded by Core and the Medical Research Council and is published in Gastroenterology.

An interesting new piece of research about the genetics of Crohn’s Disease Diabetes and other diseases.
http://www.nature.com/nature/journal/v447/n7145/pdf/nature05911.pdf

I note that one of the gene patterns associated with Crohn’s relates to “autophagy”, an innate immunity mechanism which the body uses to defend itself against intracellular bacterial pathogens.

http://en.wikipedia.org/wiki/Autophagy

“”"
The study has also confirmed the importance of a process known as autophagy in the development of Crohn’s disease. Autophagy, or “self eating”, is responsible for clearing unwanted material, such as bacteria, from within cells. The may be key to the interaction of gut bacteria in health and in inflammatory bowel disease and could have clinical significance in the future.
“”"

http://www.medicalnewstoday.com/medicalnews.php?newsid=73420

“”"
One of the newly identified genes, called ATG16L1, has been thought to be required for autophagy, a process that leads to programmed cell death and is involved in the process of inflammation. When the research team used RNA interference to suppress the gene’s activity in bacterially infected cells, decreased molecular action associated with autophagy confirmed that the process depends on ATG16L1 activity.
“”"

http://www.sciencedaily.com/releases/2007/04/070415160159.htm

Autophagy is one of the most important defenses against the class of intracellular bacterial pathogens known as mycobacteria. One of the most studied organisms in relation to autophagy is mycobacterium tuberculosis

Autophagy in Immune Defense Against Mycobacterium tuberculosis
http://www.landesbioscience.com/journals/autophagy/article/2830

Mycobacterium avium subspecies paratuberculosis (MAP) is another intracellular obligate pathogen with which autophagy would be a key defense mechanism.

So it is no surprise to me that recent results indicate a defective autophagy process in Crohn’s Disease; this lends further weight to the theory that Mycobacerium paratuberculosis is a cause of Crohn’s Disease.

Leprosy is caused a member of genus Mycobacteria, M. leprae, and some forms of leprosy are treated by “tamping down the immune system” with anti-TNF agents, such as thalidomide. Steroids are also used for the treatment of leprosy[1].

FDA approves Thalidomide for Hansen’s disease side effect, imposes unprecedented restrictions on distribution.
http://www.fda.gov/bbs/topics/ANSWERS/ANS00887.html

Redeeming thalidomide
http://pubs.acs.org/hotartcl/mdd/00/jun/mddkling.html

Remicade’s anti-TNF activity might also be useful for leprosy (a known mycobacterial disease), but there haven’t been any trials of remicade/infliximab for Leprosy (Hansen’s Disease), presumably because the manufacturers know that the average third-world leper can’t afford $30K+ a year to be maintained on remicade, and thus won’t spend the huge money necessary for the relevant clinical trials.

But Thalidomide, the poor man’s anti-TNF drug, is long out of patent, and is cheap, and so gets used for the poor lepers. But there is a fightback in the leprosy medical community, primarily because of the nasty birth defects that Thalidomide causes.

No Role for Thalidomide in Leprosy
http://www.paho.org/English/AD/DPC/CD/thalidomide.htm

Thalidomide has been trialled for Crohn’s Disease.

Thalidomide reduces tumour necrosis factor {alpha} and interleukin 12 production in patients with chronic active Crohn’s disease
http://gut.bmjjournals.com/cgi/content/abstract/50/2/196

Thalidomide: New uses for notorious drug
http://www.mayoclinic.com/health/thalidomide/HQ01507

So the relative success of treatment of Crohn’s Disease with Remicade is NOT an argument against a mycobacterial cause. In fact, if the Crohn’s Disease to Leprosy analogy that has been made by some researchers[2,3] is true, then Remicade treatment in Crohn’s could be an argument FOR a mycobacterial cause.

1. Steroid prophylaxis for prevention of nerve function impairment in leprosy: randomised placebo controlled trial.
http://bmj.bmjjournals.com/cgi/content/abstract/328/7454/1459

2. Comparisons with leprosy, tuberculosis and Johne’s disease: is Crohn’s disease caused by a mycobacterium?
http://www.paratuberculosis.org/proc7/abst6_p6.htm

3. Mycobacterium avium subspecies paratuberculosis in the causation of Crohn’s disease
http://www.wjgnet.com/1007-9327/6/630.asp

I’ve just returned from Copenhagen, Denmark, where I attended the 8th International Colloquium on Paratuberculosis (at considerable personal expense: Copenhagen is EXPENSIVE!)

