-------------------- Sick since at least age 6, now 67. Decades of misdiagnosis. Numerous arthritic, neuro, psych, vision, cardiac symptoms. Been treating for 7 years, incl 8 mos on IV. Bart was missed so now treating that. Posts: 765 | From nw ct | Registered: Sep 2008
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from the Lyme Disease website of Elena Cook Lyme borrelia biofilms expert Dr. Alan Macdonald writes to the Journal of Clinical Investigation to protest as... IDSA Guidelines Author Bockenstedt Contravenes Regulations to Evade Verification of her Findings
by Elena Cook Background
Dr Linda Bockenstedt is a Yale-based author of the deeply-flawed Lyme Disease guidelines issued by IDSA in 2006. Recently, she published a study purporting to show that signs of persisting Borrelia burgdorferi in antibiotic-treated mice (and, by implication, in humans with chronic Lyme Disease), were nothing more than the remnants of dead bacteria (1). She did not explain why this "debris" should, exceptionally, resist all normal immune mechanisms for clearing dead microbe remnants in a mammalian host.
Images accompanying her publication attracted the attention of Dr Alan B. Macdonald, who is currently researching Biofilms of Borrelia along with Dr Eva Sapi and her team at University of New Haven, Connecticut. This collaboration recently resulted in the first-ever study demonstrating Biofilm formation by Borrelia burgdorferi in vitro. (2).
Biofilms are highly organised structures produced by microbes on both living and non-living surfaces. One of their main functions is protective; they are notorious for shielding bacteria from harmful environmental influences, including the presence of antibiotics.
Dr. Macdonald believes that the bacteria, clearly seen glowing green in Dr. Bockenstedt's images (due to Green Fluorescent Protein DNA recombinantly inserted into the Lyme bacteria's infectivity plasmid Cp26) represents a mixture of live and possibly dead Borrelia biofilm community members in the mouse tissue, as opposed to all completely dead, as she alleges.
The term "amorphous globs" used by Dr. Bockenstedt to describe the fluorescent material is unscientific. It has never appeared in any published article in any language describing Borreliosis in any living host.
In her article, Bockenstedt uses arrows to indicate her "amorphous globs" as constituents of "fields or carpets" of Borrelia in the tissues of the mouse, whilst ignoring the fact that spherical (atypical) forms of Borrelia can clearly be seen within!
It is well known that the matrix (non-living structural material of the living biofilm community) is derived from microbes - once living but now dead - which contribute their outer surface glycoprotein "slime layer" and their DNA, (extracellular DNA), possibly also incorporating other microbes within the extracellular matrix. (See image gallery in paper by Sapi et al, referenced below.) The Borrelia in a biofilm consist of organisms which vary in shape and structure, reflecting the specialisation that takes place within the structure.
Spherical forms, such as those visible in the photo published by Bockenstedt, have been shown to be an integral part of a Borrelia biofilm community. Yet Bockenstedt labels this material "amorphous" (ie without shape). That is not what we see when we look carefully at the image material she herself has published (see image included with Dr. Macdonald's letter below).
Because her images so strikingly reproduce the known features of a Borrelia biofilm, Dr. Macdonald wrote to her privately, requesting that she carry out a very simple, dye-based test on the material. The test is inexpensive and easily distinguishes between dead and live micro-organisms. She refused.
Dr. Macdonald then asked her to share some of the mouse tissue from her experiment so that he could perform this test in his own lab.
Once again, Dr. Bockenstedt refused - in flagrant denial of US public health agency regulations, which require taxpayer-funded scientists to make their material available to other researchers for independent verification.
Dr. Bockenstedt's study was funded by the National Institutes for Health (NIH) and the Centre for Disease Control (CDC), but also by the non-profit National Research Fund for Tick-Borne Diseases. Her refusal to share tissue or to perform the simple dye test calls into question her integrity as a scientist.
If Dr. Bockenstedt has nothing to hide,why will she not allow anyone to determine if her "dead" persisting Borrelia are truly "dead"? Is she afraid that her study will negate the validity of her own recommendations, made in conjunction with the other IDSA Lyme committee members, denying the reality of chronic Lyme Disease?
Is she worried that what her own published material shows is actually a biofilm of Borrelia (which now has a precedent in the peer- reviewed literature) and that she may now be obliged to issue a retraction of her manuscript, with its accompanying press releases releases to the national and international media?
