2 Anonymous Comments On Chronic Lyme Disease

Last year someone who identified only as "anonx" wrote some interesting comments on last January's blog post on quorum sensing, "How bacteria "talk" and how to make them shut up". (There is a fascinating video there by researcher Bonnie Bassler. I highly recommend checking it out.)

To my knowledge, comments on Blogger do not get syndicated on post rss feeds - though one can request rss comments separately. I think that anonx's comments are worth taking another look, so I have decided to repost select excerpts of them here. (Refer back to the original comment thread for the CO side of the dialog, if needed.)

Anonymous January 21, 2011 5:06:00 PM HST
persister cells
quorum sensing
round bodies
blebs
biofilms made of borrelia
biofilms made of a mix of pathogens
symbiosis in all its forms and iterations
intracellularity
the cascade of genospecies and strains
quiescence and dormancy
transfections
borrelia stuck in B cells with CLIP attached
the role of toll-like receptors&inflammation
the growing list of possible co-infections
xmrv
new view of PANDAS that goes way beyond strep...
molecular mimicry

guess I'll stop here but I could go on --you just need a little proof: not just proof it is happening but proof that treating it will end the illness. that's the problem.

Anonymous January 21, 2011 11:03:00 PM HST

Don't you think that looking at persistence in isolation could be futile, given the large number of people treated for years without cure? Persistence could be the imperceptible spark that incites an immune tsunami. What if it is easier to treat the tsunami --by its nature outsized and blatant and in your face-- than the spark which, though present and incendiary, we cannot find? Is it possible that the yin and yang nature of the fight has prevented a view of the system overall? What if a tiny amount of infection has caused a huge inflammatory response? What if that infection is hiding in B cells because a glitch in the system has prevented T cells from recognizing the foreign invader, and thus, from finishing the job? What IF to get rid of the persistent infection, you must treat immune dysfunction first? So no ...I don't think it is as simple as you say, or that by conceiving the issue along the old paradigms of the fight, raging fruitlessly for 30 years, you will get what you want ... unless of course, it is really as simple as you suggest. I mean, what if you prove persistence, but it still won't bring a cure?

Anonymous January 22, 2011 11:57:00 AM HST

regarding the four trials cited, they do in fact show benefit to retreatment for fatigue and pain --and thus, in fact do not match with the wording of the conclusions rendered or (in the case of two of three authors) forced down those authors throats by the powers that be. There is actually ample evidence in these trials to suggest a benefit to retreatment and longer treatment --and in the case of the Klempner trial, serious issues with methodology. But as you say, the outcome of these trials must be detached from pathophysiology --what is the mechanism of the benefit? That question the trials do not address.

Still, to detach infection from immune response is just plain wrong-headed. We are 10% by weight bacteria, and most of those organisms are beneficial. The question is --which are the pathogens, and how do those pathogens do us in? In every case, inflammation and cellular immunity are going to play a role.

Anonymous January 22, 2011 11:15:00 PM HST

It is a very complicated problem, as you say.

It is TRUE that if you suppress inflammation infection would spiral out of control: The murine studies on borrelia and toll-like receptors show that to be right.

But here's the thing: Most researchers, even most of the IDSA researchers, don't actually deny that organisms can persist. They just deny that those quiescent remaining organisms are driving the continued symptoms --they say these persisting borrelia are too few, and too dormant. And they would cite the research on quorum sensing to support their case.

As I see it, it is not really persistence you need to prove, but rather, the mechanism by which persistence at the low level research suggests drives the disease. Inflammation is not the only immune mechanism --cellular immunity or dysfunction thereof can play havoc, too, and persistent infection could drive it in an endless loop.

Also: Treatment studies without knowing more about pathophysiology can work against you, because --hell-- they are just empiricism on top of empiricism, more wandering in the dark.

I contend you need more basic biology to target such studies, strategically. If you were to move forward without that, you would need an elaborate methodology with many variables and large enough numbers of patients to test for many possibilities and separate the data from the noise. And with Lyme patients still so ill-defined, with no test extant for active infection ...

Makes my head spin. A hundred million dollars would help.

Anonymous January 23, 2011 1:34:00 PM HST

Other,

My comments refer specifically to the Barthold work, with which I am extremely familiar. Barthold's findings of small numbers of dormant, quiescent spirochetes within collagen across the range of mammalian species following treatment have been well-known inside the mainstream (though published only recently) for decades. There is no great rush to debunk Barthold, whose research really is beyond dispute --Barthold himself being an especially meticulous and careful scientist. What his critics say, however, is that these spirochetes are not active enough and not numerous enough to cause disease. (to wit: issue of quorum sensing.) Barthold theorizes otherwise, contending that the small numbers of chetes may provoke an outsized --but heretofore undetected-- cytokine cascade that causes the disease. Barthold would classify this cascade under the heading of INFLAMMATORY response. This is his theory --and a powerful one that should be explored.

