0 Blog Log: Spirochetes Unwound on Flawed Study of Topical Antibiotics

Remember that article on topical azithromycin I posted earlier this month?

Our favorite spirochete blogger has some criticism about the research on which it was based here:

http://spirochetesunwound.blogspot.com/2011/09/flawed-study-claiming-prevention-of.html

A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics

Two recent papers tested the effectiveness of topical antibiotics in preventing Borrelia burgdorferi infection in mice following a tick bite. Infection by the Lyme disease spirochete was successfully halted in the Knauer et al. study from Germany1 but not in the Wormser et al. study conducted in New York.2 However a flaw in the Knauer study may have unfairly tipped the outcome in the antbiotic's favor. (I'll save the Wormser study for another post.)

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1 LDA-Columbia Lyme 2011 Conference Line-up

On October 1 and 2, there will be a combined LDA/Columbia-Lyme Conference held in Philadelphia, Pennsylvania.

Below is an outline of the conference speakers and topics...

Keynote Speaker: J. William Costerton, PhD
The Role of Biofilms in Chronic Bacterial Infections

Eva Sapi, PhD
Killing Borrelia: An impossible job?

Jason A. Carlyon, PhD
Interior Decorating: Anaplasma phagocytophilum Remodels Its Host Cell-Derived Vacuole into A Protective Niche

Richard Marconi, PhD, Co-Course Director
c-di-GMP Regulates Key Steps in the Enzootic Cycle of Tick-Borne Spirochetes

Chris Earnhart, PhD
Lyme disease vaccine: an update on recent progress

Dr. Ed Masters Memorial Lecture: Robert S. Lane, PhD
Diversity of Borrelia burgdorferi s.l. genospecies and genotypes in California, and Implications for human infection.

M. Karen Newell Rogers, PhD
A New Model of Intervention for Lyme Disease by Targeting Chronic Inflammation and Selective Aspects of Immune Activation

Robert Yolken, MD
Infections and Human Neuropsychiatric Diseases

Josep Dalmau, MD, PhD
Clinical spectrum and cellular mechanisms of autoimmunity to NMDA and other synaptic receptors

Dr. John Drulle Memorial Lecture: John Aucott, MD
Early microbiologic and immunologic events in Lyme disease

Reinhard K. Straubinger, PhD
Canine and equine Lyme borreliosis – The animals’ perspective of the disease.

Benjamin J. Luft, MD
Diagnostics: update on protein arrays and new Lyme assays

Brian Fallon, MD, Co-Course Director
What is Chronic Lyme Disease? Models and evidence

Andrew W. Walter, MD
Update on Ehrlichiosis and Hemophagocytic Lymphohistiocytosis in Children

Andrea Gaito, MD
Clinical Evaluation and treatment of Lyme Arthritis; An autoimmune perspective

Ingeborg Dziedzic, MD
What everyone should know about Eyes & Lyme Disease

Vijay Thadani, MD
Epilepsy update: Distinguishing Epileptic from Non-epileptic seizures

Steve Bock, MD
Complementary and Integrative Medical approaches to Chronic Tick-borne Disease

Elizabeth Maloney, MD
The treatment of Lyme disease - a critical review of the literature – lessons, gaps, and future research needs

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I think this conference holds more scientific weight to it than other Lyme disease-related conferences I've seen, so if anyone is going to be in Philly and attends, please take notes to share with us all.

(I won't be able to attend as I have a previous commitment scheduled before I knew this conference was going to be held at this time in October.)

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0 Round Three: Lyme Disease Research Scavenger Hunt

I gave readers an extension until midnight of Friday the 23rd for participating on Round Two of the Lyme Disease Research Scavenger Hunt, and so far, no one has completed both Part A and Part B of Round Two.

Because of this, no one qualifies for Round Two and that round is forfeit. Readers playing along at home will have to compete in the remaining rounds and complete both Part A and Part B. So far, we have one winner of Round One, Rita.

