Not so private correspondence

General or non-medical topics with information and discussion related to Lyme disease and other tick-borne diseases.
hv808ct
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Not so private correspondence

Post by hv808ct » Tue 18 Dec 2012 18:03

The odd ranting below is from LymeNut. (http://flash.lymenet.org/scripts/ultima ... p=0#000000)

It was posted by an actual nut—an unfortunate woman whose delusions and paranoia led health authorities to lock her up for a while. (See: http://www.elenacook.org/statejun06.html) Of course, not everything can be cured so she periodically pops up with some new conspiracy fantasy. The latest one—perhaps encouraged by MacDonald—is a wacky attack on Linda Bockenstedt, MD at Yale. Here’s some of it:
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

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.

[deleted]

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.

Below is the e-letter submitted to the Journal of Clinical Investigation by Dr. Alan Macdonald.
Letter Submitted to Journal of Clinical Invesigations by Dr. Alan B. Macdonald
Oct 2012
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.

[deleted because MacDonald tends to run on and on and on…]

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.

[deleted due to excessive yakking]

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
It’s not clear if MacDonald actually wrote to L.B. or to JCI. (Why he’d write to a journal about getting samples from the author of an article is a complete mystery.) It’s not clear how poor crazy Elena Cook got hold of the letter either. But I suppose the point of posting this stuff was to somehow embarrass L.B. or pressure her into giving MacDonald that to which he is not entitled.

Just because the NIH funded some of L.B.’s research does not mean she can be forced to collaborate with whoever sends her a letter. Nor does it mean she has to provide them with anything more than what is provided in the M&M section of her paper. Moreover, she might actually get into legal trouble if she were to ship off biological samples and reagents to anyone who asked for them. A retired guy tinkering in his basement is not likely to be viewed as a qualified scientist or in the employ of a legitimate research center.

L.B.’s colleagues and the journal editors found her methods and conclusions sufficiently credible to have them published. If anyone questions them, they can certainly repeat those experiments on their own.

As for MacDonald’s colorful Live-Dead assertions about Borrelia: it isn’t that simple. Davey recently noted in his minireview:

Whether we are discussing microorganisms or macroorganisms, it is usually easier to distinguish between live and killed individuals than between organisms that are alive and those which have recently died of natural causes. In the case of staining, while control live and dead samples may be clearly separable, environmental stresses often give rise to heterogeneous populations, with some cells showing an intermediate uptake of viability stains. When a population of cells is exposed to stress, depending on the magnitude of the stress, there may be a heterogeneous response in which some cells are killed, others are damaged, and yet others may show no observable phenotypic change. It has recently been shown, even for well-characterized, eukaryotic laboratory organisms when cells are under stress, that propidium iodide (PI) may enter cells during or immediately after application of the stress but that a short period of recovery will allow membrane damage to be repaired such that PI cannot enter the cells. (Hazel M. Davey HM. Life, Death, and In-Between: Meanings and Methods in Microbiology. Appl Environ Micro, 2011;77(16):5571-76.)

More specifically,

The commercially available LIVE/DEAD BacLight kit (Invitrogen) has enjoyed increasing popularity among researchers in various fields since it was released about 10 years ago. The kit consists of two stains, propidium iodide (PI) and SYTO9, which both stain nucleic acids. Green fluorescing SYTO9 is able to enter all cells and is used for assessing total cell counts, whereas red fluorescing PI enters only cells with damaged cytoplasmic membranes. The emission properties of the stain mixture bound to DNA change due to the displacement of one stain by the other and quenching by fluorescence resonance energy transfer. LIVE/DEAD staining was shown to work not only with (eu)bacteria but also with archaea or eukaryotic cells, such as yeast (Saccharomyces cerevisiae). Although this kit enables differentiation only between bacteria with intact and damaged cytoplasmic membranes, it is often used to differentiate between active and dead cells. While it seems accurate to assume that membrane-compromised bacterial cells can be considered dead, the reverse (that intact cells are active cells) is not necessarily true. Microscopic assessment of LIVE/DEAD-stained bacterial cells is usually simplified to either “green”-labeled (live) or “red”-labeled (dead) cells. However, experience with this dye combination and flow cytometry during the last few years by our group and others has shown that the staining of bacterial cells with SYTO9 and PI does not always produce distinct “live” and “dead” populations; intermediate states are also observed. In the kit manufacturer’s manual, the region of intermediate states is referred to as “unknown.” This can lead to difficulties in the interpretation of results and can be critical when, for example, decisions have to be made about the effectiveness of disinfection methods or the amount of viable bacteria in water distribution systems. (Berney M, et al. Appl Environ Micro, 2007;73(10):3283-90)

For MacDonald, it’s back to the basement with a pocketful of mice. As for Cook, maybe it’s time to see the nice mental health people again.

Camp Other
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Re: Not so private correspondence

Post by Camp Other » Wed 19 Dec 2012 1:42

hv808ct,

I fail to see any good reason for name calling and ad hominem attacks here. Why not just focus on the crux of the post, which is to explain why the BacLight method is not adequate for demonstrating that living spirochetes persist in the host, by only posting the below passage?
hv808ct wrote: L.B.’s colleagues and the journal editors found her methods and conclusions sufficiently credible to have them published. If anyone questions them, they can certainly repeat those experiments on their own.

