3 "Time For Lyme" Becomes "Lyme Research Alliance"

Just recently, the Lyme disease advocacy group, Time For Lyme - which is credited with creating a partnership with Columbia University Medical Center and the national Lyme Disease Association to establish and endow a Lyme and Tick-borne Disease Research Center in New York City at Columbia - changed its name to the "Lyme Research Alliance".

The name change came about as an effort to reflect the organization's growing emphasis on supporting more research into chronic Lyme disease and how to improve serological testing and treatment.

See this page to view a list of their currently funded research projects:

http://www.lymeresearchalliance.org/research_projects.html

Camp Other blog has written about a few Lyme Research Alliance funded projects such as:

Dr. Karen Newell Rogers' proposed chronic Lyme disease
treatment, VGV-L:
Viral Genetics' VGV-L Candidate For Treating Chronic Lyme Disease
Notes Posted On VGV-L 

Dr. Armin Alaedini's research on antibody response in chronic Lyme disease/PLDS patients:
Antibodies linked to long-term Lyme symptoms 

Dr. Steven Schutzer's research on biomarkers for late stage Lyme disease/PLDS:
Spinal Fluid Proteins Distinguish Lyme Disease From Chronic Fatigue Syndrome

I've checked out the Lyme Disease Alliance's new web site, and I approve of their overall mission and think it was fantastic they gave $3 million to fund the Columbia Lyme and Tick-borne Disease Research Center.

One thing I'm hoping to see with the change in name and greater commitment to research is a shift in the kind of accomplishments they had in the past, where videos, education packets, and legislation has been a focus - to a focus in assessing what kind of research projects would settle the Lyme disease controversy as well as focus on backing research on more novel treatment methods, such as they have done with VGV-L.

It is my dream that someday all the different Lyme disease organizations will unite for a single effort and come up with one large scale, fully funded Lyme disease research project that will help thousands of patients. I'd like to see focus on that project alone for a solid two or three years and see how far we can get in our understanding of Lyme disease.

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7 Did Isabel Diterich Have The Cure For Chronic Lyme Disease?

One researcher whose papers I've been reading recently is Isabel Diterich's. Several years ago she published two papers on Lyme disease which grabbed my attention because they not only revealed a hypothesis of immunosuppression caused by Borrelia burgdorferi spirochetes - but they also revealed a potential cure for chronic Lyme disease.

I say "potential" here with this caveat:

While the treatment did appear turn a man who was disabled into what sounds like the picture of health for at least eight years, he had to take filgrastim for almost two weeks. And filgrastim is an immune modulating drug which can have serious side effects in some people - there have even been a few fatalities.

However, most of the people who have suffered serious side effects from filgrastim were cancer and leukemia patients who already had serious health problems and were at greater risk for being affected by the drug. And most patients - including cancer patients - experience less dramatic effects of fatigue and joint pain from the use of filgrastim - something Lyme disease patients suffer with anyway.

Scary sounding as it is to take a drug which has the risk of serious or even fatal side effects, one has to consider that if better and safer immune modulating drugs could be developed - along with antibiotics - together they might be the cure for chronic Lyme disease.

To quote from Isabel Ditrech's 2003 thesis, "Immunomodulation and new therapeutic strategies in Lyme borreliosis":

"5.3.1 Case report

A 51 year old patient with a history of frequent exposures to tick bites presented with polyarthritis in the fingers and feet. Arthritic destruction of synovial clefts mainly in the metacarpophalangial and in the proximal interphalangial joints of fingers and feet could be demonstrated by X-ray. Low, but clearly positive, serum titers of Borrelia IgG by ELISA and immunoblot (p100 +++) and a negative IgM-ELISA (both MaxPettenkofer-Institute, Munich, Germany) corroborated diagnosis of late stage Borrelia infection.  

A standard two week i.v. treatment with 2 g/day Ceftriaxone (Rocephin,Hoffmann LaRoche, Grenzach-Whylen, Germany) led to transient improvement of symptoms, i.e. subjective decline of arthritis, that lasted for eight weeks. Then, the inflammatory symptoms returned and became progressively worse, indicating that the treatment had probably failed.  

We hypothesized that persistence of Borrelia might be due to a disabled immunocompetence of the patient. Therefore, we tested whether a complete eradication of the pathogen could be achieved by combining immunosupportive treatment with antibiosis. The experimental treatment regimen, applied with the informed consent of the patient, was as follows: First week 2 g Ceftriaxone (Rocephin ) i.v. daily, second week 480 µg s.c. Filgrastim (Neupogen, Amgen, Thousand Oaks, USA) every second day, and third week 2 g Ceftriaxone daily plus 300 µg Filgrastim every second day (Figure 5.1). Neutrophil counts were determined by a Coulter STKS counter (Coulter, Krefeld, Germany)"

So this lays out the background of this individual case report on one patient. What were the results? More quoted from the above thesis:

"5.4.1 Patient case report

The combination therapy of Ceftriaxone plus Filgrastim was well tolerated. Only after the first injection of Filgrastim the patient reported acute but moderate pain in the previously affected joints i.e. the shoulder, fingers and knees. 

Circulating neutrophil counts increased from 1400 to 17000 cells/µl within 24 h after the first Filgrastim injection. Monocyte numbers increased about two-fold, while there was little effect on lymphocytes (Figure 5.2a). The plateau of neutrophil counts at about 17000 cells/µl blood was maintained until one day after the end of treatment.  

The subjective symptoms disappeared during the following six weeks after the treatment. The patient reported that he was able to resume previously abandoned sporting activities including mountain climbing and downhill skiing. Moreover, fine mechanical skills needed for piano playing were restored. 

After three months, the Borrelia IgG titer was negative. The intensity of the immunoblot at this time point was significantly reduced (from +++ to +) and two years later it was negative. Eight years after treatment the patient is still free of arthritic symptoms."

Source:
http://kops.ub.uni-konstanz.de/bitstream/handle/urn:nbn:de:bsz:352-opus-9814/Diss_formated_ENDVERSION.pdf

So it seems like at least for this patient, this method of treatment changed their life so that they could return to all the things they used to do that they loved. I would have liked to know more about this patient and how he is doing today, given it has been years since this study was completed.

And I'd like to know if a similar treatment plan would work for me and everyone else suffering with chronic Lyme disease. To take ceftriaxone and filgrastim for a couple weeks - or something similar, but with fewer side effects - only to be done with this nightmare and get on with my life would be fantastic.

It would mean no more attempts at long term antibiotic treatment and experimentation with alternative medicine. I would just get treatment for three weeks and be done with it... Sounds like a plan to me.

Reflecting on this, over the years there have been anecdotes - stories I've heard passed around Lyme disease support groups - about the occasional chronic Lyme disease patient who went on to discover they had cancer, went through chemotherapy and other supportive treatment for their cancer - only end treatment not only going into remission from cancer  - but saying that they think their chronic Lyme disease is cured, too.

These stories have been around for a while, but I've never personally known anyone who went through this process. It would be great to get a confirmation from their doctors and families that after chemotherapy and supportive treatments, they had a notable and lasting improvement and feel like their old selves again. What if a drug like filgrastim played a role in their recovery?

This isn't the only example of a chronic condition where the cause has been unknown and the symptoms can be debilitating and lead to years of loss of productivity and physical pain... let's consider chronic fatigue syndrome, also known as CFS/ME or CFSIDS.

A study completed last year in Norway showed that rituximab had a profoundly positive effect on people with CFS/ME. In this study, a few people seemed to go into complete remission from their CFS and returned to work and led normal lives. It didn't work for everyone - 40% of study participants did not experience improvement from the drug. It's unknown why. But that it worked so well for the rest of treated patients deserves a closer look because it begins to reveal the mechanisms behind what causes CFS/ME.

While there has been speculation that chronic fatigue syndrome and chronic Lyme disease (CLD) are the same condition, a recent study on the different proteins found in the cerebrospinal fluid (CSF) of both CFS and CLD patients has challenged this notion. At least in terms of objective evidence, the proteins in the CSF of both groups are different. However, what if part of the underlying process behind what causes these conditions is the same?

Quoting the above well-written article from the Phoenix Rising ME web site, let's look at the mechanism behind rituximab and what it does in people with CFS/ME:

"Rituximab is believed to deplete B-cells in two ways; by recruiting other members of the immune system to attack them and by locking on a receptor on the B-cell that tells the cell to kill itself. B-cells are an integral part of the immune response. Until they are activated, B-cells quietly troll the blood, collecting and digesting molecules called antigens that appear to be suspicious. Once they are digested they place bits of them on MHC molecules for T-cells to inspect. If the T-cells decide those molecules came from a pathogen, they turn around and turn the B-cells on – transforming them into antibody producing machines (‘plasma cells’) that can generate from 100s to thousands of antibodies per second.