You may have noticed that NIH has begun to test the Barthold work with a study of xenodiagnosis, but patients are protesting that study for fear that the ticks used might not be as naive as claimed: And really, given the confusion over pathogens involved, who knows?

Other theories of persistence to pursue include the impact of round bodies AND the work of Newell, who finds Borrelia stuck inside B cells because of a dysfunction in MHC.

In the case of Newell, especially, the notion is that persistent infection can never be cured without correcting the recognition dysfunction of MHC. In other words, even though the disease is driven by persistence, Newell says you have to correct the immune problem first.

And by the way, both she and Barthold insist you can never entirely clear borrelia infection with antibiotics alone --you need the immune system to do the final kill, and so you must have an immune correct FIRST, even if the driver is persistence.

Or it could be the round bodies... but whatever it is, it is complicated --and simply fueling the fight of persistence versus immunity isn't helpful.

There is a difference between what patients use to get well right now --the ax in the form of huge quantities of endless suppressive antibiotics-- and what they should want for the future --the chisel, which could well be an immune correction that allows infection finally to be resolved. There should be a divide between the effort to protect a Lyme doc in the here and now and the direction of research for the future --but it is hard for a lot of people to understand this.

I agree with you that if we knew the mechanism we would have a target --and that is why I am so equivocal if not outright squeamish about the continued treatment trials some patients are calling for.

What are they treating? --and if they don't really know, there is a big risk that study could bury them deeper and darker than ever before.

anonx

Anonymous  January 23, 2011 7:27:00 PM HST

Given current state of knowledge immune treatments could backfire, big time --as the literature shows. If you look at the work on toll-like receptors you find a genetic curve for inflammatory response to borrelia ranging from almost nothing to off-the-charts and everything in between. That is just one immune parameter, and there are many others. These parameters could vary for every infection or strain and every person. Therefore it is possible with current state of knowledge that suppressive abx are really the best we have... there needs to be a crunching of data to understand what we are looking at --otherwise, it is just stumbling in the dark. The amazing thing is that the IDSA crew has gotten away with such a grotesquely oversimplified story of this disease for so long --and that to explain it, they perpetuate the explanation that it is a psychosis instead of a complex spectrum of infection and immune response. But by giving an oversimplified rejoinder, patients have hurt their cause, too.

In a gross way one could do a study treating infection, treating immune issues or treating both: but this would be very crude without more data up front.

anonx


Comments:

So I do have a few comments on this, now that it's nearly a year and half since these comments were posted. My thoughts on the matter have shifted over time, and after exposure to more research.

• I'd like to see evidence that Borrelia burgdorferi is hiding in B cells in vivo. That would be informative. There has been some mention of Bb being intracellular in a few in vitro studies and one in  vivo study; we need more.
  
• This anonymous author may be on to something, and it may be that the host immune response may need to be addressed in order to manage the remaining infection if it is still present. Not enough is known, but when I read about filgrastim and rixtuximab and how they have some positive effect on a patients with persisting symptoms of Lyme disease or CFS/ME,  I think that adjusting the host immune response is an avenue worth exploring. It should have been explored more years ago.
• I still think the trials must be detached from pathophysiology. My position on this has not changed. Treatment trials have done nothing to provide evidence of persistence of Borrelia burgdorferi one way or the other.

• I strongly agree we need more basic biology research. The anonymous author's comments on the role the immune system plays in infection are noteworthy. But we also need to know what is going on if the infection does persist in some form. Is there a persister phenotype? Ongoing research into persister phenotypes should not be neglected. But I wouldn't leave all research at that, because there are other hypotheses to consider.

• One thing I wonder about is the issue of quorum sensing and efficiency sensing, and if blebs or vesicles play any role in the dissemination and pathogenesis of Borrelia burgdorferi. Blebs as a form of communication are observed in other bacterial species, and perhaps Bb uses blebs and vesicles in a different manner and they are not part of cells undergoing apoptosis. Plasmid DNA and outer surface lipoproteins have been found within blebs; there is some suggestion of blebs containing adhesins... I think there's more to blebs than meets the eye.

• Anonymous said, "There is a difference between what patients use to get well right now --the ax in the form of huge quantities of endless suppressive antibiotics-- and what they should want for the future --the chisel, which could well be an immune correction that allows infection finally to be resolved. There should be a divide between the effort to protect a Lyme doc in the here and now and the direction of research for the future --but it is hard for a lot of people to understand this." I keep reflecting on what they wrote, and its implications. Is there a more targeted approach which could be designed to help treat patients?" I think they are right - there is no reason why patients cannot ask for protection for the treatment they are receiving now while promoting research on new and different treatments. I think VGV-L is one effort in this direction, but I am not sure it will work. There is a lot of complexity involved.