Current thinking among researchers in the field is that MAP itself is mostly not the culprit in the tissue damage. MAP’s role is to initiate an inflammatory response in the bowel, through some unknown process/antigen/inflammatory-pathway. This immune response results in inflammation of bowel tissue, whereby the cells of the intestine physically separate, to allow immune cells, primarily macrophages and CD4 + CD8 T-cells, to reach the site of infection. This leads to “leaky gut” syndrome , which permits the contents of the bowel (most accurately described as being similar to the contents of a sewer), to leak through the bowel wall, resulting in a *massive* inflammatory response against the many microbes present in the fecal stream.

At this point, the inflammatory process becomes self re-inforcing, with consequent runaway production of inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-alpha), various Interleukins, Interleukin-antagonists, etc.

In some patients, this results in granuloma formation, as the massively overstimulated immune system tries to contain the original MAP infection, present inside macrophages, which are unable to kill the “phagocytosed” (i.e. “eaten”) MAP, which have immune evasion techniques to avoid being killed by macrophages. The infected macrophage goes into overdrive, producing copious quantities of inflammatory cytokines, which cause it to be surrounded by a layer of CD4 T-cells, and then another layer of CD8 T-cells: a granuloma is formed, which contains the mycobacterial infection and cuts off the flow of inflammatory cytokines, but also has the unfortunate consequence of causing significant permanent scarring, thus leading to stenosis/narrowing of the bowel, and eventually strictures.

In other patients, for reasons unknown (although possibly related to the failure of the Th1 inflammatory response), this granuloma formation does not take place. Instead, the flood of antigens from the bowel contents infects the bowel “transmurally”, i.e. through the entire thickness of the bowel, leading to all kinds of scarring, and in some unfortunates, the formation of fistulas, as well as all manner of secondary infections. It is this sub-group of patients that responds best to treatment with wide-spectrum antobiotics, since it is secondary microbial infections that are primarily responsible for the tissue damage, with these secondary non-mycobacterial microbes being susceptible to non-mycobacterial antibiotic treatment.

There is an established body of clinical research which backs up the claims that GM-CSF is an effective treatment for a significant proportion of Crohn’s patients.

Treatment of active Crohn’s disease with recombinant human granulocyte-macrophage colony-stimulating factor.
http://www.ncbi.nlm.nih.gov/pubmed/12433518

GM-CSF treatment for Crohn’s disease: a stimulating new therapy?
http://www.ncbi.nlm.nih.gov/pubmed/12498002

Crohn’s disease: an immunodeficiency?
http://www.ncbi.nlm.nih.gov/pubmed/12840672

Sargramostim for active Crohn’s disease.
http://www.ncbi.nlm.nih.gov/pubmed/15917384

This therapy does fly in the face of current thinking in regards to how to treat Crohn’s Disease. Most attempts to treat Crohn’s Disease are targetted at suppressing the immune system, not boosting it. Here are a couple of references that discuss the importance of *reducing* GM-CSF levels.

Increased production of granulocyte-macrophage colony-stimulating factor in Crohn’s disease–a possible target for infliximab treatment.
http://www.ncbi.nlm.nih.gov/pubmed/15201577

Infliximab: mechanism of action beyond TNF-alpha neutralization in inflammatory bowel disease.
http://www.ncbi.nlm.nih.gov/pubmed/15201575

How to resolve the apparent paradox, i.e. whether to treat Crohn’s by boosting or by suppressing the immune system? The answer to that probably lies in the fact Crohn’s may be several different diseases presenting the same or a similar profile of symptoms: Crohn’s is not a disease but a SYNDROME. Until we can differentiate between the different underlying disease causes, we’ll be stuck with the current situation of one-size-fits-all therapy. It is to be hoped that the Sargramostim/Leukine/GM-CSF trials will not only seek to treat CD, but also to determine *why* a significant percentage (~40%) of Crohn’s patients experience improvement when their immune systems are so boosted, while the rest experience insignificant change. If some clear indicator can be found that differentiates between the two groups, then that would be a huge leap forward in the clinical treatment of Crohn’s Disease.

