Failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen brain from patients with Multiple Sclerosis
Main Article Content
Abstract
Objectives: Multiple Sclerosis is a chronic, enigmatic, progressive central nervous system “inflammatory” disease, with no know etiology. As with other “inflammatory” diseases there is the possibility that a cryptic infectious trigger may play a role in initiating Multiple Sclerosis. M. avium subspecies paratuberculosis causes Johne’s disease in ruminants and may be an infectious trigger in Crohn’s disease.
In this study, frozen archived brains from patients with Multiple Sclerosis, pure culture of multiple bacteria and circulating WBCs were assayed with proprietary (AffymetrixÔ RNA view®) Tissue and Cell fluorescent in situ hybridization assay for MAP RNA.
Results: Repetitively, false positive signal was observed in the “No-Probe” negative control. Despite advice from the technical staff at Affymetrix, multiple experimental modifications could not prevent positive signal in the “No-Probe” negative control.
When studying human white blood cells under specific storage conditions, we observe positive signal with human house-keeping genes, when no signal is seen in the No-Probe controls.
Conclusions: We conclude, that when performed according to manufactures instructions and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the AffymetrixÔ RNA view® TISSUE assay cannot be used to detect M. avium subspecies paratuberculosis in pre-frozen brains of humans with Multiple Sclerosis. In contrast, using the AffymetrixÔ RNA view Cell fluorescent in situ hybridization system, evaluating human white blood cells, we reliably identify human house-keeping genes. This indicates that the Cell fluorescent in situ hybridization assay may be useful when evaluating circulating cells for specific pathogens.
Keywords:
In situ hybridization, multiple sclerosis, Mycobacterium avium subspecies paratuberculosis, Mycobacteria, Crohn disease, Johne disease
Article Details
How to Cite
GREENSTEIN, Robert J.; FAM, Peter S.; BROWN, Sheldon T..
Failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen brain from patients with Multiple Sclerosis.
Medical Research Archives, [S.l.], v. 12, n. 11, nov. 2024.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/5853>. Date accessed: 12 dec. 2024.
doi: https://doi.org/10.18103/mra.v12i11.5853.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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9. Rotstein DL, Marrie RA, Maxwell C, et al. MS risk in immigrants in the McDonald era: A population-based study in Ontario, Canada. Neurology 2019;93(24):e2203-e2215. Doi: 10.1212/WNL.0000000000008611.
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15. Pierrot-Deseilligny C, Souberbielle JC. Is hypovitaminosis D one of the environmental risk factors for multiple sclerosis? Brain : a journal of neurology 2010;133(Pt 7):1869-88. (In eng). Doi: 10.1093/brain/awq147.
16. Smolders J, Torkildsen Ø, Camu W, Holmøy T. An Update on Vitamin D and Disease Activity in Multiple Sclerosis. CNS Drugs 2019;33(12):1187-1199. (In eng). Doi: 10.1007/s40263-019-00674-8.
17. Bruce D, Ooi JH, Yu S, Cantorna MT. Vitamin D and host resistance to infection? Putting the cart in front of the horse. Exp Biol Med (Maywood) 2010;235(8):921-7. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20660091).
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21. White JH. Vitamin D deficiency and the pathogenesis of Crohn's disease. The Journal of steroid biochemistry and molecular biology 2018;175:23-28. Doi: 10.1016/j.jsbmb.2016.12.015.
22. Talafha MM, Qasem A, Naser SA. Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis. Nutrients 2024;16(9) (In eng). Doi: 10.3390/nu16091358.
23. Greenstein RJ, Su L, Brown ST. Vitamins A & D inhibit the growth of mycobacteria in radiometric culture. PLoS ONE 2012;7(1):e29631. (In eng). Doi: 10.1371/journal.pone.0029631.
24. Greenstein R. Human genetic defects and misinterpreted pharmacological data indicate that Crohn disease is consequent to a mycobacterial infection. Medical Research Archives 2024;12(7). Doi: 10.18103/mra.v12i7.5541.
25. Johne HA, Frothingham L. Ein eigenthumlicher fall von tuberculose beim rind ( A particular case of tuberculosis in a cow). Dtsch Zeitschr Tiermed, Vergl Pathol 1895;21:438-454.
26. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn's disease? N Engl J Med 2009;361(27):2666-8. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20018963).
27. Greenstein RJ, Collins MT. Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic? Lancet 2004;364(9432):396-7. Doi: 10.1016/S0140-6736(04)16781-0.