(The answers to Round Two will be posted in a separate upcoming entry.)

Now I'll present those who wish to play along with our basic game instructions and round three of the scavenger hunt:

This is an online scavenger hunt to determine which Lyme disease research being conducted in which universities and colleges involves or has involved members of the 2006 Lyme disease guidelines group.

How to play:

I'm going to list Lyme disease related research either completed or currently being done in Column A, and in Column B, list the educational institution where the research was (or is) being conducted.

Match the research in Column A with the correct educational institution in Column B.

THEN determine if members of the department involved are A) currently doing research with a member of the 2006 Lyme disease guideline authors or B) have worked on any research in a past with said guideline author(s).

Write your matches and mentions of any guideline authors in a comment and submit your comment for posting.

You can use google, Wikipedia, and any on and offline tools for your answers.

Roughly two weeks after I post a round, I'll post the correct answers as well as post the next round of the game. I intend to run the game for several weeks - end date to be announced later.

If anyone wins all rounds, after that win is confirmed, the next post I write will be based on the winner's selected topic of choice and include hand-drawn illustrations by me.

Round Three: Lyme Disease Scavenger Hunt

Research Description	Educational Institution
1) Lyme arthritis is an inflammatory disease with periods of inflammation and resolution. Eicosanoids are powerful lipid mediators of inflammatory responses which may be involved in disease processes. Cyclooxygenase-2 (COX-2) is upregulated during injury or infection and catalyzes the production of prostaglandins from arachidonic acid. Several commercially available drugs block this response (celebrex, vioxx) and act to suppress the symptoms of chronic inflammation (pain and swelling), but their effect on underlying disease processes is currently unknown. Treatment of mice infected with B. burgdorferi with these compounds does not inhibit their ability to develop Lyme arthritis, but it does prevent its natural resolution. Current experiments are exploring the mechanism for this examining: altered prostaglandin production; increased leukotriene production; and decreased lipoxin production.	A) University of Tennessee
2) iNKT cells play an important immunoregulatory role within the immune system. This function is regulated by endogenous and exogenous glycolipid antigen presentation by CD1d molecules. Thus, by using Borellia burgdorferi, the agent of Lyme disease, as the model system, we are working to elucidate the molecules involved with and the mechanism(s) of CD1d-antigen assembly. Additionally, by understanding how antigens load onto CD1d we can determine the molecular and structural features of the iNKT cell receptor-antigen interface. Elucidating glycolipid antigen processing and presentation and its recognition by iNKT cells will allow insights into how different responses are induced and how these immunoregulatory T lymphocytes function. This insight will advance our understanding of the physiological role of the CD1d antigen presentation system and iNKT cells within the context of the immune system.	B) University of Missouri
3) One major project is the development of a Reservoir Target Vaccine for the Control of Lyme Borreliosis. Borrelia burgdorferi causes Lyme disease (LD) and is the most common vector borne infectious disease in the United States. This spirochete is maintained in endemic areas of LD by cycling between wildlife reservoirs (i. e. white-footed mice, shrews, etc) and the Ixodes scapularis tick vector. Human disseminated infection can cause permanent damage to the nervous and musculoskeletal systems and currently, there is no vaccine approved for prevention of this disease. A promising method to reduce human LD incidence is to break the mouse-tick transmission cycle by eliminating the spirochete from the reservoir and from the ticks that feed on them. To accomplish this we have developed a wildlife oral bait vaccine based in OspA and are currently testing its efficacy in a field trial.	C) Vanderbilt University

There is much to learn from playing this game in and of itself that you gain something whether you win or lose. (I also have a point to make in playing it, and I'll reveal it at the end of the series... It might not be the point you suspect I'm going to make.)

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1 The Curious Case Of Cholesterol

This article got passed on to me:

Scientists Disarm AIDS Virus’ Attack on Immune System
http://www.voanews.com/english/news/health/-Scientists-Disarm-AIDS-Virus-Attack-on-Immune-System-130313993.html

And you might be looking at that title, wondering what HIV has to do with Lyme disease.

