Reviewer's report
Title:Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
Version: 1Date:29 November 2010
Reviewer: Robert Greenstein
Reviewer's report:
Reviewer. Robert Greenstein
Journal: BMC Gastroenterology
Title: Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
By: Timo Rath et al.
Thank you for requesting my review of this well performed study of the presence
of several antibodies in a variety of gastro-intestinal diseases.
The authors had a dual intent in this study:
Objective #1 was to;
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
Objective #2 was;
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection (Matrix Metalloproteinases (MMPs)
are a family of Zn 2+-dependent endopeptidases that are considered to be the
most potent proteases in the turnover of the extracellular matrix)
For Objective #1, the authors:
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
For Objective #1, the authors the authors additionally attempted to culture MAP
from some samples
Samples for cultural investigation were not frozen at any time.
For Objective #2, the authors:
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection.
The data for Objective #1 show a non-statistically significant:
more frequent detection of MAP DNA in UC patients (20%) and controls (33%)
compared to patients with CD (7%)
The data for the secondary Objective #1: Culture of MAP from samples are not
presented, or if they are I could not find them!!!! In attempting do so I searched
the pdf file sent to me using the word “culture.” I found the word “Culture” a total
of 12 times. Six times in Methods for “Culture”, Twice in the Methods “DNA
extraction” section, twice in the Discussion section and twice in the References.
There are NO results that I could find!
The data for Objective #2 are much more difficult, if not impossible, to interpret.
This is because of the finding presented from Objective #1:
As MAP DNA was detected in only one CD patient, we were unable to analyze
MMP expression with regard to MAP status in the CD patient cohort.
Reviewer’s overview of the data presented and it significance:
The authors have spent considerable time and effort on this protocol and the
manuscript. They have been diligent and performed a complex set of studies and
experiments. They are obviously applying the Null hypotheses and are to be
commended.
As presented this manuscript perpetuates a series of publications on the
potential zoonosis of MAP that lack clarity and simply muddy the issue.
I have spent a considerable time on this review, as I believe that the effort
already expended by these authors merit such a commitment.
I will suggest what I think are the potential enormous payback that might be
achieved. However, these suggestions will require more analyses and
experiments.
Reviewer’s comment on the global design and concept of this study:
It should be made clear at the outset of these comments, that this reviewer is
seriously concerned that MAP is zoonotic and that CD may merely represent the
tip of the iceberg of the diseases that MAP causes in humans.
Whilst accepting that this is an iconoclastic viewpoint, it is no longer out of the
mainstream of academic perspective. A recent editorial in the NEJM stated that:
"It is tempting to speculate that these common genetic signatures support, albeit
indirectly, the proposal that a proportion of Crohn’s disease cases may have a
mycobacterial cause." 1
This comment in the NEJM are consequent to data showing a commonality in
genetic defects in multibacillary leprosy and Crohn’s disease. 2 Additionally,
there is parallism in the host immune response between multibacillary and
paucibacillary leprosy with perforating and non-perforating Crohn’s disease.3
There are important implications of these observations to this manuscript.
It is my opinion that there are two major reasons that the zoonosis of MAP has
been missed.
First, in humans, MAP exists in the cell wall deficient from and therefore will not
be detected by the 1882/3 mycobacterial cell wall staining methods of Ziehl 4 and
Neelsen. 5
The authors of the manuscript under review cite, but apparently have not
understood, the significance of one of Ramon Juste’s manuscripts 6 (their
reference # 36.) Either that is because the paper is incomprehensible, they have
not read it or they did read it but did not understand it.
I hope that the first explanation is not the reason, as I wrote the Juste paper.
Ramon contacted me with his unexpected observation of the prevalence of MAP
DNA in the BLOOD of controls and patients with IBD. The published results in 6
are very similar to those found for MAP DNA in the TISSUE of the presented in
this manuscript.
The clue to how I suggest a further analysis of data may prove valuable is found
in final segment of Table 3 of this manuscript (Anti-inflammatory therapy: AIT) I
previously suggested to Ramon that these analyses be expanded. Similarly I now
suggest that these authors should also expand this medication analysis.
This is because the authors have ignored a new body of literature about IBD. I
have suggested that, unknowingly, the medical profession has been treating
MAP since Svartz introduced 5-ASA in 1942.7
There are now un-refuted data showing that agents referred to as
“antiinflammatories” 8, “immune-modulators” 9-12 and “immunosuppressants” 13
all cause dose dependent inhibition of MAP in culture. In the Just manuscript, we
were able to show that concurrent OR PRIOR treatment with these agents
decreased or ELIMINATED the prevalence of MAP DNA in blood (depending on
which therapeutic agents were used.)
If the authors can retrieve the detailed pharmaceutical history of the patients that
they have studied, they may find compelling data. Additionally, to my knowledge
this will be the first time that such a correlation has been made with TISSUE,
rather than as Ramon did, with blood 6.