28. Dalziel TK. Chronic intestinal enteritis. British Medical Journal 1913;ii:1068-1070.
29. Crohn BB, Ginzberg L, Oppenheimer GD. Regional Ileitis. J Amer Med Assoc 1932;99:1323-1328.
30. Mishina D, Katsel P, Brown ST, Gilberts EC, Greenstein RJ. On the etiology of Crohn disease. Proceedings of the National Academy of Sciences of the United States of America 1996;93(18):9816-9820. (http://www.ncbi.nlm.nih.gov/pubmed/8790414).
31. Greenstein RJ. Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease. The Lancet infectious diseases 2003;3(8):507-14. (http://www.ncbi.nlm.nih.gov/pubmed/12901893).
32. Alonso R, Fernandez-Fernandez AM, Pisa D, Carrasco L. Multiple sclerosis and mixed microbial infections. Direct identification of fungi and bacteria in nervous tissue. Neurobiol Dis 2018;117:42-61. Doi: 10.1016/j.nbd.2018.05.022.
33. Minuk GY, Lewkonia RM. Possible familial association of multiple sclerosis and inflammatory bowel disease. N Engl J Med 1986;314(9):586. Doi: 10.1056/NEJM198602273140921.
34. Cossu D, Cocco E, Paccagnini D, et al. Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients. PLoS One 2011;6(4):e18482. Doi: 10.1371/journal.pone.0018482.
35. Cossu D, Masala S, Cocco E, et al. Association of Mycobacterium avium subsp. paratuberculosis and SLC11A1 polymorphisms in Sardinian multiple sclerosis patients. Journal of infection in developing countries 2013;7(3):203-7. (In eng). Doi: 10.3855/jidc.2737.
36. Cossu D, Masala S, Frau J, Cocco E, Marrosu MG, Sechi LA. Anti Mycobacterium avium subsp. paratuberculosis heat shock protein 70 antibodies in the sera of Sardinian patients with multiple sclerosis. Journal of the neurological sciences 2013;335(1-2):131-3. Doi: 10.1016/j.jns.2013.09.011.
37. Cossu D, Masala S, Frau J, et al. Antigenic epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly recognized in multiple sclerosis Sardinian patients. Molecular immunology 2014;57(2):138-40. Doi:10.1016/j.molimm.2013.09.001.
38. Frau J, Cossu D, Coghe G, et al. Role of interferon-beta in Mycobacterium avium subspecies paratuberculosis antibody response in Sardinian MS patients. Journal of the neurological sciences 2015;349(1-2):249-50. Doi: 10.1016/j.jns.2015.01.004.
39. Masala S, Cossu D, Palermo M, Sechi LA. Recognition of zinc transporter 8 and MAP3865c homologous epitopes by Hashimoto's thyroiditis subjects from Sardinia: a common target with type 1 diabetes? PLoS One 2014;9(5):e97621. Doi: 10.1371/journal.pone.0097621.
40. Mameli G, Cossu D, Cocco E, et al. Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients. J Neuroimmunol 2014;270(1-2):51-5. Doi: 10.1016/j.jneuroim.2014.02.013.
41. Chiodini RJ. Mycobacterium paratuberculosis. JClinMicrobiol 1987;25:796-801.
42. Greenstein RJ, Su L, Fam PS, Stabel JR, Brown ST. Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay. BMC Res Notes 2018;11(1):498. Doi: 10.1186/s13104-018-3601-5.
43. Greenstein RJ, Su L, Fam PS, Gurland B, Endres P, Brown ST. Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease. BMC Res Notes 2020;13(1):96. Doi: 10.1186/s13104-020-04947-0.
44. Green EP, Tizard ML, Moss MT, et al. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic acids research 1989;17(22):9063-73. (http://www.ncbi.nlm.nih.gov/pubmed/2555783).
45. Greenstein RJ, Su L, Grant IR, et al. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut pathogens 2021;13(1):30. Doi: 10.1186/s13099-021-00425-5.
2. Anonymous. Guy’s Hospital: Case of Insular Sclerosis of the Brain and Spinal Cord (under the care of Dr. Moxon). Lancet 1873;1:236.
3. Thompson AJ, Baranzini SE, Geurts J, Hemmer B, Ciccarelli O. Multiple sclerosis. Lancet 2018;391(10130):1622-1636. Doi: 10.1016/S0140-6736(18)30481-1.
4. Libbey JE, Cusick MF, Fujinami RS. Role of pathogens in multiple sclerosis. Int Rev Immunol 2014;33(4):266-83. (In eng). Doi: 10.3109/08830185.2013.823422.