• Last but not least, my anonymous commenter said this: "The amazing thing is that the IDSA crew has gotten away with such a grotesquely oversimplified story of this disease for so long --and that to explain it, they perpetuate the explanation that it is a psychosis instead of a complex spectrum of infection and immune response. But by giving an oversimplified rejoinder, patients have hurt their cause, too." 

I think there's a lot of truth in that last statement. Now, what does one do about it?

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2 Wormser et al Criticism Launched at Embers' Rhesus Macaque Study

Wormser et al have recently published a critique of Embers' study, "Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection".

Vector Borne Zoonotic Dis. 2012 May 23. [Epub ahead of print]

Critical Analysis of Treatment Trials of Rhesus Macaques Infected with Borrelia burgdorferi Reveals Important Flaws in Experimental Design.
Wormser GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, Shapiro ED.

Abstract

A critical analysis of two treatment trials of Chinese rhesus macaques infected with Borrelia burgdorferi indicates that insufficient attention was placed on documenting the blood levels, pharmacokinetics, and pharmacodynamic parameters of the antibiotics used in this host. Consequently, it is impossible to conclude that the findings have validity in judging the efficacy of doxycycline or ceftriaxone for the treatment of Borrelia burgdorferi in this animal model.

PMID: 22620495 [PubMed - as supplied by publisher]

Full text of this critique is available here:
http://online.liebertpub.com/doi/full/10.1089/vbz.2012.1012

The full text of the original study which is the focus of this critique is here:
http://www.plosone.org/article/fetchArticle?articleURI=info%3Adoi%2F10.1371%2Fjournal.pone.0029914


Related material on Camp Other blog:

• Paper: Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection
• Debate About Embers Non-Human Primate Chronic Lyme Disease Study Continues
• Embers et al Rhesus Macaque Study Correction Issued
• Embers et al Issues Statement On PLoSONE
• Why Aren't Persisting Spirochetes Enough Evidence Of Infection?

Comments:

For now I am sharing the news that the free full text this critique is available online. Further comments to be made at another time. Comments by readers are welcome.

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1 Abstract: Delays and Diversions Mark the Development of B Cell Responses to Bb Infection

Another insightful paper on the immune response to infection with Borrelia burgdorferi has been published. "Delays and Diversions Mark the Development of B Cell Responses to Borrelia burgdorferi Infection" by Hastey et al is closely related to previous research completed by Tunev et al on the immune response found in murine lymph nodes which were invaded by Borrelia burgdorferi.

A well-written blog article on Tunev et al's previous research on B cells and plasma cells and their associated (lack of) T cell response in reaction to Bb infection can be found here:

Spirochetes Unwound Blog - Does Borrelia burgdorferi cause an inadequate antibody response by altering B cell activation in the lymph node?

I recommend reading that link first before proceeding to the following abstract.

Christine J. Hastey, Rebecca A. Elsner, Stephen W. Barthold and Nicole Baumgarth. Delays and Diversions Mark the Development of B Cell Responses to Borrelia burgdorferi Infection. The Journal of Immunology. April 30, 2012

Abstract

B cell responses modulate disease during infection with Borrelia burgdorferi, the causative agent of Lyme disease, but are unable to clear the infection.

Previous studies have demonstrated that B. burgdorferi infection induces predominantly T-independent B cell responses, potentially explaining some of these findings. However, others have shown effects of T cells on the isotype profile and the magnitude of the B. burgdorferi-specific Abs.

This study aimed to further investigate the humoral response to B. burgdorferi and its degree of T cell dependence, with the ultimate goal of elucidating the mechanisms underlying the failure of effective immunity to this emerging infectious disease agent.

Our study identifies distinct stages in the B cell response using a mouse model, all marked by the generation of unusually strong and persistent T-dependent and T-independent IgM Abs.

The initial phase is dominated by a strong T-independent accumulation of B cells in lymph nodes and the induction of specific Abs in the absence of germinal centers.

A second phase begins around week 2.5 to 3, in which relatively short-lived germinal centers develop in lymph nodes, despite a lymph node architecture that lacks clearly demarcated T and B cell zones.

This response failed, however, to generate appreciable numbers of long-lived bone marrow plasma cells.

Finally, there is a slow accumulation of long-lived Ab-secreting plasma cells in bone marrow, reflected by a strong but ultimately ineffective serum Ab response.