Finally, please remember that M. leprae has NEVER been cultured 14 and thus
Koch’s postulates cannot be met for M. leprae and leprosy.15 MAP culture is not
routinely possible in humans, as vanishingly few laboratories are able to culture
MAP from humans.16,17 Additionally, when MAP is successfully cultured from
humans, as much as 18 months may be necessary for the isolated bacterium to
manifest a cell wall.16
Specific Suggestions:
#1.) The manuscript lacks a logical progression. What is mentioned first in the
Methods section is not mentioned first in the Results or the Discussion. The
authors should rearrange their manuscript so there is a consistent sequence
throughout.
#2.) Just because you performed a lot of experiments, does NOT mean that you
have to publish everything. For example, you present NO Results on the MAP
culture component of the study. Accordingly, you should delete the Culture
section from Methods.
#3.) Perform an analysis of the medications (concurrent and historical if possible)
prescribed to your IBD cohort. You may find interesting data and (to my
knowledge) for the first time show a detection effect of any antiMAP medications
in TISSUE from an IBD cohort.
#4.) The authors have the possibility of presenting a MAP DNA/RNA comparison
from IBD tissue.
As the authors state, there are differences in the detection, or inability to detect
MAP DNA in IBD. Data from my laboratory found MAP RNA in all patients with
UC and CD. 18 Subsequently, in unpublished data, we did NOT find MAP DNA in
the same specimens in which we find MAP RNA. I have suggested technical
reasons why this may be so. 15 To my knowledge, no other investigator has
looked for MAP RNA.
Since the authors already have isolated RNA from the specimens that the
studied for MAP DNA, they can also look for MAP RNA (REMEMBER you have
to use Random Primers, NOT oligo dT primers as prokaryotic RNA does not
have a poly A tail!)
#5.) MMP data
When I first reviewed the Juste data, I deleted all of his immunological data that
he subsequently published 19 (Your Ref # 47.) To my mind it only made sense to
perform such an analysis on his data when we had clarified the status in
relationship to unanticipated antiMAP therapies 8-13 (see above.) However,
Ramon felt that his analysis was sufficient and went ahead without considering a
reanalysis (I declined to review the manuscript.)
You find no relevant pattern in your MMP data.
I suggest that you perform the analyses that I have outlined (#3 & 4 above.) If
you have a story, tell it. THEN in light of those data reanalyze your MMP data
and see if you THEN get a pattern that tells a story.
HOWEVER: IF YOU DO FIND COMPELLING MAP DNA/RNA data when
analyzed from the antiMAP activity, I suggest that for the sake of clarity that you
DO NOT PUBLISH in the same manuscript. The story will get lost and yet
another paper that confuses, rather than illuminates, will be added to the miasma
that is the MAP/ IBD conundrum.
#6.) Mention that amongst the most important insights that may be found in
understanding the susceptibility to a MAP infection are the presence or absence
of genetic diseases associated with CD. These include NOD2 20,21 and Nramp1
22 amongst others.23 Such evaluations were not performed in this study. In my
opinion, in future, the presence or absence of such mutations will need to be
identified in order to better understand the response, or lack of a response, by
the host to a MAP infection.
The authors should comment on the need to identify genetic defects in future
studies.
Overall, this was a well-performed study. In my opinion, the results do not
confirm the hypotheses tested because of the lack of understanding of the
antiMAP activity of various medications.
Good luck.
Robert Greenstein
Citations:
1. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn's
disease? N Engl J Med 2009;361(27):2666-8.
2. Zhang FR, Huang W, Chen SM, et al. Genomewide association study of
leprosy. N Engl J Med 2009;361(27):2609-18.
3. Gilberts EC, Greenstein AJ, Katsel P, Harpaz N, Greenstein RJ. Molecular
evidence for two forms of Crohn disease. Proc Natl Acad Sci USA
1994;91(126):12721-12724.
4. Ziehl F. Zur Farbung des Tuberkelbacillus. Dtsch, med. Wschr, 1882;8:451.
5. Neelsen FCA. Ein casuistischer Bietrag zur Lehre von der Tuberkulose. Zbl.
med. Wiss. 1883;21(497-501).
6. Juste RA, Elguezabal N, Garrido JM, et al. On the prevalence of M. avium
subspecies paratuberculosis DNA in the blood of healthy individuals and patients
with inflammatory bowel disease. PLoS ONE 2008;3(7):e2537.
7. Svartz N. Salazopyrin, a new sulfanilamide preparation. A. Therapeutic
Results in Rheumatic Polyarthritis. B. Therapeutic Results in Ulcerative Colitis. C.
Toxic Manifestations in Treatment with Sulfanilamide Preparations. Acta Medica
Scandinavica 1942;110:577-598.
8. Greenstein RJ, Su L, Shahidi A, Brown ST. On the Action of 5-Amino-Salicylic
Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis. PLoS
ONE 2007;2:e516.
9. Greenstein RJ, Su L, Brown ST. On the effect of thalidomide on
Mycobacterium avium subspecies paratuberculosis in culture. Int J Infect Dis
2009;13:e254-263.
10. Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the Action of
Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis.
PLoS ONE 2007;2:e161.
11. Shin SJ, Collins MT. Thiopurine drugs (azathioprine and 6-mercaptopurine)
inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents
Chemother 2008;52(2):418-26.
12. Krishnan MY, Manning EJ, Collins MT. Comparison of three methods for
susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11
antimicrobial drugs. J Antimicrob Chemother 2009;64(2):310-6.
13. Greenstein RJ, Su L, Juste RA, Brown ST. On the Action of Cyclosporine A,
Rapamycin and Tacrolimus on M. avium including Subspecies paratuberculosis.
PLoS ONE 2008;3(6):e2496.
14. Stewart-Tull DES. Mycobacterium leprae - The bacteriologist's enigma. In:
Ratledge C, Stanford J, eds. The Biology of the Mycobacteria, Volume 1:
Physiology, Identification, and Classification. 1 ed. New York: Academic Press,
1982: 273-307.
15. Greenstein RJ. Is Crohn's disease caused by a mycobacterium?
Comparisons with leprosy, tuberculosis, and Johne's disease. Lancet Infect Dis
2003;3(8):507-14.
16. Chiodini RJ, Van Kruiningen HJ, Thayer WR, Merkal RS, Coutu JA. Possible
role of mycobacteria in inflammatory bowel disease. I. An unclassified
Mycobacterium species isolated from patients with Crohn's disease. Dig Dis Sci
1984;29(12):1073-9.
17. Naser SA, Ghobrial G, Romero C, Valentine JF. Culture of Mycobacterium
avium subspecies paratuberculosis from the blood of patients with Crohn's
disease. Lancet 2004;364(9439):1039-1044.
18. Mishina D, Katsel P, Brown ST, Gilberts EC, Greenstein RJ. On the etiology
of Crohn disease. Proc Natl Acad Sci U S A 1996;93(18):9816-9820.
19. Juste RA, Elguezabal N, Pavon A, et al. Association between Mycobacterium
avium subsp. paratuberculosis DNA in blood and cellular and humoral immune
response in inflammatory bowel disease patients and controls. Int J Infect Dis
2009;13(2):247-54.
20. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich
repeat variants with susceptibility to Crohn's disease. Nature
2001;411(6837):599-603.
21. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2
associated with a susceptability to Crohn's disease. Nature
2001;411(6837):603-606.
22. Kojima Y, Kinouchi Y, Takahashi S, Negoro K, Hiwatashi N, Shimosegawa T.
Inflammatory bowel disease is associated with a novel promoter polymorphism of
natural resistance-associated macrophage protein 1 (NRAMP1) gene. Tissue
Antigens 2001;58(6):379-84.
23. Abraham C, Cho JH. Inflammatory Bowel Disease. N Engl J Med
2009;361(21):2066-78.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I have submitted Provisional patents that are the consequence of my publications
since 2005. The most relevant publications have been cited in my review.
Patents applied for include a MAP vaccine patent, a MAP detection in blood use
patent and a potential antiMAP medication patent. These applications have been
funded out of pocket. There is no other outside financial support.
Reviewer's report
Title:Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
Version: 1Date:29 November 2010
Reviewer: Robert Greenstein
Reviewer's report:
Reviewer. Robert Greenstein
Journal: BMC Gastroenterology
Title: Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
By: Timo Rath et al.
Thank you for requesting my review of this well performed study of the presence
of several antibodies in a variety of gastro-intestinal diseases.
The authors had a dual intent in this study:
Objective #1 was to;
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
Objective #2 was;
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection (Matrix Metalloproteinases (MMPs)
are a family of Zn 2+-dependent endopeptidases that are considered to be the
most potent proteases in the turnover of the extracellular matrix)
For Objective #1, the authors:
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
For Objective #1, the authors the authors additionally attempted to culture MAP
from some samples
Samples for cultural investigation were not frozen at any time.
For Objective #2, the authors:
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection.
The data for Objective #1 show a non-statistically significant:
more frequent detection of MAP DNA in UC patients (20%) and controls (33%)
compared to patients with CD (7%)
The data for the secondary Objective #1: Culture of MAP from samples are not
presented, or if they are I could not find them!!!! In attempting do so I searched
the pdf file sent to me using the word “culture.” I found the word “Culture” a total
of 12 times. Six times in Methods for “Culture”, Twice in the Methods “DNA
extraction” section, twice in the Discussion section and twice in the References.
There are NO results that I could find!
The data for Objective #2 are much more difficult, if not impossible, to interpret.
This is because of the finding presented from Objective #1:
As MAP DNA was detected in only one CD patient, we were unable to analyze
MMP expression with regard to MAP status in the CD patient cohort.
Reviewer’s overview of the data presented and it significance:
The authors have spent considerable time and effort on this protocol and the
manuscript. They have been diligent and performed a complex set of studies and
experiments. They are obviously applying the Null hypotheses and are to be
commended.