Although the mainstream medical community may be surprised by the GM-CSF results, I for one am not. After all, one of the primary effects of Granulocyte-Macrophage Colony-Stimulating-Factor (GM-CSF) is to boost the activity of Macrophages, the key anti-microbial component of the innate immune system. Ineffectiveness of macrophages to phagocytose mycobacteria is the key failure of the immune system that leads to granuloma formation in mycobacterial disease.

MECHANISMS OF BACTERIAL PATHOGENICITY: Bacterial Defense Against Phagocytes
http://textbookofbacteriology.net/antiphago.html

Immune Evasion by bacteria
http://crohn.ie/archive/primer/imunevad.htm

It is quite likely that in the short term, both suppressive and booster therapies will work in relieving Crohn’s Disease symptoms. However, in the longer term, the outcomes would be very different.

1. Therapy which boosts the immune system would ideally result in elimination of the infection causing inflammation.

2. Suppression, on the other hand, would result in a failure of the Th1 immune response, and thus failure to eliminate the causative infection. This would result in the immune system response changing to a Th2 response, mediated by the humoral arm of the immune system, which is
almost always ineffective in controlling mycobacterial infections.

Isolation of Mycobacterium paratuberculosis from milk by immunomagnetic separation.
http://www.ncbi.nlm.nih.gov/pubmed/9726853

Improved detection of Mycobacterium avium subsp. paratuberculosis in milk by immunomagnetic PCR.
http://www.ncbi.nlm.nih.gov/pubmed/11118722

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk.
http://www.ncbi.nlm.nih.gov/pubmed/15214726

To read about culture methods that can determine MAP metabolism, see MT Collins excellent johnes.org site.

http://www.johnes.org/general/diagnosis.html#bactec

It will be great to finally see published the results of that double-blind, placebo-controlled and multi-center clinical trial: reliable data are desperately needed in this area.
http://www.crohns.org/treatment/austrial.htm

I expect to see results that show that RMATC (the Pharmacia trial is using RMAT + Clofazimine) is equally or more effective than existing treatments for Crohn’s Disease, and in a minority subgroup of patients, induces significantly longer clinical remission periods than existing treatments.

However, even when the results are published, we will still be in the following dissatisfactory position

1. We will not know in advance who will benefit from anti-MAP treatment and who will not.

2. If the treatment achieves a successful clinical outcome in any given patient, we will not know why.

3. If the treatment fails to achieve a successful clinical outcome, we will not know why. Which means that we will be unable to tell if the same treatment can be used again in the same patient, or if an alternative antibiotic treatment is required (e.g. due to antibiotic resistance).

4. We will not know the parameters which will define a successful end of the treatment period, e.g. organism clearance, antibody reduction, etc, etc. The currently proposed 2-year treatment period is essentially a guesstimate, based on experience with the treatment of related mycobacterial infections such as M avium subsp. avium. Since these antibiotics are expensive, and can be toxic, we need to be able to much more clearly define clinical outcome parameters. If 2 years is too short, then we risk antibiotic resistance. If 2 years is too long, then we need to reduce it. But we have little data to base conclusions upon.

5. We will not know if the treatment is microbiologically working in a patient with physical stricturing symptoms. RMAT treatment can accelerate stricture formation in a (hard to identify) subgroup of patients, due to fibrotic tissue formation, just as with other mycobacterial diseases. Physicians inexperienced with the protocol can misread the consequent temporary disimprovement in the patient’s clinical status as a treatment failure and take the patient off treatment, thus risking antibiotic resistance. We will have no tools, other than physician experience and intuition, to differentiate success from failure.

Or as Chiodini puts it, we will still be “at the whim of the individual physician”.

I believe that the bottom line is that most Gastroenterologists simply don’t know enough about infectious diseases, are not taught enough about infectious diseases: it’s not a large enough part of their educational, medical, or clinical culture. For example, some of the research published by gastroenterologists about anti-MAP antibiotic treatment for Crohn’s Disease displays fundamentally flawed reasoning and lack of microbiology experience.