These antibodies or immunoglobulins are specifically manufactured to attach to a pathogen and physically stop it from locking onto our cells. The antibodies also alert macrophages to come gobble up the pathogen and they turn on other parts of the immune system. B-cells are key players in the immune response but if they go too far; if they get too zealous, they can mistakenly attack our own cells and overactive B-cell activity has been implicated in many auto-immune disorders."

If this sounds familiar to you, then you might have been reading about Viral Genetics' targeted peptide therapy, VGV-L, for treating chronic Lyme disease.

Viral Genetics' patent states the following about treating chronic Lyme disease:

"[0116] It is believed according to the invention that Borrelia burgdorferi also produces a Toll ligand for TLR2. Replacement of the CLIP on the surface of the B cell by treatment with a thymus derived peptide with high affinity for the MHC fingerprint of a particular individual, would result in activation of the important Tregs that can in turn cause reduction in antigen-non-specific B cells. Thus treatment with thymus derived peptides could reactivate specific Tregs and dampen the pathological inflammation that is required for the chronic inflammatory condition characteristic of Lyme Disease. With the appropriate MHC analysis of the subject, a specific thymus derived peptide can be synthesized to treat that subject. Thus individuals with all different types of MHC fingerprints could effectively be treated for Lyme disease."

An easier-to-understand explanation can be found elsewhere - this research report revealed how VGV-L is used to treat HIV. In this instance, just substitute "chronic Lyme Disease" for "HIV" and you can get a picture of what VGV-L does:

"The conventional approach to HIV vaccines, for example, is to develop therapeutic vaccines to stimulate immune system response. The problem with the conventional approach is that the infected cells are camouflaged and not visible to the body’s immune system. The body’s powerful T-cells are unable to seek out and destroy the infected camouflaged cells because they cannot recognize that the cell is infected.

To understand the issue, think of the Klingon space ship on Star Trek that has its cloaking device activated. The U.S.S. Enterprise has no way of knowing where the enemy is in space. The only hope it has in winning the battle is for the Klingon vessel to be de-cloaked and, once revealed, use their ammunition to destroy it. What’s worse in the case of HIV is that while the infected cell is cloaked, it is also effectively setting off an alarm that triggers the immune system to create inflammation. Why is this important? It turns out that this inflammation is critical for allowing the HIV virus to spread to even more cells.

Many other viruses and bacteria also trigger inflammation but, unlike HIV, the inflammation does not necessarily allow or facilitate the spread of the virus or bacteria itself. However, in these cases, the inflammation itself is harmful because it creates a hostile and inflamed environment that provides the necessary components for a potential autoimmune reaction that can cause the immune system to attack and damage one’s own body. Viral believes that diseases such as Lyme Disease, Multiple Sclerosis and others involve this inflammatory mechanism.

To use the Star Trek metaphor, what Dr. Newell Rogers has developed with TPT is a de-cloaking device for the body’s immune system to use in its pursuit of invaders. Through the development and use of computational biology programs and databases, Dr. Newell Rogers and her team have created a way to remove the camouflage that is cloaking the infected cells, flagging them with custom peptides that allow the body’s immune system to seek out and destroy them.

The key discovery of the TPT platform is that a self-peptide (in other words, one that is naturally produced and a healthy part of one’s normally functioning immune system) called ―CLIP2 that was until now thought only to exist primarily inside certain immune system cells, is sometimes displayed on the outside of cells, thus leading to harmful inflammation. Dr. Newell Rogers discovered that the products of some pathogen invaders such as viruses and bacteria, when picked up on the surface of certain immune system cells, sometimes incorrectly cause those cells to display CLIP externally (i.e. ―ectopically).

Normally, when an invader strikes, this process may promote needed inflammation early in infection, but it is quickly controlled when a more specific, immune response takes over, allowing a highly-targeted immune response to be marshaled against the pathogen. However, when CLIP is improperly displayed, displayed for too long or displayed chronically, the immune system is marshaled to promote a broad and unspecified inflammation without the specific targeting, leaving open the possibility that this inflammation actually turns against one’s own cells. Replacing CLIP is the focus of Viral’s Targeted Peptides because it turns off the harmful alarm."

Read more from the source - including about individual MHC genetic profiles here: http://www.viralgenetics.com/investors/press-releases/Research_2.0_Report_Feb1_2011.pdf

They're using Star Trek metaphors to describe this... I think that's pretty geeky. Awesome.

So, it seems that whether there is current infection or not, VGV-L may be one way to effectively treat chronic Lyme disease and lower inflammation due to runaway immune dysregulation. And if infection is currently present, then it looks like VGV-L will trigger T cells that recognize the infection and summon functional B-cells to fight it.

Now, getting back to Isabel... Remember Isabel, the researcher who used filgrastim and ceftriaxone to treat a patient with chronic Lyme disease about a decade ago? Yes, that Isabel.

Well, she wrote another paper, along with Rauter, Kirshning, and Hartung: "Borrelia burgdorferi-Induced Tolerance as a Model of Persistence via Immunosuppression"

The abstract states:

"If left untreated, infection with Borrelia burgdorferi sensu lato may lead to chronic Lyme borreliosis. It is still unknown how this pathogen manages to persist in the host in the presence of competent immune cells. It was recently reported that Borrelia suppresses the host's immune response, thus perhaps preventing the elimination of the pathogen (I. Diterich, L. Härter, D. Hassler, A. Wendel, and T. Hartung, Infect. Immun. 69:687-694, 2001). Here, we further characterize Borrelia-induced immunomodulation in order to develop a model of this anergy. 

We observed that the different Borrelia preparations that we tested, i.e., live, heat-inactivated, and sonicated Borrelia, could desensitize human blood monocytes, as shown by attenuated cytokine release upon restimulation with any of the different preparations. Next, we investigated whether these Borrelia-specific stimuli render monocytes tolerant, i.e. hyporesponsive, towards another Toll-like receptor 2 (TLR2) agonist, such as lipoteichoic acid from gram-positive bacteria, or towards the TLR4 agonist lipopolysaccharide. Cross-tolerance towards all tested stimuli was induced. Furthermore, using primary bone marrow cells from TLR2-deficient mice and from mice with a nonfunctional TLR4 (strain C3H/HeJ), we demonstrated that the TLR2 was required for tolerance induction by Borrelia, and using neutralizing antibodies, we identified interleukin-10 as the key mediator involved."

Source: http://iai.asm.org/content/71/7/3979.full

Where have I heard something like this before? Oh, Dr. Karen Newell Rogers - that's right - she discussed this at a recent Lyme disease research conference:

"[...]Some researchers would argue that chronic inflammation requires the continuous presence of bacteria, whereas others would suggest that continuous presence of bacteria does not always result in inflammation and that exacerbations of chronic symptoms could result from infection with a different organism--or that chronic symptoms could re-cur from unrelated pro-inflammatory events. Potentially reconciling these seemingly conflicting perspectives on the mechanism of Lyme disease may be the effect of Borrelia burgdoreri’s bacterial by-products on Toll Like Receptors, (TLR)-mediated immune activation. 

TLR appear to be the “gate-keepers” of an inflammatory response. Bacteria, including Borrelia, produce products that, by binding to TLRs on the cell surface, promote leukocyte activation, cytokine production, and acute inflammation. In some genetic backgrounds of mice, acute inflammation is sufficient to fight off infection and resolve disease. In other mouse strains, the pathogens, or in this case the bacteria, get past TLR-induced inflammation and remain symptomatically undetectable in cells and tissues (Barthold, etc); Barthold et al. have found that no matter how severe or mild the disease in any of the genetically inbred strains of mice, there was no more inflammatory disease when the bacteria were eliminated."

And where else have I heard about IL-10 production before? Oh, right - Rituximab, and research on gender differences in antibody response to Borrelia burgdorferi...

From the previously mentioned Phoenix Rising ME article:

"While Rituximab is busy destroying B-cells there is also evidence that it may actually be turning on NK cells – which, of course, habitually underperform in CFS. Rituximab also appears to increase production of IL-10 – a key anti-inflammatory cytokine that may be a protective agent in ME/CFS – and reduces levels of the powerful pro-inflammatory cytokine tumor necrosis factor. A review article suggested that Rituximab was able restore Th1/Th2 balance in the immune system. These results suggest Rituximab could be working as an immunodulator helping to re-balance the immune response by turning down the over-activated parts of it and bumping up the under-active ones."

All this ties together quite nicely, it seems, with other research I have listed here - forming a master hypothesis with different pieces. Does it hold up to scrutiny? Tell me - I'd love to hear your ideas.