As presented this manuscript perpetuates a series of publications on the
potential zoonosis of MAP that lack clarity and simply muddy the issue.
I have spent a considerable time on this review, as I believe that the effort
already expended by these authors merit such a commitment.
I will suggest what I think are the potential enormous payback that might be
achieved. However, these suggestions will require more analyses and
experiments.
Reviewer’s comment on the global design and concept of this study:
It should be made clear at the outset of these comments, that this reviewer is
seriously concerned that MAP is zoonotic and that CD may merely represent the
tip of the iceberg of the diseases that MAP causes in humans.
Whilst accepting that this is an iconoclastic viewpoint, it is no longer out of the
mainstream of academic perspective. A recent editorial in the NEJM stated that:
"It is tempting to speculate that these common genetic signatures support, albeit
indirectly, the proposal that a proportion of Crohn’s disease cases may have a
mycobacterial cause." 1
This comment in the NEJM are consequent to data showing a commonality in
genetic defects in multibacillary leprosy and Crohn’s disease. 2 Additionally,
there is parallism in the host immune response between multibacillary and
paucibacillary leprosy with perforating and non-perforating Crohn’s disease.3
There are important implications of these observations to this manuscript.
It is my opinion that there are two major reasons that the zoonosis of MAP has
been missed.
First, in humans, MAP exists in the cell wall deficient from and therefore will not
be detected by the 1882/3 mycobacterial cell wall staining methods of Ziehl 4 and
Neelsen. 5
The authors of the manuscript under review cite, but apparently have not
understood, the significance of one of Ramon Juste’s manuscripts 6 (their
reference # 36.) Either that is because the paper is incomprehensible, they have
not read it or they did read it but did not understand it.
I hope that the first explanation is not the reason, as I wrote the Juste paper.
Ramon contacted me with his unexpected observation of the prevalence of MAP
DNA in the BLOOD of controls and patients with IBD. The published results in 6
are very similar to those found for MAP DNA in the TISSUE of the presented in
this manuscript.
The clue to how I suggest a further analysis of data may prove valuable is found
in final segment of Table 3 of this manuscript (Anti-inflammatory therapy: AIT) I
previously suggested to Ramon that these analyses be expanded. Similarly I now
suggest that these authors should also expand this medication analysis.
This is because the authors have ignored a new body of literature about IBD. I
have suggested that, unknowingly, the medical profession has been treating
MAP since Svartz introduced 5-ASA in 1942.7
There are now un-refuted data showing that agents referred to as
“antiinflammatories” 8, “immune-modulators” 9-12 and “immunosuppressants” 13
all cause dose dependent inhibition of MAP in culture. In the Just manuscript, we
were able to show that concurrent OR PRIOR treatment with these agents
decreased or ELIMINATED the prevalence of MAP DNA in blood (depending on
which therapeutic agents were used.)
If the authors can retrieve the detailed pharmaceutical history of the patients that
they have studied, they may find compelling data. Additionally, to my knowledge
this will be the first time that such a correlation has been made with TISSUE,
rather than as Ramon did, with blood 6.
Finally, please remember that M. leprae has NEVER been cultured 14 and thus
Koch’s postulates cannot be met for M. leprae and leprosy.15 MAP culture is not
routinely possible in humans, as vanishingly few laboratories are able to culture
MAP from humans.16,17 Additionally, when MAP is successfully cultured from
humans, as much as 18 months may be necessary for the isolated bacterium to
manifest a cell wall.16
Specific Suggestions:
#1.) The manuscript lacks a logical progression. What is mentioned first in the
Methods section is not mentioned first in the Results or the Discussion. The
authors should rearrange their manuscript so there is a consistent sequence
throughout.
#2.) Just because you performed a lot of experiments, does NOT mean that you
have to publish everything. For example, you present NO Results on the MAP
culture component of the study. Accordingly, you should delete the Culture
section from Methods.
#3.) Perform an analysis of the medications (concurrent and historical if possible)
prescribed to your IBD cohort. You may find interesting data and (to my
knowledge) for the first time show a detection effect of any antiMAP medications
in TISSUE from an IBD cohort.
#4.) The authors have the possibility of presenting a MAP DNA/RNA comparison
from IBD tissue.
As the authors state, there are differences in the detection, or inability to detect
MAP DNA in IBD. Data from my laboratory found MAP RNA in all patients with
UC and CD. 18 Subsequently, in unpublished data, we did NOT find MAP DNA in
the same specimens in which we find MAP RNA. I have suggested technical
reasons why this may be so. 15 To my knowledge, no other investigator has
looked for MAP RNA.
Since the authors already have isolated RNA from the specimens that the
studied for MAP DNA, they can also look for MAP RNA (REMEMBER you have
to use Random Primers, NOT oligo dT primers as prokaryotic RNA does not
have a poly A tail!)