5. Libbey JE, Fujinami RS. Potential triggers of MS. Results Probl Cell Differ 2010;51:21-42. Doi: 10.1007/400_2008_12.
6. Marrodan M, Alessandro L, Farez MF, Correale J. The role of infections in multiple sclerosis. Mult Scler 2019;25(7):891-901. Doi: 10.1177/1352458518823940.
7. Alelyani M, Gameraddin M, Alshahrani R, et al. Assessment of vitamin D status and associated risk factors in high-altitude populations affected by multiple sclerosis: A case-control study. Medicine 2024;103(22):e38369. (In eng). Doi: 10.1097/md.0000000000038369.
8. Gombash SE, Lee PW, Sawdai E, Lovett-Racke AE. Vitamin D as a Risk Factor for Multiple Sclerosis: Immunoregulatory or Neuroprotective? Front Neurol 2022;13:796933. Doi: 10.3389/fneur.2022.796933.
9. Rotstein DL, Marrie RA, Maxwell C, et al. MS risk in immigrants in the McDonald era: A population-based study in Ontario, Canada. Neurology 2019;93(24):e2203-e2215. Doi: 10.1212/WNL.0000000000008611.
10. Sabel CE, Pearson JF, Mason DF, Willoughby E, Abernethy DA, Taylor BV. The latitude gradient for multiple sclerosis prevalence is established in the early life course. Brain : a journal of neurology 2021;144(7):2038-2046. Doi: 10.1093/brain/awab104.
11. Alter M, Leibowitz U, Speer J. Risk of multiple sclerosis related to age at immigration to Israel. Archives of neurology 1966;15(3):234-7. (In eng). Doi: 10.1001/archneur.1966.00470150012002.
12. Dean G, Kurtzke JF. On the risk of multiple sclerosis according to age at immigration to South Africa. Br Med J 1971;3(5777):725-9. (In eng). Doi: 10.1136/bmj.3.5777.725.
13. Munger KL, Zhang SM, O'Reilly E, et al. Vitamin D intake and incidence of multiple sclerosis. Neurology 2004;62(1):60-5. (In eng). Doi: 10.1212/01.wnl.0000101723.79681.38.
14. Mowry EM. Vitamin D: evidence for its role as a prognostic factor in multiple sclerosis. Journal of the neurological sciences 2011;311(1-2):19-22. (In eng). Doi: 10.1016/j.jns.2011.06.035.
15. Pierrot-Deseilligny C, Souberbielle JC. Is hypovitaminosis D one of the environmental risk factors for multiple sclerosis? Brain : a journal of neurology 2010;133(Pt 7):1869-88. (In eng). Doi: 10.1093/brain/awq147.
16. Smolders J, Torkildsen Ø, Camu W, Holmøy T. An Update on Vitamin D and Disease Activity in Multiple Sclerosis. CNS Drugs 2019;33(12):1187-1199. (In eng). Doi: 10.1007/s40263-019-00674-8.
17. Bruce D, Ooi JH, Yu S, Cantorna MT. Vitamin D and host resistance to infection? Putting the cart in front of the horse. Exp Biol Med (Maywood) 2010;235(8):921-7. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20660091).
18. Hewison M. Vitamin D and the intracrinology of innate immunity. Mol Cell Endocrinol 2010;321(2):103-11. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20156523).
19. Hewison M. Vitamin D and immune function: an overview. The Proceedings of the Nutrition Society 2012;71(1):50-61. (In eng). Doi: 10.1017/S0029665111001650.
20. Wang TT, Dabbas B, Laperriere D, et al. Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease. J Biol Chem 2010;285(4):2227-31. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19948723).
21. White JH. Vitamin D deficiency and the pathogenesis of Crohn's disease. The Journal of steroid biochemistry and molecular biology 2018;175:23-28. Doi: 10.1016/j.jsbmb.2016.12.015.
22. Talafha MM, Qasem A, Naser SA. Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis. Nutrients 2024;16(9) (In eng). Doi: 10.3390/nu16091358.
23. Greenstein RJ, Su L, Brown ST. Vitamins A & D inhibit the growth of mycobacteria in radiometric culture. PLoS ONE 2012;7(1):e29631. (In eng). Doi: 10.1371/journal.pone.0029631.
24. Greenstein R. Human genetic defects and misinterpreted pharmacological data indicate that Crohn disease is consequent to a mycobacterial infection. Medical Research Archives 2024;12(7). Doi: 10.18103/mra.v12i7.5541.