But here is the master hypothesis, in its infancy:

1) Host genetics play a role in the ability of mice (and possibly people!) in clearing Borrelia burgdorferi infections. See:

http://campother.blogspot.com/2011/08/immune-infection-hla-dr-alleles.html

The host's genetic background in developing chronic infection is, however, open to debate - and may not play as big a role in disease as Borrelia burgdorferi s.l.'s genetic diversity/VlsE recombination on different plasmids:

http://campother.blogspot.com/2011/08/do-different-genetic-haplotypes-matter.html
http://campother.blogspot.com/2011/08/more-on-genetic-haplotypes-and-lyme.html

2) The genetics of Borrelia burgdorferi strains play a role in how quickly they disseminate into host tissues and also how well they can generate inflammation - which leads to overstimulation of the immune system in production of poor quality plasma b-cells, but also, ironically, immune suppression because of the mechanisms Isabel Diterich and Karen Newell Rogers describe. Refer, also, to Tunev and Barthold et al's research, "Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced Specific B Cell Activation":

http://campother.blogspot.com/2011/06/paper-borrelia-burgdorferi-rst1-ospc.html
http://spirochetesunwound.blogspot.com/2011/07/does-borrelia-burgdorferi-cause.html
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002066
http://spirochetesunwound.blogspot.com/2010/07/antigen-presentation-in-bloodstream-how.html (refer to other research on relationship between b-cells/plasma cells and T cells)

It could also be that not having enough iNKT cells is an issue:
http://www.pnas.org/content/105/50/19863.full.pdf

2a) The changing pattern of antigenic variation during this time may also be why patients produce an undulating immune response in measured antibodies which echo a more drawn-out response similar to relapsing fever:

http://campother.blogspot.com/2012/02/paper-course-of-antibody-response-in.html
http://campother.blogspot.com/2011/08/antibodies-linked-to-long-term-lyme.html (read comments, too)
http://www.ncbi.nlm.nih.gov/pubmed/9108482
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772371/
http://www.ncbi.nlm.nih.gov/pubmed/11544329
http://campother.blogspot.com/2011/07/lyme-disease-western-blots-and-antigen.html

It may not be that the tests are lousy for measuring antibodies which are present to Borrelia burgdorferi. It may be that the antibodies are not present because they are tied up in immune complexes.

2b) There is also the possibility that Borrelia burgdorferi is occasionally intracellular in nature, though there is not enough in vivo evidence to support this. If so, it would also explain why an undulatory immune response might be present:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067508/?tool=pubmed
http://campother.blogspot.com/2011/07/fibroblasts-and-lyme-disease-sample.html

Whether or not items #2a and #2b are relevant here remains to be seen - the main point is that Borrelia burgdorferi can lead to both overstimulation of the immune system as well as immune suppression.

Based on this, I surmise that may not be that blood tests are so lousy at detecting antibodies produced by the presence Borrelia burgdorferi. It may be that there is no reliable way to detect the presence of infection by correlating them with the presence of antibody responses (seronegative Lyme disease).

3) What gender you are and your hormone levels and metabolism may play a role in persisting symptoms and prolonged infection as well, so there is ALSO a metabolic cause behind chronic Lyme disease. How well the immune system can respond to initial infection to begin with seems to play a role in developing chronic Lyme disease, as even 10% of acute cases of Lyme disease result in treatment failure.

http://campother.blogspot.com/2012/03/lyme-disease-presents-differently-in.html
http://campother.blogspot.com/2012/01/two-new-hypotheses-for-chronic-lyme.html (read comments, too)
http://www.ncbi.nlm.nih.gov/pubmed/17438273 (this may provide the scientific link for the anecdotes that people who develop chronic Lyme disease generally were under more stress when they contracted the disease)

4) If there are persister cells, this is an additional consideration - throwing more antibiotics at a pathogen which is antibiotic tolerant when it is a persister cell will, at most, keep the infection from getting worse but it won't eliminate it.

http://campother.blogspot.com/2012/01/paper-persistence-of-borrelia.html
http://campother.blogspot.com/2012/02/blog-log-spirochetes-unwound-on.html

See also:
The research of Kim Lewis on persister cells: www.bu.edu/abl/files/killing_persisters.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145328/

And it may be that persister cells are more likely to be on the scene earlier, depending on how appropriate a given antibiotic is for treating specific genospecies - refer to item #2 above, but also:

http://campother.blogspot.com/2011/05/abstract-evaluation-of-in-vitro.html

5) Because the host has a sub-optimal immune system, even with long term antibiotics, a subset of the population will have trouble clearing the remaining spirochetes after antibiotics are stopped. Additional antibiotics plus a treatment which eliminates low quality plasma b-cells and promotes the activity of Treg cells which recognize current infection could overturn the dysregulated immune system.

What does this boil down to?

Easy: The argument of "is it a chronic infection or is it an immune disorder, possibly autoimmune" is a false dichotomy and too simplistic.

The circumstances which give rise to chronic Lyme disease are more complex than that, and if people want to solve the chronic Lyme problem, they have to roll up their sleeves and look at more puzzle pieces and how they fit together.

Image credit: 
Original image by Muns on Wikimedia Commons; derived image above by Schlurcher.

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11 Why Aren't Persisting Spirochetes Enough Evidence Of Infection?

On the heels of Embers et al having published their statement on PLoSONE, a number of patients are already questioning its content.

Some are claiming that Embers et al statement about how their findings should not be used to oppose current IDSA treatment guidelines for Lyme disease is something they were asked to write - rather than something the authors included on their own.

I don't know. For this claim - whether it's true or not - I have no evidence. However, one thing I do know is that there are solid scientific reasons which back the need for more research on spirochetes which survive after prolonged antibiotic treatment.

The question, of course, which weighs heavily on every patient's mind has been this one:

Why aren't persisting spirochetes enough evidence of infection?

It's become a political hot button question, and it's a scientific question. But most people think that as long as the spirochetes Embers found are alive and metabolically active, that is enough evidence to state that yes, Lyme disease is a chronic infection - let's stop all this nonsense right now and change the treatment guidelines!

Given my own experience and how longer than standard treatment helped me improve, I totally get this. I've been there, done that - and I think that a standard course of antibiotic treatment does not work for everyone. Particularly if there is a delay in proper diagnosis and treatment. Particularly if a coinfection is present. Particularly if there is some abnormality in one's immune system.

But if you are a scientist and you are researching this phenomenon of persistence - whether you as a scientist suspect these spirochetes can cause persisting infection or not; whether the above claim by other patients is true or not - you will be called upon by other scientists to support your findings.

It isn't just going to be the IDSA or the ALDF or other organizations which deny the possibility of persistent infection as a cause of chronic Lyme disease which are going to want to know the outcome of your study.

It's going to be the American Society for Microbiology (ASM) that wants to know the outcome. It's going to be researchers in Europe like the Brorsons who study the "cyst" form of Borrelia burgdorferi and want confirmation of their own findings about persistence.

It's also going to be universities and health departments and many different organizations which may not have any particular position on whether or not Lyme disease can be chronic who will want to know the outcome of your study.

They're all going to want to know the outcome of a study such as Embers et al, so these researchers must be certain about what they found and its significance, and conduct additional research related to their findings in order to confirm them.

They must find evidence that no one can argue against - even the most skeptical - if they are to support their own hypotheses. And it may be that at this stage they genuinely do not know what to make of these persistent spirochetes and not only their ability to cause disease - but how they cause it.

I can easily imagine that Embers et al is being very cautious about the interpretation of their results and wanting further studies as easily as it is for other people to imagine that Embers at al were somehow instructed to downplay the significance of their spirochetes surviving antibiotic treatment.

Why do I say this? I say this because I have learned a few things about these stealthy bacteria and think there is good reason for Embers et al to be cautious about the interpretation and approaching their results either way.

Borrelia burgdorferi spirochetes, plasmids, and infectivity

After doing some research on this issue, the issue of whether or not these spirochetes were infectious and pathogenic or not is a more complex issue than it at first appears.

First, here's a refresher of some basic microbiological definitions. (Bear with me, I'll try to get through this part quickly.)

Infection = the replication of organisms in the tissue of a host; when defined in terms of infection, disease is overt clinical manifestation. In an inapparent or subclinical infection, an immune response can occur without overt clinical disease.

Colonization = A carrier (colonized individual) is a person in whom organisms are present and may be multiplying, but who shows no clinical response to their presence.

Pathogenicity = The pathogenicity of an agent is its ability to cause disease; pathogenicity is further characterized by describing the organism's virulence and invasiveness.

Virulence = refers to the severity of infection, which can be expressed by describing the morbidity (incidence of disease) and mortality (death rate) of the infection.

Invasiveness = invasiveness of an organism refers to its ability to invade tissue.

Now that we're past these definitions, I'll cut to the chase and say there are two important things to know upfront:

1) During in vitro passage or certain stressors, Borrelia burgdorferi can lose some of their plasmids. How soon this happens varies depending on the strain and particular isolates of Borrelia. 