#5.) MMP data
When I first reviewed the Juste data, I deleted all of his immunological data that
he subsequently published 19 (Your Ref # 47.) To my mind it only made sense to
perform such an analysis on his data when we had clarified the status in
relationship to unanticipated antiMAP therapies 8-13 (see above.) However,
Ramon felt that his analysis was sufficient and went ahead without considering a
reanalysis (I declined to review the manuscript.)
You find no relevant pattern in your MMP data.
I suggest that you perform the analyses that I have outlined (#3 & 4 above.) If
you have a story, tell it. THEN in light of those data reanalyze your MMP data
and see if you THEN get a pattern that tells a story.
HOWEVER: IF YOU DO FIND COMPELLING MAP DNA/RNA data when
analyzed from the antiMAP activity, I suggest that for the sake of clarity that you
DO NOT PUBLISH in the same manuscript. The story will get lost and yet
another paper that confuses, rather than illuminates, will be added to the miasma
that is the MAP/ IBD conundrum.
#6.) Mention that amongst the most important insights that may be found in
understanding the susceptibility to a MAP infection are the presence or absence
of genetic diseases associated with CD. These include NOD2 20,21 and Nramp1
22 amongst others.23 Such evaluations were not performed in this study. In my
opinion, in future, the presence or absence of such mutations will need to be
identified in order to better understand the response, or lack of a response, by
the host to a MAP infection.
The authors should comment on the need to identify genetic defects in future
studies.
Overall, this was a well-performed study. In my opinion, the results do not
confirm the hypotheses tested because of the lack of understanding of the
antiMAP activity of various medications.
Good luck.
Robert Greenstein
Citations:
1. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn's
disease? N Engl J Med 2009;361(27):2666-8.
2. Zhang FR, Huang W, Chen SM, et al. Genomewide association study of
leprosy. N Engl J Med 2009;361(27):2609-18.
3. Gilberts EC, Greenstein AJ, Katsel P, Harpaz N, Greenstein RJ. Molecular
evidence for two forms of Crohn disease. Proc Natl Acad Sci USA
1994;91(126):12721-12724.
4. Ziehl F. Zur Farbung des Tuberkelbacillus. Dtsch, med. Wschr, 1882;8:451.
5. Neelsen FCA. Ein casuistischer Bietrag zur Lehre von der Tuberkulose. Zbl.
med. Wiss. 1883;21(497-501).
6. Juste RA, Elguezabal N, Garrido JM, et al. On the prevalence of M. avium
subspecies paratuberculosis DNA in the blood of healthy individuals and patients
with inflammatory bowel disease. PLoS ONE 2008;3(7):e2537.
7. Svartz N. Salazopyrin, a new sulfanilamide preparation. A. Therapeutic
Results in Rheumatic Polyarthritis. B. Therapeutic Results in Ulcerative Colitis. C.
Toxic Manifestations in Treatment with Sulfanilamide Preparations. Acta Medica
Scandinavica 1942;110:577-598.
8. Greenstein RJ, Su L, Shahidi A, Brown ST. On the Action of 5-Amino-Salicylic
Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis. PLoS
ONE 2007;2:e516.
9. Greenstein RJ, Su L, Brown ST. On the effect of thalidomide on
Mycobacterium avium subspecies paratuberculosis in culture. Int J Infect Dis
2009;13:e254-263.
10. Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the Action of
Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis.
PLoS ONE 2007;2:e161.
11. Shin SJ, Collins MT. Thiopurine drugs (azathioprine and 6-mercaptopurine)
inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents
Chemother 2008;52(2):418-26.
12. Krishnan MY, Manning EJ, Collins MT. Comparison of three methods for
susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11
antimicrobial drugs. J Antimicrob Chemother 2009;64(2):310-6.
13. Greenstein RJ, Su L, Juste RA, Brown ST. On the Action of Cyclosporine A,
Rapamycin and Tacrolimus on M. avium including Subspecies paratuberculosis.
PLoS ONE 2008;3(6):e2496.
14. Stewart-Tull DES. Mycobacterium leprae - The bacteriologist's enigma. In:
Ratledge C, Stanford J, eds. The Biology of the Mycobacteria, Volume 1:
Physiology, Identification, and Classification. 1 ed. New York: Academic Press,
1982: 273-307.
15. Greenstein RJ. Is Crohn's disease caused by a mycobacterium?
Comparisons with leprosy, tuberculosis, and Johne's disease. Lancet Infect Dis
2003;3(8):507-14.
16. Chiodini RJ, Van Kruiningen HJ, Thayer WR, Merkal RS, Coutu JA. Possible
role of mycobacteria in inflammatory bowel disease. I. An unclassified
Mycobacterium species isolated from patients with Crohn's disease. Dig Dis Sci
1984;29(12):1073-9.
17. Naser SA, Ghobrial G, Romero C, Valentine JF. Culture of Mycobacterium
avium subspecies paratuberculosis from the blood of patients with Crohn's
disease. Lancet 2004;364(9439):1039-1044.
18. Mishina D, Katsel P, Brown ST, Gilberts EC, Greenstein RJ. On the etiology
of Crohn disease. Proc Natl Acad Sci U S A 1996;93(18):9816-9820.