Overall, the study indicates that B. burgdorferi might evade B cell immunity by interfering with its response kinetics and quality.

This work was supported in part by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant AI073911 (to N.B. and S.W.B.) and T32 Training Grant AI060555 (to C.J.H. and R.A.E.).

Full text is available behind pay wall here: http://www.jimmunol.org/content/early/2012/04/30/jimmunol.1103735.full.pdf+html

Comments:

This is an interesting development in the ongoing process of trying to understand how Borrelia burgdorferi evades the immune system. What we know is what starts out looking like the host mounting a strong immune response to infection ends up looking like a poorly differentiated immune response where plasma cells are inadequate and not engaging in the right immune class switching to fight infection - and where T cells are not fully participating in B cell activation.

This study indicated that not only is the immune response inadequate and ill-directed in its early phase in lymph nodes (which Tunev et al studied) but that in later stages antibody response is inadequate as well.

These studies indicate that the host immune response fails to clear Borrelia burgdorferi and somehow the bacteria is able to evade it. More details on specifically how is likely available in the pay-for-view full text of the paper (until the six month NIH/NIAID publication embargo is over).

Questions remain as to how this research applies to human hosts. Does the same immune response occur in humans that occurs in mice? How does the introduction of antibiotics affect this response? Knowing how both the host immune system and antibiotics work together in combatting this infection would be useful.

One thing I would like to see Tunev, Hastey, Barthold, and others doing this work is to somehow detect which outer surface proteins are upregulated during the time they are invading the lymph nodes and generating a lot of inflammation. In particular, I am wondering if OspA is being expressed in the lymph nodes as much as it has been proposed as being expressed in the CNS in neuroborreliosis.

I leave those reading to consider this paper which was published in Nature, and the following excerpt from it:

OspA-CD40 dyad: ligand-receptor interaction in the translocation of neuroinvasive Borrelia across the blood-brain barrier

"Some authors have suggested downregulation of OspA in early phase of the infection 21, 22, while others have reported expression of OspA in the unique environment of the brain and CSF, but not in the serum 23, 24. Therefore, it was essential to determine whether OspA is expressed in borreliae that are present in the brain vasculature in vivo in infected laboratory animals. PCR analysis of the brain and brain microvasculature of Wistar rats infected with SKT-7.1, revealed not only the presence but also the augmented expression of OspA (Fig. 3). This finding is crucial to support a role of OspA as an adhesive molecule in the transient tethering of Borrelia."

"OspA is undoubtedly a multifunctional protein that is absolutely necessary in the various stages of borrelial lifecycle and pathogenesis. OspA is abundantly expressed in tick gut as an important adhesive molecule 29. To avoid an inflammatory response, expression of OspA is downregulated in the early stages of Lyme disease. However, OspA expression in vivo can be significantly induced if the spirochetes are kept in an inflammatory environment 46. OspA plays an important role in binding to neuronal cells. These data indicate that OspA must be upregulated during the CNS invasion and acts as an important adhesion factor, which is essential in the pathogenesis of Lyme neuroborreliosis 23. It is also well known that Borrelia can bind plasminogen via OspA on their surface 47. OspA also upregulates membrane urokinase-type plasminogen activator receptor (uPAR) 48. Plasminogen can be activated to plasmin 47, 48 leading to degradation of the extracellular matrix. The mammalian plasminogen-plasmin proteolytic system plays a crucial role in extracellular matrix degradation (intercellular junctions) and cell migration 49. Binding of host-derived proteinases (like plasminogen and MMPs) via OspA supports the theory that Borrelia exploits these proteinases to degrade the intercellular tight junctions. Owing to the hypervariability of OspA among several Borrelia strains, it is important to note that only expression of OspA is not sufficient, but its ability to interact with host's receptors is crucial in the invasion processes."

After reading a passage like this - plus these studies on B cell activation during Bb infection - I have to ask if OspA plays a role in in vivo infection not only inside the CNS in neuroborreliosis - but also in dissemination to other parts of the body. Would this account for the widespread pain patients experience from inflammation, since OspA is highly immunogenic? What is OspA's degradability?
See: http://en.wikipedia.org/wiki/Immunogenicity

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6 Paper On Borrelia burgdorferi, RpoS, And Formation of Round Bodies or "Cysts"

Dr. MacDonald just posted information on Lymenet Europe concerning a new paper by Dunham-Emsl et al about the round body aka "cyst" form of Borrelia burgdorferi as part of the Lyme disease life cycle within the tick.

The indication here is that Borrelia burgdorferi assumes a round body form within the tick in order to survive until circumstances for transmission to the host are present. There is no confirmation here, however, of its formation in vivo within a host mammal...