2) When Borrelia burgdorferi loses specific plasmids with specific genes on them, it can lose infectivity and pathogenicity. It should be noted that specific genes for specific purposes can show up on different plasmids on different strains. (For example: Bb strain N40's VlsE locus is different from the one found on commonly studied B31, and it shows up on a different plasmid than on B31.) 

The essential bit of information here is that the loss of a particular gene or set of genes can affect spirochetes' ability to cause infection - and even if these genes are lost, spirochetes may still survive for a while. They can become attentuated or less infectious.

Numerous studies on Borrelia burgdorferi's plasmids have shown that lp28-1 is a linear plasmid which makes Borrelia burgdorferi infectious. VlsE genes found on lp28-1 are thought to be essential for mammalian infection with Borrelia burgdorferi.

When the lp28-1 and yet a different plasmid, lp25, are missing from spirochetes, they are unable to infect mice. The lack of lp25 completely abolishes infectivity since this plasmid encodes a gene (bbe22) which is essential for Borrelia burgdoferi's survival in mice.

Spirochetes which lose lp28-1 plasmids will still live for a while - but the immune system tends to mop them up in a few weeks without antibiotic usage.

Specific research on mutant spirochetes with a lack of the lp28-1 plasmid has shown the following:

"While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1− mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1− mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1− mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis."

To translate the above:

Normal Bb spirochetes infected C3H/HeN (mice which are specifically bred for the ability to demonstrate joint swelling and arthritic symptoms similar to those found in the average person who gets Lyme disease) mice and these spirochetes could not be cleared by the immune system despite the fact that these mice had a strong humoral response.

However, mutant Bb spirochetes which did not contain linear plasmid 28-1 were completely cleared by these C3H/HeN mice.

What's fascinating about this study is even though the mutant Bb spirochetes lacked lp28-1, these spirochetes could still disseminate. Only in severely compromised immune deficient mice (scid mice) could the spirochetes both disseminate and persist - acquired immunity must be functional in animals infected with such mutants in order to clear the spirochetes.

So, here is one example of how you can have spirochetes which are alive and metabolically active and  can even disseminate - yet they are no longer causing disease. In this instance, they were cleared by the immunocompetent mice without the use of any antibiotics within a mere 18 days. (I wish I were that lucky!)

More recently, other plasmids have been found to contribute to infectivity in mammalian hosts - such as lp36. lp36 is viewed as being another major contributor to persistent infection in mice, and spirochetes become attenuated when lp36 is removed.

Linear plasmid 28-1 and lp25 have a much longer history of their role in infectivity and pathogenicity, and they are two of the most studied linear plasmids thus far - lp28-1 the most because of its VlsE genes in strain B31.

So, keep this in mind when you think of the Embers et al study, and realize why this part of their paper on Rhesus macaques caught my attention:

"A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after  > 9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals. We also hypothesized that persistent spirochetes may lose linear plasmid 28-1 (lp28-1), which encodes the VlsE antigen bound by the anti-C6 antibody. Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D).

In the case of Embers et al study on Rhesus macaques, one antibiotic treated animal was found to have evidence of transcription of a lp28-1 gene (bbf26 - protein; purpose unknown) from a sample taken from heart tissue (Fig. 6D) and that transcription should only be able to occur if the lp28-1 plasmid is intact and functional. lp28-1 is a linear plasmid which is very specific to infection both in vitro and in vivo, whether a tick or needle inoculation is used.

In Embers study, in addition to transcription of a gene from lp28-1, OspA transcription from lp54 was found in three treated animals. OspA transcription was detected in two tissue samples taken from the bladder and one tissue sample taken from the spleen. Additional OspA transcription was found in different organs in two out of three of the same animals using organ tissue culture pellets.

Overall, this sounds interesting and points to the possibility of chronic infection after antibiotic treatment.

 But if I have seemed cautiously optimistic about this study, it's because of a few factors*:

1) Only one treated animal had evidence of a infection where lp28-1 transcription was taking place - had more treated animals shown evidence of transcription on this plasmid, I would have been more excited. How long could spirochetes maintain these plasmids while being treated? What about lp25?

2) It is unknown to me if the genetic background and/or immune system of the treated Rhesus macaques somehow played a role in their inability to clear the spirochetes which remain after antibiotic treatment. (Refer to this post on HLA-DR types, read what's before and after the "=" signs, and you'll see what I mean.)

3) it is unknown to me how different the results would be if the Rhesus macaques had been infected using ticks instead of needle inoculations. It seems to make sense to me to do this study again using ticks because that mimics what happens in nature.

On the other hand, I find it very interesting that three animals showed evidence of transcription of OspA. Given how much inflammation people experience during Lyme disease - plus evidence of later stage antibody reactivity to OspA - it at least gives me pause to think about how often OspA has been a culprit for my own symptoms, directly or indirectly.

The only kind of spirochete
you don't mind getting close to.

So it's a mixed bag how I look at the results of the Embers Rhesus macaque study. I think it's a positive step in the right direction establishing what happens with spirochetes in their host after antibiotic treatment. And yet the unanswered questions for me seem related to the same unanswered questions the researchers themselves wrote in their paper.


Is There Anything Positive To Glean From Dr. Baker?


Of Dr. Baker's two major stated issues with the Embers study, the only one now left is whether or not the spirochetes which were transmitted by ticks to new hosts (xenodiagnosis) were in fact infectious. His other concern was over the use of ceftiofur in the study rather than ceftriaxone - however, the authors of the study have since posted a correction to PLoSONE stating that ceftriaxone - not ceftiofur - was used throughout the entire study.

If there are any remaining minor issues he has with the study, he has yet to share them on the Lyme Policy Wonk blog. Mostly, he seemed to reiterate his concern about these two issues and focused on the single mention of ceftiofur in the paper repeatedly.

About the most positive response I heard from Dr. Baker on that blog thus far was about his view of how Lyme disease research should be conducted:

"...I favor a multi-disciplinary approach that moves the field in a different direction, rather than solutions based on the assumed yet to be proved existence of a persistent infection that can only be cured by antibiotics. I don’t really discount such a view; rather, I feel we are neglecting other possibilities that may provide the answers we all are looking for. A case in point, would be the recent work of good friend, Armin Alaedini — who I helped support when I was at the NIH– using specimens collected by Mark Klempner as part of his clinical trial. These valuable specimens are being maintained by Mark in a specimen repository for use in just such cutting-edge research. They are available free of charge on request."

Like Pamela Weintraub, I agree that a multi-disciplinary approach to research on Lyme disease is important. And while Dr. Baker also supports a multi-disciplinary approach to research on Lyme disease and he states he doesn't discount the view of persistent infection in the above paragraph - his direct responses to patients suffering with CLD/PTLDS state that most patients are suffering from some other non-Lyme disease related condition - something I find particularly unhelpful to my situation. That and a lack of sufficient research on other treatment approaches has been an issue for ages.

In my opinion, Dr. Baker's response to the Embers Rhesus macaque study was more negative than it warranted. I wouldn't have viewed it negatively at all - I see it as a stepping stone in getting a better understanding about Lyme disease.  And just because it leaves unanswered questions does not mean it was inherently flawed - which was what Dr. Baker seemed to suggest.

To quote someone else on that blog:

"My question to Dr. Baker is why don’t you and your colleagues offer some expert advice, according to your best opinions and hunches if science really has proven inadequate for your epistemic standards of validity, without having to officially disclose any sensitive data that might get you in trouble with your career, that could actually HELP these affected people lessen their pain and disability? Just disparaging some controversial or technically flawed research as being invalid does not seem helpful enough to me."

Yes. This.

Regardless of anyone's opinion - Dr. Baker, or LLMDs, or my friends and family - researchers will be expected to provide evidence to the world that these remaining spirochetes are pathogenic. They will need to provide evidence that that they can cause infection and reproduce - even if they are already proven to be alive.

Researchers who are trying to work without bias will want to cover all the bases and check their postulates twice to be 100% certain that Borrelia burgdorferi either causes a chronic infection or it does not after standard antibiotic treatment.

This may be so - but I'm impatient about it.


References:

The Absence of Linear Plasmid 25 or 28-1 of Borrelia burgdorferi Dramatically Alters the Kinetics of Experimental Infection via Distinct Mechanisms. Maria Labandeira-Rey, J. Seshu, and Jonathan T. Skare. Infect Immun. 2003 August; 71(8): 4608–4613. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC166013/

Correlation between plasmid content and infectivity in Borrelia burgdorferi. Purser JE, Norris SJ. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13865-70. http://www.ncbi.nlm.nih.gov/pubmed/11106398

High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Norris, SJ, Howell, JK, Garza, SA, Ferdows, MS, and Barbour, AG. Infect. Immun. 63:2206-2212.