19. Juste RA, Elguezabal N, Pavon A, et al. Association between Mycobacterium
avium subsp. paratuberculosis DNA in blood and cellular and humoral immune
response in inflammatory bowel disease patients and controls. Int J Infect Dis
2009;13(2):247-54.
20. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich
repeat variants with susceptibility to Crohn's disease. Nature
2001;411(6837):599-603.
21. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2
associated with a susceptability to Crohn's disease. Nature
2001;411(6837):603-606.
22. Kojima Y, Kinouchi Y, Takahashi S, Negoro K, Hiwatashi N, Shimosegawa T.
Inflammatory bowel disease is associated with a novel promoter polymorphism of
natural resistance-associated macrophage protein 1 (NRAMP1) gene. Tissue
Antigens 2001;58(6):379-84.
23. Abraham C, Cho JH. Inflammatory Bowel Disease. N Engl J Med
2009;361(21):2066-78.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I have submitted Provisional patents that are the consequence of my publications
since 2005. The most relevant publications have been cited in my review.
Patents applied for include a MAP vaccine patent, a MAP detection in blood use
patent and a potential antiMAP medication patent. These applications have been
funded out of pocket. There is no other outside financial support.
Please review this paper.
user
Reviewer's report
Title:Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
Version: 1Date:29 November 2010
Reviewer: Robert Greenstein
Reviewer's report:
Reviewer. Robert Greenstein
Journal: BMC Gastroenterology
Title: Presence of intestinal Mycobacterium avium subspecies paratuberculosis
(MAP) DNA is not associated with altered MMP expression in ulcerative colitis
By: Timo Rath et al.
Thank you for requesting my review of this well performed study of the presence
of several antibodies in a variety of gastro-intestinal diseases.
The authors had a dual intent in this study:
Objective #1 was to;
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
Objective #2 was;
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection (Matrix Metalloproteinases (MMPs)
are a family of Zn 2+-dependent endopeptidases that are considered to be the
most potent proteases in the turnover of the extracellular matrix)
For Objective #1, the authors:
determined the prevalence of MAP DNA in biopsy samples of patients with UC
and CD as well as in patients without IBD using highly sensitive and MAP
specific PCRs.
For Objective #1, the authors the authors additionally attempted to culture MAP
from some samples
Samples for cultural investigation were not frozen at any time.
For Objective #2, the authors:
To analyze a potential regulation of MMP expression by MAP in vivo, we further
assessed the colonic expression of a broad MMP spectrum in UC patients with
and without intestinal MAP DNA detection.
The data for Objective #1 show a non-statistically significant:
more frequent detection of MAP DNA in UC patients (20%) and controls (33%)
compared to patients with CD (7%)
The data for the secondary Objective #1: Culture of MAP from samples are not
presented, or if they are I could not find them!!!! In attempting do so I searched
the pdf file sent to me using the word “culture.” I found the word “Culture” a total
of 12 times. Six times in Methods for “Culture”, Twice in the Methods “DNA
extraction” section, twice in the Discussion section and twice in the References.
There are NO results that I could find!
The data for Objective #2 are much more difficult, if not impossible, to interpret.
This is because of the finding presented from Objective #1:
As MAP DNA was detected in only one CD patient, we were unable to analyze
MMP expression with regard to MAP status in the CD patient cohort.
Reviewer’s overview of the data presented and it significance:
The authors have spent considerable time and effort on this protocol and the
manuscript. They have been diligent and performed a complex set of studies and
experiments. They are obviously applying the Null hypotheses and are to be
commended.
As presented this manuscript perpetuates a series of publications on the
potential zoonosis of MAP that lack clarity and simply muddy the issue.
I have spent a considerable time on this review, as I believe that the effort
already expended by these authors merit such a commitment.
I will suggest what I think are the potential enormous payback that might be
achieved. However, these suggestions will require more analyses and
experiments.
Reviewer’s comment on the global design and concept of this study:
It should be made clear at the outset of these comments, that this reviewer is
seriously concerned that MAP is zoonotic and that CD may merely represent the
tip of the iceberg of the diseases that MAP causes in humans.
Whilst accepting that this is an iconoclastic viewpoint, it is no longer out of the
mainstream of academic perspective. A recent editorial in the NEJM stated that:
"It is tempting to speculate that these common genetic signatures support, albeit
indirectly, the proposal that a proportion of Crohn’s disease cases may have a
mycobacterial cause." 1
This comment in the NEJM are consequent to data showing a commonality in
genetic defects in multibacillary leprosy and Crohn’s disease. 2 Additionally,
there is parallism in the host immune response between multibacillary and
paucibacillary leprosy with perforating and non-perforating Crohn’s disease.3
There are important implications of these observations to this manuscript.
It is my opinion that there are two major reasons that the zoonosis of MAP has
been missed.