Full Text Source: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002532

Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission

Star M. Dunham-Ems, Melissa J. Caimano, Christian H. Eggers, Justin D. Radolf

Abstract

While the roles of rpoSBb and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable.

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2 Paper: Course of Antibody Response In Lyme Borreliosis Patients Before And After Therapy

ISRN Immunology Volume 2012 (2012), Article ID 719821, 4 pages doi:10.5402/2012/719821 Research Article

Course of Antibody Response in Lyme Borreliosis Patients before and after Therapy

Elisabeth Aberer¹ and Gerold Schwantzer<sup>2

1</sup>Department of Dermatology and Venereology, Medical University of Graz, Auenbruggerplatz 8, 8036 Graz, Austria

²Institute for Medical Informatics, Statistics and Documentation, Graz, Medical University of Graz, 8036 Graz, Austria

Received 27 September 2011; Accepted 27 October 2011

Academic Editors: A. Clayton and S. Devi

Copyright © 2012 Elisabeth Aberer and Gerold Schwantzer. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The early immune response (IR) in European Lyme borreliosis patients has not yet been studied in detail. The aim of the study was to analyse retrospectively the antibody development in 61 erythema migrans (EMs) patients depending on the duration of infection from tick bite by using a whole-cell lysate B. garinii immunoblot. The evolution of antibodies proved to be undulatory in untreated patients with two peaks for IgM at weeks 5 and 9 and for IgG at weeks 4 and 8. The analysis of IR courses after therapy identified patients constantly seropositive or seronegative and patients with repeated seroconversions with a switch, disappearance, or reappearance of anti-23 kD or anti-39 kD antibodies during the one-year period. We suggest that the antibody production in EM patients may be missed due to an undulatory IR. This phenomenon might be an as yet insufficiently researched aspect in Lyme borreliosis.

1. Introduction

Serological testing aids in the diagnosis of Lyme borreliosis (LB). IgM antibodies develop during the first 3 weeks after infection followed by IgG antibodies after 4–6 weeks [1]. However, to our knowledge, the early immune response (IR) in European patients has not yet been studied in detail.

In routine clinical practice, we observed patients whose IR switched from positive to negative and vice versa in the course of time. The aim of this study was to analyse the development and the course of the IR in European erythema migrans (EMs) patients with known duration of disease from tick bite and to follow up their antibody profile during a 12-month period after treatment.

2. Patients, Materials, and Methods

One hundred and two patients with EM were enrolled for clinical and serological diagnosis. Their mean age was 50 (SD16) years. Forty-seven patients were male and 55 female. The mean duration of EM was 23 (1–210) days. Eighty-seven patients had EM without and 15 patients EM with extracutaneous symptoms. As sixty-one (28 males and 33 females aged between 22 and 78 years) remembered their tick bite, the onset of their infection was known and only these patients were included in the study. These patients received antibiotic treatment with phenoxymethylpenicillin 1500 000 IE threetimes a day, 26 patients for 14 days and 35 patients for 20 days. No statistical difference in the clinical outcome of the differently treated groups was observed during the 12-month observation period as described in a previous study [2].

Blood samples were obtained from all 61 patients for serological testing at the initial visit before therapy and then thereafter at 3 weeks after first blood withdrawal. Sera were available from 46 patients 3 months after therapy, from 52 after 6 months, and from 53 after 12 months (Table 1).

Table 1: Number of investigated patients’ sera.

Patients’ sera that had been stored at −20°C were retested at the same time and antibody response was measured by a whole-cell lysate immunoblot (IB) according to the manufacturer’s instruction (Borrelia garinii, MRL Diagnostics, Cypress, CA). Interpretation of the IB bands was performed by a single technician. Criteria for a positive IB, as indicated in the manufacturer’s manual, were 1 of 2 bands (23 and 39 kD) for IgM and 4 of 7 bands (21, 23, 37, 39, 41, 45, and 93 kD) for IgG. Data obtained were entered in a computerized database and graphically displayed.

3. Results and Discussion

3.1. Development of Immune Response

Twenty-five of 61 (41%) sera showed IgM antibodies in IB before therapy. The IR started in week 1 after tick bite and peaked in week 5, when 5 of the 7 tested sera (71%) reacted positively (Figure 1).

In the following weeks there was a wavelike decrease of seropositive reactions. Furthermore, from week 8 there was a second increase in seropositivity peaking at week 9. IgG antibodies were positive in 13/60 (21%) patient sera (one serum could not be analysed because of a smear). The IR started from week 2 and peaked at week 4, where 75% (3 of the 4 tested sera) reacted positively (Figure 1).

Similar to the IgM antibody trend, the IgG IR decreased in the following weeks. However, there was a second increase of IgG IR that peaked in week 8.