Plasmid Stability during In Vitro Propagation of Borrelia burgdorferi Assessed at a Clonal Level. Dorothee Grimm, Abdallah F. Elias, Kit Tilly and Patricia A. Rosa. Infect. Immun. June 2003 vol. 71 no. 6 3138-3145 http://iai.asm.org/content/71/6/3138.full

Experimental assessment of the roles of linear plasmids lp25 and lp28-1 of Borrelia burgdorferi throughout the infectious cycle. Grimm D, Eggers CH, Caimano MJ, Tilly K, Stewart PE, Elias AF, Radolf JD, Rosa PA. Infect Immun. 2004 Oct;72(10):5938-46. http://www.ncbi.nlm.nih.gov/pubmed/15385497

The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mollie W Jewett, Kevin Lawrence, Aaron C Bestor, Kit Tilly, Dorothee Grimm, Pamela Shaw, Mark VanRaden, Frank Gherardini, and Patricia A Rosa. Mol Microbiol. 2007 June 1; 64(5): 1358–1374. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1974800/?tool=pubmed

Basic Epidemiology. Beaglehole R, Bonita R, Kjellstrom T. World Health Organization, Geneva, Switzerland, 1993

* Factors which concern others but I did not originally think of are included in comments below.

[Edited March 9, 2012 - Removed item above about brain tissue after reviewing Embers paper again - multiple brain samples were taken; one treated animal was positive for B. burgdorferi RNA in both heart and brain.]

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0 Comments On 2011 Lyme and TBD Conference Summaries

I have a few comments to make as they come up on October's 2011 conference.

I may add and repost to this entry a few times - stay tuned.

1) I looked at this part of the summary on Dr. Reinhard K. Straubinger's talk on “Canine and Equine Lyme Borreliosis” focused on Lyme borreliosis in animals, especially in dogs and horses:

"The highly variable surface protein VlsE is, according to current knowledge, exclusively expressed in the mammalian host. The invariable region IR6, and even a shorter peptide sequence of IR6 called C6 were found having a high potential as specific antigenic components in serologic test systems. This was shown by evaluating sera from infected humans, dogs, monkeys and mice. In experimentally infected dogs, C6-specific IgG antibodies appeared 3 weeks post infection; hence almost one week earlier than antibodies detected with ELISA based whole-cell preparations. Additionally, another benefit became clear when testing sera of people and dogs before and after antibiotic treatment. Contrary to antibodies against whole-cell components, research demonstrates that C6-antibodies declined substantially a few months after treatment. However, in animals with low C6-antibody levels prior to treatment, the decline obviously was minimal post treatment. Despite their high specificity for borrelial contact, C6-antibodies do not necessarily correlate with clinical signs in dogs and false-positive results may result from maternal antibodies in puppies born to infected bitches."

It brought to mind the following paper and a passage within it:

Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced Specific B Cell Activation. Stefan S. Tunev, Christine J. Hastey, Emir Hodzic, Sunlian Feng, Stephen W. Barthold, Nicole Baumgarth.

Source: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002066

"We did not include the VlsE protein in our studies, a surface-protein thought to subvert the immune response to B. burgdorferi through extensive genetic variation within the host. However, the N40 strain of B. burgdorferi, which we have used here, does not seem to express this protein, based on transcriptional analysis of the IR6 region of vlsE. Moreover, we found no evidence of seroconversion to the C6 antigen of vlsE from strain B31 (S. W. Barthold, unpublished). Recent sequence analysis of the N40 genome has confirmed that N40 vlsE and BBK01 are on different plasmids and that the vlsE locus is indeed significantly different compared to B31, the commonly used VlsE-expressing Borrelia-strain."

So what do we know about C6 tests, briefly?

C6 Lyme ELISAs are based on the synthetic peptide C6, which corresponds to the invariable region IR6 of the surface antigen VLsE.

IR6 is supposed to be highly preserved in all pathogenic strains of Borrelia, and is only expressed in vivo. It evokes a strong immune response.

As a diagnostic antigen, the C6 peptide shows only minor cross-reactivity to other pathogens (e.g. spirochetes for syphilis).

Knowing this, it's interesting to point out that using the C6 ELISA may not work as well for testing a host which is infected with a strain of N40 Borrelia. How can it, when this strain of N40 does not express the vlsE protein?

I have to wonder how many other strains may have vlsE on different plasmids - and not only that - a different locus?

2) Dr. John Aucott’s talk on “Early Lyme disease” reported from the SLICE prospective cohort and his Maryland studies. I want to point out this part of the summary:

"He emphasized that the classic description of a “bull’s eye rash” occurs only 20% of the time – it is not the most common manifestation of the Lyme rash. Rather, a uniformly red or reddish-blue rash, round or oval in shape, with sharply demarcated borders is most common. Most often the rash develops in places such as the knee, groin, or arm pit, occurring at prime tick season, such as the late spring and early summer."

It's important to know that these varied descriptions of so-called "bull's eye" or EM rashes found in Lyme disease are not the only variations found in Lyme disease culture positive lesions.

In an earlier entry about Dr. Vijay Sikand's testimony at an FDA Lymerix Vaccine Review, Dr. Vikand reported this about EM rashes:

"[...] However, erythema migrans is not a single beast. Certainly this is the one which we easily recognize and which I just referred to.Before I continue with further slides, let me point out that the erythema migrans lesions you are about to see are all biopsy lesions which were laboratory proven to be caused by Borrelia burgdorferi.

Sometimes erythema migrans can present as a pustular lesion as is this one in the popliteal fossa inviting the scalpel of a surgeon.

Sometimes the lesions are vesicular in nature, inviting a diagnosis perhaps of herpes simplex infection.

Sometimes our round lesion is actually triangular.

Sometimes it doesn't even look round or red at all and invites a diagnosis of an intertriginous fungal infection in the groin of this patient who was biopsied and proven to have Lyme disease.

Sometimes the lesion is more plaque-like, inviting diagnosis of nummular eczema, psoriasis, or other similar lesions.

Sometimes it is in unusual locations.

Sometimes it is large like this one. Sometimes it is small with satellite areas. Sometimes it is multiple, appearing almost like urticaria or erythema multiform.

Sometimes, as in this individual who was a placebo recipient in the Lyme 008 SmithKline Beecham trial, it presents with other manifestations of early dissemination. This individual came in mainly because he was concerned about his face and it felt kind of funny and it was weak on one side. When I asked him whether he had had any unusual rashes, he said oh do you mean this one, and he showed me his arm with that EM. This is simply to illustrate the infranuclar 7th nerve palsy with which he presented. This patient, by the way, had no history of a tick bite or any unusual antecedent illness which he could remember."

If Sikand's testimony is anything to go on, one has to wonder about the reliability of the "bull's eye rash" as a diagnostic marker.

(For what it's worth, my own EM rash was large, oval, dark red, slightly elevated, and expanding for several days after the bite.)

3) Dr. Karen Newell Rogers presented a talk about novel ways to target chronic inflammation and chronic immune activation among patients with chronic Lyme disease.

Now this part below - especially in bold - really caught my eye:

"[...]Some researchers would argue that chronic inflammation requires the continuous presence of bacteria, whereas others would suggest that continuous presence of bacteria does not always result in inflammation and that exacerbations of chronic symptoms could result from infection with a different organism--or that chronic symptoms could re-cur from unrelated pro-inflammatory events. Potentially reconciling these seemingly conflicting perspectives on the mechanism of Lyme disease may be the effect of Borrelia burgdoreri’s bacterial by-products on Toll Like Receptors, (TLR)-mediated immune activation. TLR appear to be the “gate-keepers” of an inflammatory response. Bacteria, including Borrelia, produce products that, by binding to TLRs on the cell surface, promote leukocyte activation, cytokine production, and acute inflammation. In some genetic backgrounds of mice, acute inflammation is sufficient to fight off infection and resolve disease. In other mouse strains, the pathogens, or in this case the bacteria, get past TLR-induced inflammation and remain symptomatically undetectable in cells and tissues (Barthold, etc); Barthold et al. have found that no matter how severe or mild the disease in any of the genetically inbred strains of mice, there was no more inflammatory disease when the bacteria were eliminated."

Reading this, I reflect back on an earlier entry I made about the combination of genetics effect on individual immune systems AND persistent infection as both leading to ongoing symptoms in hosts:

Immune + Infection = HLA-DR alleles determine responsiveness to Borrelia burgdoferi:
http://campother.blogspot.com/2011/08/immune-infection-hla-dr-alleles.html

Perhaps part of the answer to what is happening with ongoing symptoms lies both in Dr. Newell Rogers' work and Bettina Panagiota Iliopoulou, Mireia Guerau-de-Arellano, and Brigitte T. Huber's research?

It's certainly thought-provoking.

And Barthold's statement implies that ongoing inflammation is intimately tied to persisting infection.

How does one provide evidence this is the case?

Could the answer lie in longer term in vivo GFP and/or iRFP studies on mice and other mammals? Could it lie in maltodextrin enhanced imaging studies? Or something else?