First, in humans, MAP exists in the cell wall deficient from and therefore will not
be detected by the 1882/3 mycobacterial cell wall staining methods of Ziehl 4 and
Neelsen. 5
The authors of the manuscript under review cite, but apparently have not
understood, the significance of one of Ramon Juste’s manuscripts 6 (their
reference # 36.) Either that is because the paper is incomprehensible, they have
not read it or they did read it but did not understand it.
I hope that the first explanation is not the reason, as I wrote the Juste paper.
Ramon contacted me with his unexpected observation of the prevalence of MAP
DNA in the BLOOD of controls and patients with IBD. The published results in 6
are very similar to those found for MAP DNA in the TISSUE of the presented in
this manuscript.
The clue to how I suggest a further analysis of data may prove valuable is found
in final segment of Table 3 of this manuscript (Anti-inflammatory therapy: AIT) I
previously suggested to Ramon that these analyses be expanded. Similarly I now
suggest that these authors should also expand this medication analysis.
This is because the authors have ignored a new body of literature about IBD. I
have suggested that, unknowingly, the medical profession has been treating
MAP since Svartz introduced 5-ASA in 1942.7
There are now un-refuted data showing that agents referred to as
“antiinflammatories” 8, “immune-modulators” 9-12 and “immunosuppressants” 13
all cause dose dependent inhibition of MAP in culture. In the Just manuscript, we
were able to show that concurrent OR PRIOR treatment with these agents
decreased or ELIMINATED the prevalence of MAP DNA in blood (depending on
which therapeutic agents were used.)
If the authors can retrieve the detailed pharmaceutical history of the patients that
they have studied, they may find compelling data. Additionally, to my knowledge
this will be the first time that such a correlation has been made with TISSUE,
rather than as Ramon did, with blood 6.
Finally, please remember that M. leprae has NEVER been cultured 14 and thus
Koch’s postulates cannot be met for M. leprae and leprosy.15 MAP culture is not
routinely possible in humans, as vanishingly few laboratories are able to culture
MAP from humans.16,17 Additionally, when MAP is successfully cultured from
humans, as much as 18 months may be necessary for the isolated bacterium to
manifest a cell wall.16
Specific Suggestions:
#1.) The manuscript lacks a logical progression. What is mentioned first in the
Methods section is not mentioned first in the Results or the Discussion. The
authors should rearrange their manuscript so there is a consistent sequence
throughout.
#2.) Just because you performed a lot of experiments, does NOT mean that you
have to publish everything. For example, you present NO Results on the MAP
culture component of the study. Accordingly, you should delete the Culture
section from Methods.
#3.) Perform an analysis of the medications (concurrent and historical if possible)
prescribed to your IBD cohort. You may find interesting data and (to my
knowledge) for the first time show a detection effect of any antiMAP medications
in TISSUE from an IBD cohort.
#4.) The authors have the possibility of presenting a MAP DNA/RNA comparison
from IBD tissue.
As the authors state, there are differences in the detection, or inability to detect
MAP DNA in IBD. Data from my laboratory found MAP RNA in all patients with
UC and CD. 18 Subsequently, in unpublished data, we did NOT find MAP DNA in
the same specimens in which we find MAP RNA. I have suggested technical
reasons why this may be so. 15 To my knowledge, no other investigator has
looked for MAP RNA.
Since the authors already have isolated RNA from the specimens that the
studied for MAP DNA, they can also look for MAP RNA (REMEMBER you have
to use Random Primers, NOT oligo dT primers as prokaryotic RNA does not
have a poly A tail!)
#5.) MMP data
When I first reviewed the Juste data, I deleted all of his immunological data that
he subsequently published 19 (Your Ref # 47.) To my mind it only made sense to
perform such an analysis on his data when we had clarified the status in
relationship to unanticipated antiMAP therapies 8-13 (see above.) However,
Ramon felt that his analysis was sufficient and went ahead without considering a
reanalysis (I declined to review the manuscript.)
You find no relevant pattern in your MMP data.
I suggest that you perform the analyses that I have outlined (#3 & 4 above.) If
you have a story, tell it. THEN in light of those data reanalyze your MMP data
and see if you THEN get a pattern that tells a story.
HOWEVER: IF YOU DO FIND COMPELLING MAP DNA/RNA data when
analyzed from the antiMAP activity, I suggest that for the sake of clarity that you
DO NOT PUBLISH in the same manuscript. The story will get lost and yet
another paper that confuses, rather than illuminates, will be added to the miasma
that is the MAP/ IBD conundrum.
#6.) Mention that amongst the most important insights that may be found in
understanding the susceptibility to a MAP infection are the presence or absence
of genetic diseases associated with CD. These include NOD2 20,21 and Nramp1
22 amongst others.23 Such evaluations were not performed in this study. In my
opinion, in future, the presence or absence of such mutations will need to be
identified in order to better understand the response, or lack of a response, by
the host to a MAP infection.
The authors should comment on the need to identify genetic defects in future
studies.