Figure 1: Development of immune response from tick bite (weeks).

The development of the IR to an infection with B. burgdorferi (Bb) s.s. in EM patients from the United States was studied by Aguero-Rosenfeld and showed that 43% of the patients had positive IgM antibodies before therapy [3].

Due to the heterogeneity of borrelia strains in Europe, differences between IR on the two continents are expected since the Bb s.s. species provokes a stronger immune reaction than the dominant European species B. afzelii. Patients with culture-confirmed B. burgdorferis.s. erythemas from the USA were more often seropositive (35.3%) at presentation compared with 22.4% culture-confirmed B. afzelii erythemas [4].

In another European study with recombinant ELISA, German Lyme borreliosis patients yielded positive IgG antibodies in 22% and IgM antibodies in 61.5% for phase I of LB [5].

Both the IgM and IgG IR showed an undulatory distribution with antibodies coming and going in untreated patients over weeks. A similar periodicity is known in other bacterial infections, like relapsing fever with recurrent bacteraemias [6].

Syphilis can be reactivated by different conditions like HIV infection, showing seroconversion of the unspecific VDRL [7].

3.2. Course of Immune Response after Treatment

With respect to the course of IR after therapy, 21 of 61 (34%) patients did not show IgM seroconversion (constantly negative), whereas 12 (20%) were constantly positive. In the remaining 28 patients, different kinds of IgM seroconversion occurred. Nine patients (15%) (Figure 2, patients 1–9) seroconverted from positive to negative and 6 (11%) (Figure 2) (Figure 2, patients 16–22) seroconverted to positive and than back to seronegative during the 12 months. Six patients (10%) (Figure 2, patients 23–28) showed repeated seroconversions of IgM antibodies that presented as a switch of anti-23 kD to anti-39 kD antibodies and vice versa or the dis- or reappearance of either anti-23 kD or anti-39 kD antibodies.

 Figure 2: IgM antibody response in immunoblot before and  up to 12 months after therapy. Filled circles: positive IgM antibody response; empty circles: negative IgM antibody response; blank spaces: no serum available. BT: before therapy; AT: after therapy; 3, 6, 12 months after start of therapy.

Thirty-eight of the 60 patients (63%) were constantly IgG negative and 3 patients (5%) constantly positive in IB. Nineteen patients seroconverted within the observation period. Their data are presented in Figure 3. Six patients (10%) (patients 1–6) seroconverted from initially positive to negative and 2 (3%) (Figure 3, patients 7 and 8) from negative to positive.

Seven patients (12%) (Figure 3, patient 9–15) who were primarily negative seroconverted to positive and then back to seronegative during the observation period.

Figure 3: IgG antibody response in immunoblot before and up to 12 months after therapy. Filled circles: positive IgG antibody response; empty circles: negative IgG antibody response; blank spaces: no serum available. BT: before therapy; AT: after therapy; 3, 6, 12 months after start of therapy.

Four patients (7%) (Figure 3, patients 16–19) showed repeated seroconversion with at least 2 changes. The seroconversion itself presented as a loss of detectable antibodies from a previous maximum of up to 5 bands (23, 37, 39, 41, 45, and 93 kD) to 1 band.

When observed closely, none of the patients with a repeated seroconversion had any features that could distinguish them from the other patients with either persistent antibodies or seronegative individuals. There was also no relation between IgM and IgG seroconversion. Clinically, no correlation to the duration of treatment or to the presence of extracutaneous signs could be drawn.

A recent survey on the development of the IR, measured by ELISA, in Austrian EM patients over a minimum of 1 year after treatment showed 3 distinct courses: persistent positive, persistent negative, and positive to negative patients [8]. In this study, there were also patients with a change from negative to positive antibodies (3% IgM, 4% IgG), but this was not attributed to the original EM as seroconversion occurred after a median of 350 (for IgM) and 349 days (for IgG) after EM. In our study, IB was found to be more sensitive than ELISA; therefore only IB results were analyzed and the IR could be studied in more detail.

In our patients, the appearance of IgM antibodies only detected after 6 months (patients 21 and 22) and IgG antibodies only at 3 months (patients 9 and 10) cannot be related to the previous infection. Moreover, an isolated IgM seroreaction has been observed to be unspecific [9, 10].

Isolated positive serum IgM titers were seen in about 20% of children with a febrile illness, enterovirus meningitis, or headache [9]. In another article 2,6% of sera submitted for B. burgdorferi serology expressed unspecific anti-p41 IgM antibodies by ELISA. Confirmation test with IB showed that some sera also reacted with p39 or OspC antigen. No conclusive evidence for borrelia infection could be drawn [10].