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12 Antibodies linked to long-term Lyme symptoms

Nature has just published an article today on research which shows a relationship between post-Lyme disease syndrome and persistent infection.

Armin Alaedini at Weill Cornell Medical College in New York and his colleagues have found that patients diagnosed with post-Lyme disease syndrome have antibodies that suggest they carried the infection for an unusually long time. The finding, published in Clinical Immunology, might help the syndrome to be better understood, diagnosed and treated.

Bockenstedt is quoted in the article, saying, "This is the first study I've seen that shows some immunologic difference between someone who resolves their Lyme and someone who develops post-Lyme disease syndrome."

I highly recommend everyone checks it out, supporters and naysayers alike: The finding suggests that patients with chronic symptoms have experienced a prolonged infection.

Original Research:
Epitope mapping of antibodies to VlsE protein of Borrelia burgdorferi in post-Lyme disease syndrome. Abhishek Chandra, Norman Latov, Gary P. Wormser, Adriana R. Marques, and Armin Alaedini. Clin. Immunol. http://dx.doi.org/10.1016/j.clim.2011.06.005 (2011)

Comments:

I'm withholding any further comments on the content of the paper until I read the full text, but am open to discussing the contents and context of the article from Nature in comments.

READ MORE: http://www.nature.com/news/2011/110805/full/news.2011.463.html

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13 Books: Borrelia. Plus a lesson in terminology.

Borrelia: Molecular Biology, Host Interaction and Pathogenesis. If you have GB £159 or US $310 on hand, and you want to know the state of the science on Borrelia including Borrelia burgdorferi, afzelii, garinii, and its relapsing fever relatives - this is a good book to get.

If you would prefer to review the book before purchasing - or don't want to purchase it at all - check out your local library's interlibrary loan program.


See a detailed chapter outline at this link: http://www.horizonpress.com/borrelia

I have yet to do a review on this book as it is information dense and takes some time to read through - but I pulled some highlights from the chapter outlines (it is pretty robust for an outline, I have to admit)  and provided some vocabulary translation in which some readers may take an interest:

"B. burgdorferi strain B31, the B. burgdorferi type strain, has been studied in the most detail and harbors twelve linear and nine circular plasmids that comprise about 612 kbp. The plasmids are unusual, as compared to most bacterial plasmids, in that they contain many paralogous sequences, a large number of pseudogenes and, in some cases, essential genes. In addition, a number of the plasmids have features suggesting that they are prophages. Some correlations between genome content and pathogenicity have been deduced and comparative whole genome analyses promise future progress in this arena. [CO comment: Refer to Ben Luft's and Steven Norris' research on Bb strains and their ability to cause infection in human hosts.]"

In general, the whole plasmid thing is just weird. Having plasmids that take up one third of your genome is interesting.

Circular plasmids? No problem. You find them in bacteria all the time.

Linear plasmids? That is a bit unusual. Linear plasmids used to be thought of as only in eukaryotic organisms - organisms with a cell nucleus - not prokaryotic organisms like bacteria. Linear plasmids are also found in viruses. But they are found in few bacteria - Borrelia burgdorferi being one of them.

But prophages...  These are very interesting, and what makes some Borrelia strains more harmful to their hosts. That viral genes end up in Borrelia plasmids is pretty fascinating to me, even though this sort of thing happens with other bacteria too - it's fascinating and adds to Borrelia's existing complexity.

Translation for biology/genetics beginners:

(You might want to check this out sometime: http://en.wikipedia.org/wiki/Introduction_to_genetics .)

chromosome = A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences.

plasmid  = Usually described as a double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Three of the plasmids found in all Borrelia burgdorferi isolates have been described as "mini-chromosomes".

Your handy little Bb plasmid cheat sheet:
cp = circular plasmid
lp = linear plasmid
cp26 = needed for viability, encodes OspC.
lp25 and lp28-1 = needed for infectivity in mice; lp28-1 involved in antigenic variation.
cp32 = contains prophage material; encodes a BpaB protein, which appears to play a role in replication and segregation.
lp54 = encodes OspA/B operon and decorin-binding protein operon dpbBA

Other Borrelia plasmids (relapsing fever):
cp32 = described in B. hermsii; does not contain an OspE/F/Elp lipoprotein (unlike Bb)

Plasmids make up about a third of Borrelia burgdorferi's entire genome. (Fraser et al, 1997; Casiens et al, 2000). Linear plasmids are the most genetically diverse part of Bb's genome, and rearrangements and deletions inside them contribute to their uniqueness.

In Bb, there is a division of labor between the chromosome and extrachromosomal elements or “plasmids”. Genes encoded on the chromosome tend to do housekeeping - housekeeping genes are present in single copy - while genes on the plasmids tend to be Borrelia-specific, of unknown function or involved in the infective cycle, and present in multiple, related copies.

Source material:
The Prokaryotes: A Handbook on the Biology of Bacteria (Vol. 7) by Martin Dworkin and Stanley Falkow.
The Linear Hairpin Replicons of Borrelia burgdorferi. Kerri Kobryn. Microbiol Monogr (7) Universite de Sherbooke, Sherbrooke, QC, Canada

genome = the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA.

pseudogene = dysfunctional relatives of known genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell. Also known as mutationally damaged or "Junk DNA" - it is not always necessarily "junk" - its function is simply unknown at this time.

essential gene = genes that are indispensable to support cellular life. These genes constitute a minimal gene set required for a living cell - if they don't work, the organism dies.

paralogous = This one takes a little more explaining...

Homologous gene sequences = the same, where "homo" means "like, similar" and "hetero" means "different, divergent".

For example, in general, if two or more genes have highly similar DNA sequences, it is likely that they are homologous. "Paralogous" means they are homologous gene sequences which are similar but they occupy different positions or locations in the same genome.

Sequence similarity is often seen in organisms that evolved from the same common ancestor. However, sequence similarity may also arise without common ancestry - short sequences may be similar by chance, and sequences may be similar because both were selected to bind to a particular protein, such as a transcription factor. Such sequences are similar but not homologous.

prophage = Okay... This is where things get weird. A prophage is a phage (viral) genome inserted as part of the linear structure of the DNA chromosome of a bacterium. A temperate phage is integrated into the host chromosome or existing as an extrachromosomal plasmid. This is a latent form of a bacteriophage, in which the viral genes are incorporated into the bacterial chromosomes without causing disruption of the bacterial cell.

In other words, a bacteriophage is a virus that lives inside bacteria, and a prophage is viral genetic material that becomes part of the chromosome in the bacteria (see virus to left, injecting its genetic material inside a non-spirochetal bacteria). So, in this case, at some point in time, a phage known as phiBB-1 (also spelled φBB-1) made its viral genes part of Borrelia burgdorferi's plasmids.

In prophages in general, if the host bacteria is damaged, the prophage is excised from the bacterial chromosome in a process called prophage induction. After induction, viral replication begins via the lytic cycle.

Prophages are important agents of horizontal gene transfer, and are considered part of the mobilome.

In many bacterial species, prophages figure prominently in the biology of these cells, often conferring key phenotypes that can convert a non-pathogenic strain into a pathogen. In other words, while it's residing inside the bacteria, the prophage can turn bacteria that is harmless into harmful bacteria.

Such phenotypic changes can include prophage-encoded toxins, bacterial cell surface alterations, or resistance to the human immune system.

Prophage integration into the host genome can inactivate or alter the expression of host genes. In addition to these direct genetic alterations associated with the addition or inactivation of genes, prophages can also alter the phenotype of bacteria at the population level by facilitating the spread of favorable genes through transduction. (transduction = the process by which DNA is transferred from one bacterium to another by a virus.)

Its significance in Borrelia burgdorferi:

It's not discussed much on Lyme disease patient forums, but some strains of Borrelia burgdorferi have had their plasmids invaded by prophages. The presence of those phages - their viral genetic material - can make Borrelia burgdorferi more pathogenic.

phage particles in Borrelia burgdorferi
from Eggers et al

The phiBB-1 prophage is capable of transducing a cp32 between cells of the same isolate and between different Bb isolates. This means this prophage could play a role in the genetic diversity of different Bb isolates.

The structural proteins for phiBB-1 have not yet been identified, and a number of proteins encoded on cp32 have no homologs in any databases outside of Bb.

cp32 expresses a few outer surface or membrane proteins, of which ospE is the only one of which its function is known. It binds to complement factor H and helps Bb evade the complement system of the host.

A number of different bacteriophages have been observed in association with spirochetes and are being researched now. It may contribute to Lyme disease's ability to persist.