Overall, this was a well-performed study. In my opinion, the results do not
confirm the hypotheses tested because of the lack of understanding of the
antiMAP activity of various medications.
Good luck.
Robert Greenstein
Citations:
1. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn's
disease? N Engl J Med 2009;361(27):2666-8.
2. Zhang FR, Huang W, Chen SM, et al. Genomewide association study of
leprosy. N Engl J Med 2009;361(27):2609-18.
3. Gilberts EC, Greenstein AJ, Katsel P, Harpaz N, Greenstein RJ. Molecular
evidence for two forms of Crohn disease. Proc Natl Acad Sci USA
1994;91(126):12721-12724.
4. Ziehl F. Zur Farbung des Tuberkelbacillus. Dtsch, med. Wschr, 1882;8:451.
5. Neelsen FCA. Ein casuistischer Bietrag zur Lehre von der Tuberkulose. Zbl.
med. Wiss. 1883;21(497-501).
6. Juste RA, Elguezabal N, Garrido JM, et al. On the prevalence of M. avium
subspecies paratuberculosis DNA in the blood of healthy individuals and patients
with inflammatory bowel disease. PLoS ONE 2008;3(7):e2537.
7. Svartz N. Salazopyrin, a new sulfanilamide preparation. A. Therapeutic
Results in Rheumatic Polyarthritis. B. Therapeutic Results in Ulcerative Colitis. C.
Toxic Manifestations in Treatment with Sulfanilamide Preparations. Acta Medica
Scandinavica 1942;110:577-598.
8. Greenstein RJ, Su L, Shahidi A, Brown ST. On the Action of 5-Amino-Salicylic
Acid and Sulfapyridine on M. avium including Subspecies paratuberculosis. PLoS
ONE 2007;2:e516.
9. Greenstein RJ, Su L, Brown ST. On the effect of thalidomide on
Mycobacterium avium subspecies paratuberculosis in culture. Int J Infect Dis
2009;13:e254-263.
10. Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the Action of
Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis.
PLoS ONE 2007;2:e161.
11. Shin SJ, Collins MT. Thiopurine drugs (azathioprine and 6-mercaptopurine)
inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents
Chemother 2008;52(2):418-26.
12. Krishnan MY, Manning EJ, Collins MT. Comparison of three methods for
susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11
antimicrobial drugs. J Antimicrob Chemother 2009;64(2):310-6.
13. Greenstein RJ, Su L, Juste RA, Brown ST. On the Action of Cyclosporine A,
Rapamycin and Tacrolimus on M. avium including Subspecies paratuberculosis.
PLoS ONE 2008;3(6):e2496.
14. Stewart-Tull DES. Mycobacterium leprae - The bacteriologist's enigma. In:
Ratledge C, Stanford J, eds. The Biology of the Mycobacteria, Volume 1:
Physiology, Identification, and Classification. 1 ed. New York: Academic Press,
1982: 273-307.
15. Greenstein RJ. Is Crohn's disease caused by a mycobacterium?
Comparisons with leprosy, tuberculosis, and Johne's disease. Lancet Infect Dis
2003;3(8):507-14.
16. Chiodini RJ, Van Kruiningen HJ, Thayer WR, Merkal RS, Coutu JA. Possible
role of mycobacteria in inflammatory bowel disease. I. An unclassified
Mycobacterium species isolated from patients with Crohn's disease. Dig Dis Sci
1984;29(12):1073-9.
17. Naser SA, Ghobrial G, Romero C, Valentine JF. Culture of Mycobacterium
avium subspecies paratuberculosis from the blood of patients with Crohn's
disease. Lancet 2004;364(9439):1039-1044.
18. Mishina D, Katsel P, Brown ST, Gilberts EC, Greenstein RJ. On the etiology
of Crohn disease. Proc Natl Acad Sci U S A 1996;93(18):9816-9820.
19. Juste RA, Elguezabal N, Pavon A, et al. Association between Mycobacterium
avium subsp. paratuberculosis DNA in blood and cellular and humoral immune
response in inflammatory bowel disease patients and controls. Int J Infect Dis
2009;13(2):247-54.
20. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich
repeat variants with susceptibility to Crohn's disease. Nature
2001;411(6837):599-603.
21. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2
associated with a susceptability to Crohn's disease. Nature
2001;411(6837):603-606.
22. Kojima Y, Kinouchi Y, Takahashi S, Negoro K, Hiwatashi N, Shimosegawa T.
Inflammatory bowel disease is associated with a novel promoter polymorphism of
natural resistance-associated macrophage protein 1 (NRAMP1) gene. Tissue
Antigens 2001;58(6):379-84.
23. Abraham C, Cho JH. Inflammatory Bowel Disease. N Engl J Med
2009;361(21):2066-78.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I have submitted Provisional patents that are the consequence of my publications
since 2005. The most relevant publications have been cited in my review.
Patents applied for include a MAP vaccine patent, a MAP detection in blood use
patent and a potential antiMAP medication patent. These applications have been
funded out of pocket. There is no other outside financial support.
assistant