The first appearance of antibodies after 6 and 12 months in our study (patients 14 and 15 for IgM, and 7 and 8 for IgG) might indicate a new infection in these patients.

Although the undulatory character of the IR before therapy in our patients could not be determined in every single patient, the findings after treatment might reflect a similar situation also in untreated patients before therapy. So, we suggest that a single serological finding is a snap shot and gives evidence of an infection. On the other hand, the true infection might be missed by negative IR, as might be the case in the ~40% seronegative EM patients.

Serological findings do not distinguish between active and previous disease. Borrelia DNA can persist in urine for even 1 year after treatment [11], and antibodies to Bb may persist for up to 20 years after appropriate therapy [12]. Bearing in mind the characteristics of cyclic patterns in other bacterial infections, the undulatory IR noted in our study may be an as yet insufficiently researched aspect in Lyme borreliosis.

Conflict of Interests

The authors declare that there is no conflict of interests.

Acknowledgments

The authors greatly acknowledge Jasmina Custovic, M.D., for collecting and analysing the data which were also partly shown in her thesis (development of the immune response in erythema migrans with special significance of the p18 antigen) and Mrs. Ingrid Krainberger for her excellent laboratory support during the study.

References

1. B. Wilske, “Serodiagnosis of lyme borreliosis,” Zeitschrift fur Hautkrankheiten, vol. 63, no. 6, pp. 511–514, 1988.
2. E. Aberer, P. Kahofer, B. Binder, T. Kinaciyan, H. Schauperl, and A. Berghold, “Comparison of a two- or three-week regimen and a review of treatment of erythema migrans with phenoxymethylpenicillin,” Dermatology, vol. 212, no. 2, pp. 160–167, 2006.
3. M. E. Aguero-Rosenfeld, J. Nowakowski, S. Bittker, D. Cooper, R. B. Nadelman, and G. P. Wormser, “Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans,” Journal of Clinical Microbiology, vol. 34, no. 1, pp. 1–9, 1996.
4. F. Strle, R. B. Nadelman, J. Cimperman et al., “Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia,” Annals of Internal Medicine, vol. 130, no. 1, pp. 32–36, 1999.
5. K. P. Hunfeld, M. Ernst, P. Zachary, B. Jaulhac, H. H. Sonneborn, and V. Brade, “Development and laboratory evaluation of a new recombinant ELISA for the serodiagnosis of Lyme disease,”Wiener Klinische Wochenschrift, vol. 114, no. 13-14, pp. 580–585, 2002.
6. C. Larsson, M. Andersson, J. Pelkonen, B. P. Guo, A. Nordstrand, and S. Bergström, “Persistent brain infection and disease reactivation in relapsing fever borreliosis,” Microbes and Infection, vol. 8, no. 8, pp. 2213–2219, 2006.
7. A. McMillan, H. Young, and J. F. Peutherer, “Influence of human immunodeficiency virus infection on treponemal serology, in patients who have been treated for syphilis,” Journal of Infection, vol. 21, no. 1, pp. 95–103, 1990.
8. M. Glatz, M. Golestani, H. Kerl, and R. R. Müllegger, “Clinical relevance of different IgG and IgM serum antibody responses to Borrelia burgdorferi after antibiotic therapy for erythema migrans: long-term follow-up study of 113 patients,” Archives of Dermatology, vol. 142, no. 7, pp. 862–868, 2006.
9. R. Bennet, V. Lindgren, and B. Zweygberg Wirgart, “Borrelia antibodies in children evaluated for Lyme neuroborreliosis,” Infection, vol. 36, no. 5, pp. 463–466, 2008.
10. E. Ulvestad, A. Kanestrøm, L. J. Sønsteby et al., “Diagnostic and biological significance of anti-p41 IgM antibodies against Borrelia burgdorferi,” Scandinavian Journal of Immunology, vol. 53, no. 4, pp. 416–421, 2001.
11. E. Aberer, A. R. Bergmann, A. M. Derler, and B. Schmidt, “Course of Borrelia burgdorferi DNA shedding in urine after treatment,” Acta Dermato-Venereologica, vol. 87, no. 1, pp. 39–42, 2007.
12. R. A. Kalish, G. McHugh, J. Granquist, B. Shea, R. Ruthazer, and A. C. Steere, “Persistence of immunoglobulin M or immunoglobulin G antibody responses to Borrelia burgdorferi 10–20 years after active Lyme disease,” Clinical Infectious Diseases, vol. 33, no. 6, pp. 780–785, 2001.

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0 Paper: Vector Competence of the Tick Ixodes ricinus for Transmission of Bartonella birtlesii

This paper's abstract was recently posted to Lymenet Europe. For those not familiar with one of the debates over the issue of tickborne coinfections, for some time some researchers have claimed that Bartonella can not be transmitted by ticks - while others claim it can be.