Well-known research on this includes this study:
Eggers et al http://www.ncbi.nlm.nih.gov/pubmed/11466280

Source material:
Wikipedia: http://en.wikipedia.org/wiki/Prophage
Wikipedia: http://en.wikipedia.org/wiki/Introduction_to_genetics
Borrelia: Molecular Biology, Host Interaction and Pathogenesis.
The Prokaryotes: A Handbook on the Biology of Bacteria (Vol. 7) by Martin Dworkin and Stanley Falkow.

pathogenicity = the ability of a pathogen to create infectious disease in an organism.

One of the relevant points to be gleaned from the above is that the genetic makeup of the plasmids are related to the virulence and pathogenicity of Borrelia. (virulence = the degree of pathogenicity within a group or species of microorganisms or viruses as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host.)

"The highly unusual segmented genomes of Borrelia species can contain over 20 autonomously replicating DNA molecules. Many of the molecules, including the chromosome, are linear with covalently closed hairpin ends. Current knowledge of the replication and maintenance of DNA molecules will be reviewed, including the process of telomere resolution, whereby the covalently closed hairpin ends are generated from replicative intermediates. Finally, the proposal that reverse telomere resolution is the driving force shaping the ongoing rearrangements and telomere exchanges in the linear replicons of Borrelia species will be discussed."

Translation for biology/genetics beginners:

chromosome = A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences.

nucleotide = molecules that when joined together make up the structural units of RNA and DNA. In addition, nucleotides play central roles in metabolism.

covalently closed hairpin ends = strands of genetic material curve back on itself in a hairpin pattern.

telomere = a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. The telomere shortening mechanism normally limits cells to a fixed number of divisions, so a shorter telomere means there are fewer divisions left for that cell and it will die sooner. Animal studies suggest that this is responsible for aging on the cellular level and sets a limit on lifespans.

telomere resolution = when the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same DNA molecule.

reverse telomere resolution = when linear DNA cleaves (splits) and joins hairpin telomeres on unrelated (rather than related) DNA molecules. The new linked plasmid structure could fuse to another linear plasmid in the future, and this explains how Borrelia can engage in telomere exchanges. This process may play a major role in the development of weird linear plasmids in Borrelia and their ability to confuse the immune system.

replicon = a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication. For most prokaryotic chromosomes, the replicon is the entire chromosome.

This part, I think most readers will know about the last sentence - but suspect they will be less familiar with the first half:

Borrelia are not Gram-positive...  But... they are not exactly Gram-negative bacteria, either.

"Although Borrelia spirochetes are often, but mistakenly described as Gram-negative bacteria due to their diderm, i.e. double-membrane envelopes, a closer examination reveals significant differences in composition and architecture. Probably most striking is the lack of LPS, the presence of major surface lipoproteins at the host-pathogen interface during transmission, persistence and ensuing pathogenic processes and the additional function of periplasmic flagella in defining cell shape. While surface lipoproteins such as the Osps interact with a variety of ligands in different organ tissues, they are also targets of the immune response and several have emerged as vaccine candidates."

I found information confirming the fact that Borrelia is not Gram-negative or Gram-positive. It's its own special thing:

Borrelia were thought to be Gram negative because of their double membrane structure, but genetic analysis places them - along with other spirochetes - into a separate eubacterial phylum. Ultrastructural molecular and biochemical studies have emphasized the wide taxonomic gap between spirochetes and Gram-negative bacteria. ( The Genus Borrelia. Melissa Caimano. Prokaryotes (2006) 7:235-293.)

Translation for biology/genetics beginners:

Gram-negative = Gram-Negative Bacteria are simply called this because of their detection by the Gram’s Stain test in which they do not retain the crystal violet color (dye) in their cell wall. The Gram-Negative bacteria cell-wall holds the pink or reddish dye once a counterstain chemical is used. This is characteristic of bacteria that have a cell wall composed of a thin layer of a particular substance (called peptidoglycan).

diderm = double-membrane envelope consisting of an inner cytoplasmic membrane and outer membrane found in Gram-negative bacteria.

double-membrane envelopes = See the picture to the left. Monodermic bacteria (mono = one) have one membrane, and didermic bacteria have two (di = two) membranes - the inner membrance (cm) and outer membrane (om).


LPS = Lipopolysaccharides (LPS), also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals. [CO comment - a question for my readers: If Borrelia burgdorferi do not have LPS, what is producing the endotoxins everyone in the Lyme patient community says are important to detox? Keep reading, the answer is downstream...]

lipid = Lipids are a broad group of naturally occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, as structural components of cell membranes, and as important signaling molecules. In this context, lipids make up the inner membrane of Borrelia burgdorferi.

polysaccharide = Polysaccharides are polymeric carbohydrate structures, formed of repeating units (either mono- or di-saccharides) joined together by glycosidic bonds. Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin.

lipoproteins = A lipoprotein is a biochemical assembly that contains both proteins and lipids water-bound to the proteins. Many enzymes, transporters, structural proteins, antigens, adhesins and toxins are lipoproteins.

(a) external side view of Borrelia burgdorferi spirochete,
(b)  head-on view of cross-section of Bb, and (c)
side view of cross-section of Bb's diderm membranes.  

periplasmic = The periplasmic space or periplasm is a space between the inner cytoplasmic membrane and external outer membrane of Gram-negative bacteria or the equivalent space outside the inner membrane of Gram-positive bacteria. It may constitute up to 40% of the total cell volume in Gram-negative species, and is drastically smaller in Gram-positive species.

flagella = A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and functions in locomotion. (Flagella is plural of flagellum.)

Osps = Outer surface proteins. The outer membrane of Borrelia burgdorferi is composed of various unique outer surface proteins (Osp) that have been characterized (OspA through OspF). The Osp proteins are lipoproteins anchored by N-terminally-attached fatty acid molecules to the membrane. They are presumed to play a role in virulence, transmission, or survival in the tick.

ligand = Any substance (e.g. hormone, drug, functional group, etc.) that binds specifically and reversibly to another chemical entity to form a larger complex.

"Several borrelial proteins have been implicated in adherence to host cell surface proteins and extracellular matrix components and are likely to be involved in the homing of Borrelia to histologic compartments within each tissue, penetration of blood vessels and adherence to and migration through endothelial cells and tissue strata at distant sites. Activation of plasmin on the bacterial surface and induction of host proteases are thought to facilitate dissemination and/or inflammation. Most tissue damage appears to result from host inflammatory reactions. Although the mechanisms are not entirely understood, induction of cytokine/chemokine expression by bacterial lipoproteins and the resulting recruitment and activation of lymphocytes, macrophages and granulocytes play a major role in both local histopathology and constitutional symptoms. Despite their relatively low densities in tissues, Borrelia cause neurologic, cardiovascular, arthritic and dermatologic manifestations during the disseminated and persistent stages of infection by mechanisms that remain largely a mystery. Immune evasion mechanisms, including the vls antigenic variation system, complement-regulator acquiring surface proteins (CRASPs), down-regulation of highly antigenic surface proteins (such as OspC) and invasion of protective niches, permit the survival of the pathogens for months to years following infection despite robust antibody and cellular responses."

Translation for biology/immunology/genetics beginners:

host cell surface proteins = proteins on the surface of the host's cell.

extracellular matrix = Any material produced by cells and secreted into the surrounding medium, but usually applied to the noncellular portion of animal tissues. In other words - the space around the outside of cells, but not within cells.

histologic = related to cells and tissue on the microscopic level.

endothelial cells = cells that lines the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall.

plasmin = an enzyme present in blood that breaks down many blood plasma proteins, most notably, it breaks down fibrin clots (blood clots).

protease = an enzyme that breaks down proteins.

cytokine = small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication.

The term "cytokine" has been used to refer to the immunomodulating agents, such as interleukins and interferons. Biochemists disagree as to which molecules should be termed cytokines and which hormones. As we learn more about each, anatomic and structural distinctions between the two are fading.

chemokine = are a family of small cytokines, or proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. Chemotaxis is simply the act of cells changing their movement according to certain chemicals in their environment.

lymphocyte = White blood cells. Large lymphocytes include natural killer cells (NK cells). Small lymphocytes consist of T cells and B cells.

macrophage = white blood cells which phagocytose (engulf and then digest) cellular debris and pathogens, either as stationary or as mobile cells. They also stimulate lymphocytes and other immune cells to respond to pathogens.

granulocyte = a category of white blood cells characterized by the presence of granules (meaning "grains") in their cytoplasm (inside the cell membrane). These are thought of as the "phil" brothers of white blood cells: neutrophils, eosinophils, and basophils.

histopathology = the microscopic examination of tissue in order to study the manifestations or signs of disease.

vls = Gene locus in Borrelia burgdorferi which is required for encoding variable surface proteins.

vlsE = VlsE is a lipid-protein conjugate, found on the cell's outer surface during all Borrelia life stages. It is similar to a lipoprotein of the organism that causes African sleeping sickness. Unlike most proteins, VlsE is produced in many forms. It is a complicated protein with several variable regions (VRs), and six invariable regions (IRs).

