The Crohnie

Another piece of research which establishes the anti-paratuberculosis antibiotic activity of molecules which are currently thought to have an immuno-suppressant effect in Crohn’s Disease.

http://www.paratuberculosis.org/pubs/proc9/abst185f_o4.htm

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BACKGROUND:

We have shown that the “immuno-modulators” methotrexate and 6-MP and the “anti-inflammatory” 5-ASA inhibit MAP growth (www.PLoSONE.org) and concluded that their most plausible mechanism of action in several idiopathic diseases is as antiMAP antibiotics. Thalidomide is an “immunomodulator” used in multiple “auto-immune” and “inflammatory” diseases and the mycobacterial diseases leprosy and tuberculosis. We now test the hypothesis that thalidomide inhibits MAP growth.

METHODS

Thalidomide (+-) and (+) and (-) and its two components, phthalimide and 1-hydroxy 2,6 piperidine dione (HPD) were evaluated in culture of two strains each of MAP (ATCC 19698 [bovine] & Dominic [human]) and M. avium subspecies avium (ATCC 25291 & 101.) We used a radiometric (14CO2 Bactec(R)) detection system. Inhibition is indicated by “percent decrease in cumulative Growth Index” (%-DcGI) from control.

RESULTS:

Phthalimide has no dose dependent inhibition on any strain. There was no dose dependent inhibition on either M. avium strain with thalidomide or its components. With the two MAP strains, there is dose dependent inhibition with thalidomide (+1); Dominic (31%-DcGI) and ATCC 19698 (26%-DcGI) at 64microg/ml. Thalidomide (+) is more inhibitory than (-). HPD is, on a weight for weight basis, the most inhibitory agent evaluated; Dominic (46%-DcGI) and ATCC 19698 (44%-DcGI at 64microg/ml)

CONCLUSIONS:

We show in vitro heretofore-undescribed inhibition of MAP growth by racaemic thalidomide. Thalidomide (+) is more potent than (-). Of thalidomide’s two moieties, phthalimide has no antiMAP activity and HPD is the active component in inhibiting MAP growth. We suggest that since 1942, initially with 5-ASA, the medical profession has unknowingly been treating MAP infections. These data are compatible with our concern that MAP is zoonotic. We conclude that all idiopathic “autoimmune” and “inflammatory” diseases, empirically treated with medications that we show are active against MAP, should now be evaluated for MAP as the etiological agent.

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.”
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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.

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Sulfasalazine (Azulfidine) is an anti-inflammatory medication that belongs to a class of drugs called sulfa drugs. The active ingredients in sulfasalazine consist of salicylate (the main ingredient in aspirin) combined with a sulfa antibiotic.
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American College of Rheumatology
http://www.rheumatology.org/public/factsheets/sulfasalazine.asp

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[Sulfasalazine] is split by the action of bacterial azoreductases in the large intestine into sulfapyridine and mesalazine (mesalamine, 5- aminosalicylic acid),
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Sulfasalazine. A review of its pharmacological properties and therapeutic efficacy in the treatment of rheumatoid arthritis.
http://www.ncbi.nlm.nih.gov/pubmed/7588084

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Sulfapyridine is a sulfonamide antibiotic. The sulfonamides are synthetic bacteriostatic antibiotics with a wide spectrum against most gram-positive and many gram-negative organisms. However, many strains of an individual species may be resistant. Sulfonamides inhibit multiplication of bacteria by acting as competitive inhibitors of p- aminobenzoic acid in the folic acid metabolism cycle. Bacterial sensitivity is the same for the various sulfonamides, and resistance to one sulfonamide indicates resistance to all.
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DrugBank: Sulfapyridine
http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/getCard.cgi?CARD=APRD00491.txt

Note also that the antibiotic para-aminosalicylic acid (4-ASA) is closely related to the 5-ASA portion of sulfasalazine, and is used for antibiotic treatment of mycobacterial infections, particularly tuberculosis.

http://www.answers.com/topic/para-aminosalicylic-acid?cat=health
http://www.rxlist.com/cgi/generic/paser.htm

The anti-mycobacterial agent 4-ASA has also been successfully used in the treatment of IBD, particularly Crohn’s Disease.

On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.
Greenstein RJ, Su L, Shahidi A, Brown ST.
PLoS ONE. 2007 Jun 13;2(6):e516

http://www.ncbi.nlm.nih.gov/pubmed/17565369

Full Text

Abstract

BACKGROUND: Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat “inflammatory” bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth. METHODOLOGY: The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric ((14)CO(2) BACTEC(R)) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (%-DeltacGI). PRINCIPAL FINDINGS: There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%-DeltacGI at 64 microg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%-DeltacGI at 4 microg/ml) is as effective as methotrexate, our positive control (88%-DeltacGI at 4 microg/ml). CONCLUSIONS: 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine’s introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

On the Action of Cyclosporine A, Rapamycin and Tacrolimus on M. avium Including Subspecies paratuberculosis
Greenstein RJ, Su L, Juste RA, Brown ST.
PLoS ONE. 2008 Jun 25;3(6):e2496

http://www.ncbi.nlm.nih.gov/pubmed/18575598

Full Text

Abstract

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the “immuno-modulators” methotrexate, azathioprine and 6-MP and the “anti-inflammatory” 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The “immunosuppressants” Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of “autoimmune” and “inflammatory” diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth. METHODOLOGY: The effect on radiometric MAP (14)CO(2) growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as “percent decrease in cumulative GI” (%-DeltacGI.) PRINCIPAL FINDINGS: The positive control clofazimine has 99%-DeltacGI at 0.5 microg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-DeltacGI by 32 microg/ml (Dominic), Rapamycin has 74%-DeltacGI by 64 microg/ml (UCF 4) and Tacrolimus 43%-DeltacGI by 64 microg/ml (UCF 4) CONCLUSIONS: We show heretofore-undescribed inhibition of MAP growth in vitro by “immunosuppressants;” the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some “autoimmune” and “inflammatory” diseases.

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.