Below is the e-letter submitted to the Journal of Clinical Investigation by Dr. Alan Macdonald. (1)Bockenstedt L. et al Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy DOI 10.1172/JCI 58813 (2)Sapi E. et al. (2012) Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro. PLoS ONE 7(10): e48277. doi:10.1371/journal.pone.0048277 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048277
Letter Submitted to Journal of Clinical Invesigations by Dr. Alan B. Macdonald Oct 2012
Title: Biofilms as a differential diagnosis for "Amorphous Globs of Dr Linda Bockenstedt"
Letter:
Dear Dr Bockenstedt,
I remark with great interest your inclusion of the brief discussion of Biofilms of Borrelia burgdorferi in your Discussion section, in connection with possible etiologies for the GFP-emitting "amorphous blobs" of Borrelia burgdorferi in deep dermal sites closely apposed to the articular cartilage of the murine arthritic Lyme arthritis mice.
Our paper [6] describing for the very first time the entity of in vitro Biofilms of Borrelia burgdorferi, and accepted for publication release on the PLOS ONE website during the week Of October 24, 2012, is the first peer reviewed manuscript on Biofilm formation by borrelia of any species. It is indeed fortuitous that we, and you were working with borrelia burgdorferi.
We and you were curious to understand the implications of possible in vivo Borrelia biofilm formation. At the level of verbal definition to fulfill the requisite (Costerton)[Ref 5] definitions for a biofilm community are the following:
1. Specialization of member microbes within the community which set these specialized forms apart from Planktonic forms of the establishing microbe.
2. Investment of the microbial members of the biofilm community by a self generated Extracellular matrix, usually including structural constituents from once living but now dead members of the biofilm community. The presence of free Extracellular DNA derived from once living now dead members of the biofilm community.
3. The existence of microbe density which exceeds the density of Planktonic microbes on an ordinary bacteriologic solid phase medium.
4. The existence of "plumbing networks" (i.e. water channels and channels for waste product elimination) in the biofilm community. (Ref 4)
5. The Resistance to antibiotic killing among specialized members of the biofilm community. (Ref4)
Now which of items 1-5 above are or might be "in play" in your so called "Amorphous Blobs" of GFP-labeled Borrelia burgdorferi in the Lyme arthritic mice?
1. Specialization:
Morphology of the microbes within the so-called "amorphous globs" differs significantly from the corkscrew-shaped forms (Planktonic forms-motile forms) (Ref 4) of Borrelia burgdorferi, which are nicely demonstrated in your supplementary Videos and in Figures 1-5 (Especially in your figures 5A and 5B). You clearly demonstrate "Round bodies" among the members of the Amorphous blobs.
Round form metamorphosis from pre-existing spiral forms of Borrelia burgdorferi has been elegantly demonstrated in the long list of peer reviewed publications by Drs Oystein Brorson and his cousin Dr. Sverre Henning Brorson (Ref 7) using electron Microscopy and Phase contrast microscopy. Round bodies were embraced by the late Dr. Lynn Margulis.(Ref 8)
You specifically took the time and effort to demonstrate the formation of borrelia Round bodies in your attached supplementary videos in still photographs in Figure 5 (5A and 5B) in your manuscript.Non-spiral borrelia - i.e.round body borrelia are indeed legitimate shape shifted forms of the Borrelia genus, and represent a specialization by the well known spiral MOTILE form of Borreliae (Planktonic from of Borrelia). Round bodies, granular forms, Cell wall deficient forms, and finally spiral or straightened forms of Borrelia are specialized forms of the Planktonic (spiral form).
Thanks to the works of Dr Alban and colleagues (Ref 1) at the University of Rhode Island, these vital round body borreliae demonstrate a diverse protein repertoire which differ from spiral forms in their protein "fingerprint" in two-dimensional SDS PAGE gel electrophoresis.(Ref 1)
2. The overall density of borrelia microbes in your "Amorphous blobs" in the murine deep dermis,exceeds by several orders of magnitude, the density of Planktonic (spiral/motile) forms of Borrelia burgdorferi when these are grown on solid media in the Microbiology laboratory ( Preac-Mursic, V. et al)(Ref 9)
3. The assertion that All of the borrelia microbes in the "Amorphous Blobs in murine deep dermis in laboratory induced Chronic Lyme Arthritis" are ALL DEAD and merely represent "debris"....is just that: an assertion, buttressed by what you say are corroborative mRNA supportive data...( not presented in your paper).