I don’t find it at all strange that RMAT treatment was pioneered by a surgeon, John Hermon-Taylor. Fortunately, there are many excellent, open-minded and energetic gastroenterologists who are at the forefront of a fundamental rethink of Crohn’s Disease: Ira Shafran(Florida), Tom Borody(Sydney), Will Chamberlin(US Army), etc, etc. I’ve had the great fortune to work with all three: three things stand out about all of them: 1. They concern themselves deeply with the health of their patients, 2. they are not afraid to admit that they don’t know everything and are willing to learn, and 3. they are not driven by money.

Live Mycobacterium avium subspecies paratuberculosis (MAP) has been cultured from retail milk purchased from stores in California, Minnesota and Wisconsin.

This means that American consumers are being exposed to live bacteria that are known to cause Inflammatory Bowel Disease (Johne’s Disease) in a wide range of animals, including dairy and beef cattle, and is suspected of being a cause of human Crohn’s Disease.

The most important points are

1. From May 2002 through April 2003, milk was purchased from stores in California, Minnesota and Wisconsin – three of the USA’s top 5 milk-producing states. (The other two are New York and Pennsylvania.)

2. Milk was tested for presence of viable MAP, using methodologies created in the 1990′s by British researchers, to study the presence of MAP in retail milk in the UK. It has been known since 1998 that United Kingdom dairy products are contaminated with live MAP.

3. Of 702 US samples tested, 2.8 percent contained viable MAP – that is, MAP bacteria that was alive, capable of multiplying and establishing infection, and capable of causing Inflammatory Bowel Disease in susceptible species.

4. Rate of positives was similar among states, but there was a seasonal effect. More positive samples were found during July, August and September.

This study confirms what we in the Paratuberculosis Awareness and Research Association have long believed: that American consumers are eating and drinking food that contains a live and dangerous bacterium, through the medium of MAP-contaminated dairy products.

On average 2.8% of milk cartons were found to be contaminated. Assuming that the average milk consumer drinks from a single carton of milk per day, this means that the average milk consumer is exposed to live paratuberculosis on average ten times a year. Consuming from 2 different milk cartons per day, 20 times a year, etc. This applies particularly to children, who are encouraged to consume milk, for the calcium and protein content. An average American child living in Minnesota, California or Wisconsin, if they consume from one milk carton per day, will have been exposed to live paratuberculosis up to 100 times by their tenth birthday.

The published results apply only to milk. Although research has shown that the food treatment methodologies used to manufacture other dairy products, such as cheese, chocolate, whey, etc, are incapable of destroying MAP, no US research has sought to determine the percentage of these retail dairy products also contaminated with live MAP. The majority of Wisconsin milk is used for cheese manufacture. Recent scientific results have shown that the methods to manufacture cheddar cheese do not kill paratuberculosis.

To this date, the food safety regulators in the United States, the Food & Drug Admninistration (FDA), have taken no action on the presence of live paratuberculosis in milk, dairy and beef products. The time has now come for the FDA to revise its policy of inaction, and to act immediately to protect American consumers from this dangerous bacterium.

If you believe that the US Government should put the interests of the American public before the interests of American Dairy and Beef Industries, and act to eradicate MAP from human food, please visit the PARA web site for steps you can take to help.

1. Original datasheet from American retail milk study
http://www.johnes.org/newsfiles/109216471862392.html

2. Articles about live paratuberculosis contamination in human food
http://www.crohns.org/map_food

3. Articles about the relationship between Mycobacterium avium subspecies paratuberculosis and Crohn’s Disease.
http://www.crohns.org/articles
http://www.crohns.org/research
http://www.crohns.org/treatment

4. Government agencies with responsibility for regulating Food Safety.
http://www.crohns.org/governments

5. PARA’s work to get the US Congress to help address this problem.
http://www.crohns.org/congress

6. What you can do to help.
http://www.crohns.org/help

Persistence of Mycobacterium paratuberculosis during Manufacture and Ripening of Cheddar Cheese.

Donaghy JA, Totton NL, Rowe MT.

Agriculture, Food and Environmental Science Division (Food Microbiology Branch), Department of Agriculture and Rural Development for N. Ireland, Newforge Lane, Belfast BT9 5PX, N. Ireland, United Kingdom.

http://www.ncbi.nlm.nih.gov/pubmed/15294829

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10(4) to 10(5) CFU/ml) and low (10(1) to 10(2) CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log(10)) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.