Well, I'm wondering if there is any relationship...

Excerpt:

"Scientists say they have found a way to disarm the AIDS virus in research that could lead to a vaccine. Researchers have discovered that if they eliminate a cholesterol membrane surrounding the virus, HIV cannot disrupt communication among disease-fighting cells and the immune system returns to normal.

Scientists have discovered that HIV needs cholesterol, which it picks up from the first immune cells it infects, to keep the virus' outer membrane fluid. That allows it to communicate with - and disrupt - the body's immune system."

Chlamydia apparently has a membrane with cholesterol in it, as does Borrelia burgdorferi (review this entry posted on Friday). I wonder if Borrelia burgdorferi can do the same thing to the immune system that HIV does? Does it disrupt communication among disease-fighting cells, too? It certainly does evade the immune system even though the immune system gives it a huge response when present.

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2 Speculation About Borrelial Blebs And Camouflage

I've read one abstract and one blog entry that are making me wonder about Borrelia burgdorferi's survival mechanisms - in particular blebbing and camouflage.

In this article on the MicrobiologyBytes blog, the author discusses how certain bacteria use sphingomyelins from their hosts in order to create their own lipids:

"Fifteen years ago, in a series of elegant studies, Hackstadt and colleagues showed that the obligate intracellular bacteria Chlamydia trachomatis save on their lipid needs by incorporating sphingomyelins (SMs) made by their host. Shortly after, Hatch and McClarty’s teams reported that several eukaryotic glycerophospholipids are also trafficked from the host to the bacteria, which replace host-synthesized straight-chain fatty acids by their own branched-chain fatty acids. Even cholesterol, a lipid rarely found in bacteria, was shown to accumulate in Chlamydia. As a result of this intense exploitation of host lipids, the composition of the bacterial membrane is closer to that of a eukaryotic cell than to that of a prokaryote.

Throughout their developmental cycle, chlamydiae reside within a membrane-bounded compartment, the inclusion. How they acquire host lipids remains an open question. Possible mechanisms studied so far involve vesicular trafficking from host compartments, including vesicular traffic out of the Golgi apparatus, fusion with multivesicular body–derived vesicles, and engulfment of lipid droplets."

Rerouting of Host Lipids by Bacteria: Are You CERTain You Need a Vesicle? (2011) PLoS Pathog 7(9): e1002208. doi:10.1371/journal.ppat.1002208

So this got me thinking about Borrelia, too, as Borrelia burgdorferi contains free cholesterol and cholesterol glycolipids. Cholesterol 6-O-acyl-β-D-galactopyranoside and its non-acylated form are significant components of membranes of the spirochete Borrelia burgdorferi.

Then there is this research which was brought to my attention:

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077381/?tool=pubmed

Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-a stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-a responsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30-100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-a stimulation.

Similar to what happens with Mycobacterium tuberculosis, Borrelia burgdorferi engages in blebbing, where vesicles form on the surface of the bacteria and are released. Each vesicle or "bleb" has been shown to contain lipoproteins, and some research has indicated these vesicles are not just an artifact or sign of cellular injury.

Gram-negative bacteria vesicles can contain various virulence factors such as toxins, proteases, adhesins, and lipopolysaccharide, which are utilized to establish a colonization niche, modulate host defense and response, and impair host cell function.

Some bacteria may selectively create vesicles with specific lipoproteins, such as Porphyromonas gingivalis - but in this bacteria, lipopolysaccarides (LPS) are thought to do the sorting. But Borrelia burgdorferi does not have LPS, so some other mechanism determines which lipoproteins are contained in vesicles.

In earlier research it was shown that not only do Borrelia burgdorferi create vesicles which contain outer membrane proteins and lipids - but they also have been shown to contain DNase I-resistant plasmid DNa - suggesting they were at least partially derived from the inner membrane.

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So, now that I've pointed out these things, what am I wondering?