25. Johne HA, Frothingham L. Ein eigenthumlicher fall von tuberculose beim rind ( A particular case of tuberculosis in a cow). Dtsch Zeitschr Tiermed, Vergl Pathol 1895;21:438-454.
26. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn's disease? N Engl J Med 2009;361(27):2666-8. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20018963).
27. Greenstein RJ, Collins MT. Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic? Lancet 2004;364(9432):396-7. Doi: 10.1016/S0140-6736(04)16781-0.
28. Dalziel TK. Chronic intestinal enteritis. British Medical Journal 1913;ii:1068-1070.
29. Crohn BB, Ginzberg L, Oppenheimer GD. Regional Ileitis. J Amer Med Assoc 1932;99:1323-1328.
30. Mishina D, Katsel P, Brown ST, Gilberts EC, Greenstein RJ. On the etiology of Crohn disease. Proceedings of the National Academy of Sciences of the United States of America 1996;93(18):9816-9820. (http://www.ncbi.nlm.nih.gov/pubmed/8790414).
31. Greenstein RJ. Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease. The Lancet infectious diseases 2003;3(8):507-14. (http://www.ncbi.nlm.nih.gov/pubmed/12901893).
32. Alonso R, Fernandez-Fernandez AM, Pisa D, Carrasco L. Multiple sclerosis and mixed microbial infections. Direct identification of fungi and bacteria in nervous tissue. Neurobiol Dis 2018;117:42-61. Doi: 10.1016/j.nbd.2018.05.022.
33. Minuk GY, Lewkonia RM. Possible familial association of multiple sclerosis and inflammatory bowel disease. N Engl J Med 1986;314(9):586. Doi: 10.1056/NEJM198602273140921.
34. Cossu D, Cocco E, Paccagnini D, et al. Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients. PLoS One 2011;6(4):e18482. Doi: 10.1371/journal.pone.0018482.
35. Cossu D, Masala S, Cocco E, et al. Association of Mycobacterium avium subsp. paratuberculosis and SLC11A1 polymorphisms in Sardinian multiple sclerosis patients. Journal of infection in developing countries 2013;7(3):203-7. (In eng). Doi: 10.3855/jidc.2737.
36. Cossu D, Masala S, Frau J, Cocco E, Marrosu MG, Sechi LA. Anti Mycobacterium avium subsp. paratuberculosis heat shock protein 70 antibodies in the sera of Sardinian patients with multiple sclerosis. Journal of the neurological sciences 2013;335(1-2):131-3. Doi: 10.1016/j.jns.2013.09.011.
37. Cossu D, Masala S, Frau J, et al. Antigenic epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly recognized in multiple sclerosis Sardinian patients. Molecular immunology 2014;57(2):138-40. Doi:10.1016/j.molimm.2013.09.001.
38. Frau J, Cossu D, Coghe G, et al. Role of interferon-beta in Mycobacterium avium subspecies paratuberculosis antibody response in Sardinian MS patients. Journal of the neurological sciences 2015;349(1-2):249-50. Doi: 10.1016/j.jns.2015.01.004.
39. Masala S, Cossu D, Palermo M, Sechi LA. Recognition of zinc transporter 8 and MAP3865c homologous epitopes by Hashimoto's thyroiditis subjects from Sardinia: a common target with type 1 diabetes? PLoS One 2014;9(5):e97621. Doi: 10.1371/journal.pone.0097621.
40. Mameli G, Cossu D, Cocco E, et al. Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients. J Neuroimmunol 2014;270(1-2):51-5. Doi: 10.1016/j.jneuroim.2014.02.013.
41. Chiodini RJ. Mycobacterium paratuberculosis. JClinMicrobiol 1987;25:796-801.
42. Greenstein RJ, Su L, Fam PS, Stabel JR, Brown ST. Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay. BMC Res Notes 2018;11(1):498. Doi: 10.1186/s13104-018-3601-5.
43. Greenstein RJ, Su L, Fam PS, Gurland B, Endres P, Brown ST. Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease. BMC Res Notes 2020;13(1):96. Doi: 10.1186/s13104-020-04947-0.
44. Green EP, Tizard ML, Moss MT, et al. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic acids research 1989;17(22):9063-73. (http://www.ncbi.nlm.nih.gov/pubmed/2555783).
45. Greenstein RJ, Su L, Grant IR, et al. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut pathogens 2021;13(1):30. Doi: 10.1186/s13099-021-00425-5.