I have suggested to you in a personal private communication that the way to establish Live versus Dead borrelia in your "Amorphous Blobs" is to pour some Dye ( Invitrogen ::Live Dead Assay) (Ref 10)(Red=dead, and Green = alive).
You have steadfastly refused to accommodate this quality control procedure, which is fast, cheap, reliable and easily accomplished. You have refused me tissue from your "amorphous Globs" dermal tissue from murine subjects, so that I might perform this simple quality control step in my own laboratory.
Refusal to provide tissue to an outside scientist, is in violation of the guidelines of the NIH and other Federal funding agencies. Your Research activities were accomplished with the use of public funds. You have an incumbent obligation to provide tissue to outside scientists upon request,to verify your experimental findings.
4. A review of all previously published manuscripts since the 1982 NEJM articles announcing the Spirochetal (Borrelial) etiology of Lyme Disease, performed by me and by pathology colleagues (Ref3) interested in the published peer-reviewed Borrelia pathology manuscripts , which specifically demonstrate with figure illustrations Borrelia burgdorferi in diseased tissues--
Such a comprehensive 30-year literature review by Board Certified Anatomic and Clinical Pathologists--reveals that there has NEVER been published a manuscript which contains images of Borrelia burgdorferi - either Living Borrelia or Dead Borrelia - which are present in mammalian tissue in the densities which were illustrated in your figures 5A and 5B. (Ref 3) By Density criteria ALONE, these "Amorphous globs" are Biofilm communities in murine tissue, UNTIL PROVEN OTHERWISE.(Ref 4)
I therefore challenge you to voluntarily participate in Quality Control microscopic exercise -- to establish with the LIVE DEAD Invitrogen Kit that ALL of the Borrelia inside of your "Amorphous Blobs" stain Red(=Dead) and that none of the borrelia within your "Amorphous Globs" stains Green in the Invitrogen Live Dead assay.(Ref 10) If you are correct, there is nothing to lose in this quality control exercise.
Respectfully,
Alan B.MacDonald MD, FCAP, FASCP
References:
1. Microbiology. 2000 Jan: 146 ( Pt1):119-27. Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Alban PS, Johnson PW, Nelson DR. Source Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston R.I. 02881, USA 2.Burgdorfer, W,., Barbour, A.G., Hayes,S.F., Benach, J.L. et al, "Lyme Disease - A Tick borne Spirochetosis" , Science, 1982:216: 1317-9 3. Duray, Paul Harrison, MD, Complete Bibliography, Pub Med. 4. Montana State University, Center for Biofilm Studies: http://www.biofilm.montana.edu 5. Costerton, William , J. , Complete bibliography ( 600 peer reviewed Biofilm manuscripts), Pub Med. 6. Sapi, Eva, J, et al, PONE-D-12-11352R2 Characterization of biofilm formation by Borrelia burgdorferi in vitro :PLOS ONE , 2012, In Press, To be released on the Internet PLOS ONE website in the week of October 24, 2012. 7. Brorson, Oystein and Brorson, Sverre Henning, Complete bibliography, PubMED 8. Brorson, O,... Margulis, Lynn, et al, "Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline ", Oystein Brorson, Sverre-Henning Brorson, John Scythes, James MacAllister, Andrew Wiere, and Lynn Margulis http://www.pnas.org/cgi/content/full/0908236106 9. Preac-Mursic V, Wilske B, Reinhardt S. Eur J Clin Microbiol. Infect Dis. 1991 Dec;10 (12):1076-9. “Culture of Borrelia burgdorferi on six solid media.-, Source Max von Pettenkofer Institut, Ludwig-Maximillian-Universitat, Munich, Germany. 10. Invitrogen Inc- http://www.invitrogen.com/site/us/en/home/References/protocols/cell‐and‐tissue‐analysis/flowcytometry‐ protocol/cell‐viability/live‐dead‐fixable‐dead‐cell‐stain‐kits.html 11. Eisendle K, Grabner T, Zelger B.Focus floating microscopy: "Gold standard" for cutaneous borreliosis?, Am J Clin Pathol. 2007 Feb;127(2):213-22. Bockenstedt image of so-called dead Bb juxtaposed with image of borrelia biofilm
(Note: Use of boldface, extra spacing and other minor formatting changes mine -EC)
Above: Set of images included by Dr. Macdonald in his letter to Dr. Bockenstedt, sent to JCI, showing the striking resemblance between what she dismissed as shapeless globs and a known Borrelia biofilm. As some resolution may have been lost in transferring this image, interested readers can view the original published by Bockenstedt at http://www.jci.org/articles/view/58813/figure/5. There many round forms can clearly be distinguished in the so-called "glob" labelled "A". (All the green in her image is Borrelia, genetically altered to fluoresce for easy identification.)