I'm wondering:

If like Chlamydia trachomatis, Borrelia burgdorferi also has a membrane that is more like a eukaryotic membrane? If so, can this contribute to its stealthy nature?

How much Borrelia burgdorferi really relies on its host for its own needs? Compared to other bacteria, Bb has a relatively small genome and is an obligate mostly-extracellular parasite, which means it needs to take what it needs from its host in order to survive. How does it build these lipid rafts and attach to endothelial cells?

If Borrelia burgdorferi creates vesicles or these blebs which contain a mix of outer surface lipids and proteins as well as some inner membrane components, is it possible that it leaves a trail of antigenic material behind it and moves into new areas undetected because its membrane does not produce the same immunological response that blebs or vesicles do?

Somewhat Related Trivia: Chikungunya virus can hide from the immune system inside apoptotic blebs. You think something is now innocuous and on its way out? Think again - these blebs carry the virus through the blood stream to other cells. This virus also manages to replicate inside macrophages while not causing any inflammatory effect. This is a reminder that Borrelia burgdorferi is not the only pathogen that does interesting and stealthy things!

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0 Failure of Topical Antibiotics to Prevent Disseminated Borrelia burgdorferi Infection

Last week, I posted an article on how topical azithromycin could help prevent Lyme disease infection. And now, this new abstract has turned up on PubMed...

Failure of Topical Antibiotics to Prevent Disseminated Borrelia burgdorferi Infection Following a Tick Bite in C3H/HeJ Mice. Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, Pavia CS. J Infect Dis. 2011 Sep 19. [Epub ahead of print]

Abstract

A prior study in mice has shown that the timely application of topical antibiotics to the skin at the tick bite site could eradicate Borrelia burgdorferi infection. That study, however, did not evaluate antibiotic preparations that are considered suitable for use in humans. In this murine study, topical application of 2% erythromycin and 3% tetracycline preparations that are acceptable for use in humans was found to be ineffective in eliminating B. burgdorferi from the tick bite site or in preventing dissemination to other tissues. Reasons for the discrepant findings are discussed.

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I find it very odd that they would choose erythromycin as a topical antibiotic for Borrelia burgdorferi - there is research that shows that Borrelia burgdorferi is resistant to erythromycin. I would have never thought to make that a choice for treatment. Tetracycline makes more sense.

How many previous studies of topical antibiotics for treating early Lyme disease have there been, and what have the outcomes been? How many have been done using suitable human antibiotic preparations versus ones only effective in animal subjects?

It seems to me that the researchers who filed this patent might already have a good idea of what might work - is this the same formulation that was used in the study I posted last week?

Composition C
Composition [mg]
Component per unit [1 g]
Azithromycin 150
Dermacryl 79 50
Klucel MF 25
Miglyol 812 50
Ethanol 94% (w/w) ad 1 g

Or is it different?

Results: There is no infection of Lyme disease detectable when composition C is applied to the area where the ticks were allowed to feed. The topical application of Formulation C results in antibody titers, which are not different from naïve mice (KELA values between 10 and 40), whereas the infected mice show KELA values of 160 to 400.

Especially, the tissue and serum samples taken from different parts of the mice 56 days after the tick bite show no Borrelia burgdorferi organisms when cultivated and no specific antigenes are detectable. Tissue probes are taken from heart tissue, bladder, joint and ear. The group of mice topically treated with Formulation C show complete absence of B. burgdorferi , whereas B. burgdorferi is detected in untreated mice, which are exposed to tick-feed.

The recultivation conditions of B. burgdorferi are suitable to detect B. burgdorferi in any morphological form known to date. Surprisingly, Formulation C was able to eradicate B. burgdorferi in a way, that no infectious agents of B. burgdorferi are detected in the target tissues of B. burgdorferi.

How were these tissue probes completed and could they have missed any bacteria? What about the parenchyma of the brain, since this is low passage N40 they're talking about? (Problematic in rodent studies - should be studied in other animals.) I wonder about the methodology...

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