As for MacDonald’s colorful Live-Dead assertions about Borrelia: it isn’t that simple. Davey recently noted in his minireview:

Whether we are discussing microorganisms or macroorganisms, it is usually easier to distinguish between live and killed individuals than between organisms that are alive and those which have recently died of natural causes. In the case of staining, while control live and dead samples may be clearly separable, environmental stresses often give rise to heterogeneous populations, with some cells showing an intermediate uptake of viability stains. When a population of cells is exposed to stress, depending on the magnitude of the stress, there may be a heterogeneous response in which some cells are killed, others are damaged, and yet others may show no observable phenotypic change. It has recently been shown, even for well-characterized, eukaryotic laboratory organisms when cells are under stress, that propidium iodide (PI) may enter cells during or immediately after application of the stress but that a short period of recovery will allow membrane damage to be repaired such that PI cannot enter the cells. (Hazel M. Davey HM. Life, Death, and In-Between: Meanings and Methods in Microbiology. Appl Environ Micro, 2011;77(16):5571-76.)

More specifically,

The commercially available LIVE/DEAD BacLight kit (Invitrogen) has enjoyed increasing popularity among researchers in various fields since it was released about 10 years ago. The kit consists of two stains, propidium iodide (PI) and SYTO9, which both stain nucleic acids. Green fluorescing SYTO9 is able to enter all cells and is used for assessing total cell counts, whereas red fluorescing PI enters only cells with damaged cytoplasmic membranes. The emission properties of the stain mixture bound to DNA change due to the displacement of one stain by the other and quenching by fluorescence resonance energy transfer. LIVE/DEAD staining was shown to work not only with (eu)bacteria but also with archaea or eukaryotic cells, such as yeast (Saccharomyces cerevisiae). Although this kit enables differentiation only between bacteria with intact and damaged cytoplasmic membranes, it is often used to differentiate between active and dead cells. While it seems accurate to assume that membrane-compromised bacterial cells can be considered dead, the reverse (that intact cells are active cells) is not necessarily true. Microscopic assessment of LIVE/DEAD-stained bacterial cells is usually simplified to either “green”-labeled (live) or “red”-labeled (dead) cells. However, experience with this dye combination and flow cytometry during the last few years by our group and others has shown that the staining of bacterial cells with SYTO9 and PI does not always produce distinct “live” and “dead” populations; intermediate states are also observed. In the kit manufacturer’s manual, the region of intermediate states is referred to as “unknown.” This can lead to difficulties in the interpretation of results and can be critical when, for example, decisions have to be made about the effectiveness of disinfection methods or the amount of viable bacteria in water distribution systems. (Berney M, et al. Appl Environ Micro, 2007;73(10):3283-90)
[additional slams removed]

Let's focus on the spirochetes, shall we?

So what I'm getting out of this is that what we basically have is even worse than Schrödinger's spirochetes, correct? That even if one applies the BacLight kit, that one can't be sure that spirochetes which are green are definitely alive even if they are observed to be green?

The thing is, why not use this as a preliminary step for further investigation of the worse-than-Schrödinger state spirochetes? At the very least, if we observe ALL RED spirochetes, then we know they're all dead. But if there's any green at all, then there is at least some doubt - and then, wouldn't one want to run additional tests to determine their viability and metabolic state?

How does one determine that these remaining spirochetes are alive and metabolically active if not through the use of BacLight alone?

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inmacdonald
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Re: Not so private correspondence

Post by inmacdonald » Wed 19 Dec 2012 5:38

A green light signal from live bacteria
or
An entirely Red signal from entirely Dead bacteria

A cupful of Dye is all that is required for quality control of Dr Bockenstedt's experimental report.

Inexpensive in dollars and in time allocated to the study.

If Dr Bockenstedt's assertion that all of the Amorphous globs of bacteria
in the mouse deep dermis are ALL DEAD is ratified by All RED staining
in this Invitrogen Kit method, then the discussion is over.

The Expense which would necessarily follow the Green Staining result even if only focal green staining
were observed
in Dr Bockenstedt's mouse model.... Is the expense to her Credibility
if Green staining results are realized.

She would then have to retract her article, and to issue a series of
press releases attesting to her retraction.

Dr Linda Bockenstedt had no hesitation in issuing press releases to the
Lay press upon the initial publication of her paper.

Fact checking is needed to find where the truth lies.

My letter to the JCI was not published. My letter was intended to be available to
the JCI readership. Nothing private in the letter to the editor of the JCI was
intended nor implied.

Letters to the editor are public documents.

Camp Other
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Re: Not so private correspondence

Post by Camp Other » Wed 19 Dec 2012 8:12

inmacdonald wrote:A green light signal from live bacteria
or
An entirely Red signal from entirely Dead bacteria

A cupful of Dye is all that is required for quality control of Dr Bockenstedt's experimental report.

Inexpensive in dollars and in time allocated to the study.