When synthesizing VlsE, Borrelia periodically replace the VRs with new sequences. This replacement presents fresh surface antigens, and helps Borrelia remain invisible to the immune system. Within four days of being transferred to a mammalian host, VlsE will be produced with more than one VR suite, reducing the strength of the immune response. In ticks, VlsE does not modify the VRs. (Credit to Dr. Albert Burchsted, retired Field Biologist, for this description.)

antigenic variation = the mechanism in which an infectious organism alters its surface proteins in order to evade a host immune response. This change in antigenic profile may occur as the pathogen passes through a host population (also called "antigenic diversity") or may take place in the originally infected host.

The strategy is particularly important for organisms that a.) target long-lived hosts, b.) repeatedly infect a single host, and c.) are easily transmitted. Pathogens that express these characteristics and undergo antigenic variation have a selective advantage over their more genetically stable counterparts.

A number of bacteria use antigenic variation to evade the immune system, but Borrelia burgdorferi's method of antigenic variation is very complex and unusual compared to these.

In the case of Borrelia burgdorferi, the lp28-1 plasmid has been responsible for antigenic variation in vls locus - responsible for vlsE and the changing of Borrelia's outer surface proteins which helps it evade the immune system.

During the past decade, it was thought that removing the lp28-1 plasmid or altering its genetic material would remove Borrelia's infectivity entirely. It reduced it, but removing specific genes near the vlsE locus has done more to reduce Borrelia burgdorferi's infectivity.

The mutation of either of the genes encoding the two subunits of the RuvAB branch migrase blocked transfer of genetic information into vlsE during mouse infections, identifying the first required function for antigenic variation in the Lyme disease spirochete.

But basically, both the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion.

See:
http://www.ncbi.nlm.nih.gov/pubmed/15501789
http://www.ncbi.nlm.nih.gov/pubmed/19266024
http://www.ncbi.nlm.nih.gov/pubmed/19997508

Source material:
The Linear Hairpin Replicons of Borrelia burgdorferi. Kerri Kobryn. Microbiol Monogr (7) Universite de Sherbooke, Sherbrooke, QC, Canada
Borrelia: Molecular Biology, Host Interaction and Pathogenesis.

complement = a small protein which mediates antibody response in the host's body. This is part of a complex immune system known as the complement system.

CRASPS = Borrelia produces complement regulator-acquiring surface proteins (CRASPs) that bind host complement factor H protein. Factor H protein is a negative regulator of the complement cascade (inactivates C3b). Borrelia produces a whole family of CRASP proteins. Different family members are able to interact with different factor H proteins from divergent hosts.

If that sounds too confusing, the thing to remember is that Borrelia has surface proteins which can bind to a host's complement's protein which then inactivates part of the complement system.

down-regulation = the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable. (Up-regulation is the opposite process.)

So, as I've stated before - researchers do know that the infection can be persistent - the question and controversy has been over whether or not infection persists after one has antibiotic treatment. 

It's my opinion so far that it can persist - especially if one has neuroborreliosis and has been undertreated or untreated. Those who have been suffering from neuroborreliosis and reading this will say it definitely can persist, based on their experience. The question remains as to how one can know whether or not the infection has been eradicated. This is what needs to be known. 

Also, there is persisting, and then there is persisting... One of my nightmare scenarios has been that when the IDSA Lyme disease guidelines panel states that Lyme disease does not respond to long-term antibiotic treatments, what it really means that none of the antibiotics which exist today can effectively eradicate all of it and the immune system isn't always capable of mopping it up after antibiotic treatment.

This is why people began experimenting with a number of alternative treatments: in their experience and opinion, antibiotic treatment either failed to eradicate the infection or stop their symptoms - and sometimes the side effects or infection with C. difficile led to the decision to stop antibiotic treatment.

Onward...

Lyme Disease in Humans - from the last chapter in Borrelia:

"Lyme disease is a rapidly emerging tick-borne, complex, multi-system infectious disorder caused by the spirochetal bacterium Borrelia burgdorferi. The ailment, which affects adults and children alike, is widespread in the Northern Hemisphere and it continues to expand as humans encroach on the sylvatic habitat of the spirochete's mammalian reservoirs."

"Since first identified in the 1970s the incidence of Lyme disease has increased more than 30-fold and it is now considered the most prevalent arthropod-transmitted infection in both the United States and Europe."

This is why I say doctors should look at the possibility of ruling it in, more than ruling it out...

"B. burgdorferi is transmitted by ticks of the Ixodes ricinus complex, including I. scapularis, I. ricinus and I. persulcatus. In North America, B. burgdorferi sensu stricto is the only species proven to be pathogenic for humans [CO note: Relapsing fever Borrelia are pathogenic to humans too, and some species of Borrelia have yet to be determined for their pathogenicity in humans.]. In Europe, both B. afzelii and B. garinii are most commonly associated with human disease.

The spirochete's genomic features, as well as its unique molecular architecture, are considered to have a seminal role not only in how it is transmitted from ticks to humans, but also how it triggers immune responses in afflicted individuals. Inflammatory manifestations associated with the disease result from the host's innate and adaptive immune responses to the bacterium, rather than from toxigenic molecules, which borrelia cannot produce. Indeed, the deposition of spirochetes into human dermal tissue generates a local inflammatory response that becomes manifest as erythema migrans (EM), the hallmark skin lesion of Lyme disease in North America. In Europe, two additional dermatologic disorders, borrelial lymphocytoma and acrodermatitis chronicum atrophicans (ACA) are frequently associated with infection. EM is frequently accompanied by 'flu-like' symptoms, including myalgias, arthralgias and fever, which are generally believed to be cytokine-mediated in response to hematogenous spread of the bacterium. If treated appropriately, the prognosis is excellent; however, if untreated, patients may develop a wide range of inflammatory clinical manifestations, most commonly involving the central nervous system, joints and heart. Within days of treatment, the signs and symptoms associated with the disease typically begin to subside, although in some individuals a complete recovery can take several weeks or even months. A minority of treated patients may go on to develop a poorly defined fibromyalgia-like illness, which is not responsive to prolonged antimicrobial therapy. Below we integrate current knowledge regarding the ecological, epidemiological, microbiological and immunological facets of Lyme disease into a conceptual framework that sheds light on the disorder that healthcare providers encounter."

According to this, Borrelia burgdorferi does not produce toxigenic molecules, and most symptoms are believed to be cytokine-mediated in response to hematogenous spread of the bacterium. (hematogenous = originating in or spread by the blood). They are talking about all Borrelia there.

Hm. That's interesting. Does that mean that what people think is a Herxheimer reaction due to the release of toxins is actually something else, some other process?

I wanted to know what the deal is with this, so I looked it up... Huh, this was interesting:

Borrelia have abundant glycolipids but they do not have an endotoxin-like lipopolysaccharide. And the authors' running hypothesis is that the Herxheimer reaction is due to the action of lipoproteins on toll-like receptors (TLR2) in macrophages and other cells. Apparently, if you infuse antibodies that recognize TNFa (Tumor Necrosis Factor alpha) into someone who is infected with related Borrelia recurrentis before giving them penicillin, it reduced the severity of a Herxheimer reaction (Fekade et al, 1996 - full text at preceding link). When cytokine levels get quite high, this contributes to the reaction. (Borrelia, p. 339)

So, does this mean that detoxing is useless because there is no toxin to remove? The paper goes on to state that "...lipoproteins from two other spirochetes, B. burgdorferi and Treponema pallidum, induce the biosynthesis of tumor necrosis factor in murine macrophages."

So when patients are having a Herxheimer reaction, it is a reaction to the die-off induced by the immune system - not a reaction induced by a toxin.

Regarding the bit in the last section on persistence: Researchers are somewhat divided on this, and even as I write this, studies are published on the use of long-term antibiotic treatment for some of this minority of treated patients with persistent symptoms.

Dattwyler co-wrote this chapter along with Radolf, and one has to wonder if it would have been written somewhat differently and been left open to more uncertainty had Volkman or Barthold co-written it? Something to think about there.

At any rate...the running hypothesis is that infectivity of Borrelia species depends heavily on the evasion from the host response. And this evasion can be based on multiple mechanisms.

You've gotta just love the closing quote on the end of one chapter in the book...

Not.

“If our work with Borrelia has taught us one thing, it is to expect the unexpected. So we should not be surprised to discover that this phylogenetic ancient spirochaetal microorganism has developed several solutions of  its own and that it does not always conform to the dogmatic structure and function of the cell envelope of Gram-negative bacteria. We therefore anticipate a bright future with many challenges and unsolved mysteries for several generations of Borrelia researchers.”

Yeah. Right. Your science project is my life, man. I'm not looking at this as some professional subject of interest. My focus in this is different from yours. By a longshot, baby. By a longshot.

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