Below: The same known Borrelia biofilm reproduced below showing more detail. The use of spectral imaging has clearly brought out the shapes of the Borrelia organisms in photo "A", which appear in red. A few spiral, very many round forms are distinguished from the extracellular matrix (here coloured green) which envelopes them.
IMAGES borrelia biofilm under spectral imaging shows round as well as spiral forms
A supplementary atlas of Biofilms of Borrelia and related information are available on Dr. Macdonald's two websites at the following URL's:
The articles by Elena Cook on this website may be distributed as long as they are reproduced without changes, attributing the author, and the link to the original URL is included.
Disclaimer: Material on this website is intended for informational purposes only. It is not intended as medical advice. For all questions relating to your own health, please consult a qualified medical professional.The site owner is not responsible for the content of external sites.
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Copyright � 2012-2013Elena Cook
Template design by Andreas Viklund
-------------------- Sick since at least age 6, now 67. Decades of misdiagnosis. Numerous arthritic, neuro, psych, vision, cardiac symptoms. Been treating for 7 years, incl 8 mos on IV. Bart was missed so now treating that. Posts: 765 | From nw ct | Registered: Sep 2008
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lpkayak
Honored Contributor (10K+ posts)
Member # 5230
posted
i cant pretend to understand it right now...i have in the past understood his writings. but i am still happy about it. woo hoo!!!!! dr M...my hero (i do have more than one hero these days...eva is another one-and there are more)
-------------------- Lyme? Its complicated. Educate yourself. Posts: 13712 | From new england | Registered: Feb 2004
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posted
Could anyone summarize this for me? I cant even begin to comprehend what it says either thanks
Posts: 574 | From Out there somewhere | Registered: Jul 2010
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poppy
Frequent Contributor (1K+ posts)
Member # 5355
posted
She says blobs seen are not biofilm, not alive.
He says there is test to confirm whether dead or alive.
She declines to do the test.
Posts: 2888 | From USA | Registered: Mar 2004
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posted
"The test he wants done has accuracy problems". Then it should be accepted by the IDSA, just like the rest of their lyme related tests, LOL.
Posts: 520 | From Maryland | Registered: Jan 2007
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lax mom
Frequent Contributor (1K+ posts)
Member # 38743
posted
quote:Originally posted by joysie: "The test he wants done has accuracy problems". Then it should be accepted by the IDSA, just like the rest of their lyme related tests, LOL.
quote:Originally posted by poppy: She says blobs seen are not biofilm, not alive.
He says there is test to confirm whether dead or alive.
She declines to do the test.
Not only that, she refused to give Macdonald tissue samples.
Her refusal is in violation of Federal regulations.
Macdonald knows his stuff. He knows there were live organisms in that biofilm matrix. Clearly, Bockenstedt refuses to give anyone an opportunity to prove it. That would make her evil in my book. People are dying.
-------------------- Sick since at least age 6, now 67. Decades of misdiagnosis. Numerous arthritic, neuro, psych, vision, cardiac symptoms. Been treating for 7 years, incl 8 mos on IV. Bart was missed so now treating that. Posts: 765 | From nw ct | Registered: Sep 2008
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poppy
Frequent Contributor (1K+ posts)
Member # 5355
posted
How convenient, if true.
Posts: 2888 | From USA | Registered: Mar 2004
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posted
Hi all Thank you for posting my article containing Dr McDonald's letter, but we seem to have a bit of a duplicate thread going... There was already a discussion here at LymeNet Medical which you can find at the following URL:
I wonder if we could go back to that original thread as it gets confusing when there are too many threads discussing the same thing?
Some of you said you found Dr M's letter difficult to understand. It is a letter written by one scientist to another, so it is understandably difficult for a layperson. I tried to clarify some of the content in lay terms, and some of you here have mentioned the gist of what is going on, but if anyone still has difficulty understanding it, please email me privately ([email protected]) and I'll try my best to clarify.
I'm going back to the original thread now, where I will answer the allegation from our "guest" Mr. Punter. Elena Cook
-------------------- Justice will be ours. Posts: 786 | From UK | Registered: Oct 2007
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