If Dr Bockenstedt's assertion that all of the Amorphous globs of bacteria
in the mouse deep dermis are ALL DEAD is ratified by All RED staining
in this Invitrogen Kit method, then the discussion is over.

The Expense which would necessarily follow the Green Staining result even if only focal green staining
were observed
in Dr Bockenstedt's mouse model.... Is the expense to her Credibility
if Green staining results are realized.

She would then have to retract her article, and to issue a series of
press releases attesting to her retraction.

Dr Linda Bockenstedt had no hesitation in issuing press releases to the
Lay press upon the initial publication of her paper.

Fact checking is needed to find where the truth lies.

My letter to the JCI was not published. My letter was intended to be available to
the JCI readership. Nothing private in the letter to the editor of the JCI was
intended nor implied.

Letters to the editor are public documents.
Can you and/or Dr. Sapi or someone else reproduce her experiment without using Dr. Bockenstedt's samples but the equivalent - then run BacLight on it?

Seems that wouldn't be too difficult an experiment to run?

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inmacdonald
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Re: Not so private correspondence

Post by inmacdonald » Wed 19 Dec 2012 13:30

Special Federal research regulations come into the equation regarding
the use of mouse colonies in laboratory research.

Dr Eva Sapi's laboratory at the University of New Haven is not
set up and is not funded to maintain a mouse colony.

Any other Mouse colony maintaining laboratories are encouraged to
repeat Dr Bockenstedt 's experimental methods. She was using SCID mice strains,
which add an additional cost to repeating the experiment.

She informed me that ALL of the mouse tissue had been exhausted
in her research study and therefore there was no residual tissue available
for any other laboratory to further analyze her experimental results
with a Live Dead Staining protocol.
She also stated that she had checked the nonviabiity of the amorphous Globs
in her mice using an mRNA technique [no experimental details were provided
in her published manuscript and no supplementary Image data were sent to me
to prove that such an mRNA study had actually been performed by her}

Results in the Bockenstedt mouse study indicating any viable borrelia
in her amorphous globs
would mandate that she issue a retraction of her paper
and issue press releases to notify the taxpayers that her published
study was incomplete [no proof of total DEAD borrelia in the Amorphous Globs]
and that her summation conclusions were erroneous.

This denouement would inflict personal, professional, and institutional
embarrassment on the Yale Rheumatology community and call into question
the possibility that other Yale Rheumatology research dating back decades
might also need to be revisited, possibly retracted, and removed from the
peer reviewed literature. A nightmare PUBLIC scenario.
Last edited by inmacdonald on Wed 19 Dec 2012 13:42, edited 1 time in total.

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inmacdonald
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Re: Not so private correspondence

Post by inmacdonald » Wed 19 Dec 2012 13:40

http://www.lymeneteurope.org/forum/view ... f=5&t=4289
The above link:::
A pictorial review from Oct 17,2012 to refresh the image data
and to discuss Dr Linda Bockenstedt's pre-emptive Discussion of Borrelia biofilms
in relation to her Mouse experiments.

Note:
Peer reviewed paper by Sapi, Eva et al had not yet been published in PLOS ONE
as of October 17,2012.

Why did Linda feel that a pre-emptive disclaimer statement on Borrelia biofilms
was necessary in her JCI publication??

Henry
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Re: Not so private correspondence

Post by Henry » Wed 19 Dec 2012 19:17

MacDonald: I have a better idea. Instead of devoting so much angst to disparaging the work of Linda Bockenstedt, why don't to ask Steve Barthold to do the experiment that you suggest. After all, it was HE -- not Linda-- who claimed that these were viable -- but not cultivable-- Borrelia. Or, has he already done the experiment and obtained negative results..............Perhaps such an important experiment ought to be done down in your basement under your direct supervision. You are reminded that there is as yet no evidence to indicate that Borrelia form biofilms in vivo and that they have any clinical relevance whatsoever. Until there is such evidence, your rants in that regard are shear fantasy and ypur "biofilms" are nothing more than laboratory artifacts of interest only to someone with a clear bias in such an interpretation......

X-member
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Re: Not so private correspondence

Post by X-member » Wed 19 Dec 2012 21:44

http://foreignaffairs.house.gov/112/HHR ... 120717.pdf

From the link above (Professor Stephen W Barthold):
Non-cultivable spirochetes resurge at 12 months after antibiotic treatment, with
increased BbDNA copy numbers and widespread dissemination in host tissues.

lou
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Re: Not so private correspondence

Post by lou » Thu 20 Dec 2012 22:02

Got at least one ringer on this thread.

Given the potential impact on a lot of people who have been marginalized by the medical establishment, any honest researcher would have exhausted all possibilities here to prove it one way or another. That was not done. "All of the research material had been exhausted?" Give me a break. What is exhausting is the way that crowd twists themselves into pretzels to avoid coming to an obvious conclusion that would support chronic infection. We are never going to get the truth out of these people.

nnecker
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Re: Not so private correspondence

Post by nnecker » Thu 20 Dec 2012 22:14

And what, Dr Sapi exhausted all the possibilities in her studies,give me a break.

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