Up to this point, Bartonella DNA has been found in ticks, there have been positive PCR results in questing ticks which indicate that the bacterium (or at least its DNA) can survive in the tick through the molt from one life stage to another - and there has been evidence that people who have had tick bites have produced antibodies for both Lyme disease and Bartonella concurrently. But his was not taken as evidence that the tick was the vector responsible for transmission of Bartonella.

As it's been said many times before: Correlation is not causation. There needed to be evidence that Bartonella from ticks carrying the pathogenic organism could be directly transmitted to its host.

In light of the research cited below, it appears this evidence has been found: An Ixodes tick has been shown to be a vector for Bartonella in vivo.

My practical response to this one way or the other has been that regardless of source - fleas from one's cat or dog, or tick - that if one has symptoms of Bartonella and positive test results that they definitely should be treated for Bartonella.

Treat first, and let researchers sort out the source over time...

Reis C, Cote M, Le Rhun D, Lecuelle B, Levin ML, Vayssier-Taussat M, Bonnet SI. Vector Competence of the Tick Ixodes ricinus for Transmission of Bartonella birtlesii. PLoS Negl Trop Dis. 2011;5(5):e1186.

Source:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104967/?tool=pubmed

Abstract

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii.

Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

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0 Dr. Alan MacDonald Discussing Spirochetal Biofilms on LNE

There are a series of discussions going on right now on Lymenet Europe I want to point out.

It looks like Dr. Alan Macdonald is having an involved exchange about biofilms in different spirochetes with someone (Henry) who has identified as a microbiologist in previous entries.

You might want to check out this thread now:

Biofilms of still yet spirochetal type - Treponema:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3607

Also follow the following related threads:

Structure of Biofilms of Borrelia Lecture link:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3602

Biofilms of yet another spirochetal species - Leptospira:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3606

I would like to see more doctors and microbiologists engage in discussion about Borrelia and other spirochetes more often - especially if the implications have an impact on translational medicine and clinical outcome. Obviously some of this discussion is going to be purely speculative, but it is interesting to hear different points of view.

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0 Institute of Medicine Final Report on October 2010 Tickborne Disease Workshop

Back in October 2010, the Institute of Medicine (IOM) held a workshop which was broadcast online live (and remains available at TV worldwide), Critical Needs and Gaps in Understanding: Prevention, Amelioration, and Resolution of Lyme and Other Tick-Borne Diseases: The Short-Term and Long-Term Outcomes.

The workshop participants were members of the Institute of Medicine, various researchers, doctors, and members of the Lyme disease patient advocacy community.

A preliminary summary report on the workshop was published by the IOM in April 2011. Now, an official final report has been published and is available on PubMed:

http://www.ncbi.nlm.nih.gov/pubmed/21977545

For a more detailed table of contents, try:

http://www.ncbi.nlm.nih.gov/books/NBK57020/

Editors: Committee on Lyme Disease and Other Tick-Borne Diseases: The State of the Science.

Source: Washington (DC): National Academies Press (US); 2011.
The National Academies Collection: Reports funded by National Institutes of Health.

Excerpt

It was obvious to participants at the workshop that a significant impasse has developed in the world of Lyme disease. There are conflicts within and among the science; policy; politics; medicine; and professional, public, and patient views pertaining to the subject, which have created significant misunderstandings, strong emotions, mistrust, and a game of blaming others who are not aligned with one’s views. Lines in the sand have been drawn, sides have been taken, and frustration prevails. The “walk in the woods” process of conflict resolution or a similar process seems necessary for creating a new environment of trust and a better environment for more constructive dialogue to help focus research needs and achieve better outcomes. Such a process does not imply a compromise of the science but rather is needed to shift to a more positive and productive environment to optimize critical research and promote new collaborations.

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I'd have to say this is a good report for those who are new Lyme disease and other tickborne illnesses to read in order to get an idea of what issues concern researchers and patients.

In terms of an action item plan and treatment to help patients, though, this report is lacking in either and what is sorely needed at this point in time.

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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.

---

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 On Explaining One's Position

I recently received a comment on a post in the past from someone who has identified as a skeptic, condemning this blog for offering any support for chronic Lyme disease as a concept. I'm not specifically linking to it now, but mention it on general principle because I think it reflects a misunderstanding on what I'm trying to do and my position in the controversy.

I mention in numerous places throughout this blog that I have a skeptical nature, and I have also had persisting symptoms since I contracted Lyme disease. I've stated that I think chronic Lyme disease could happen under specific circumstances, and I do not know how common that is nor is it clear to me what all such circumstances are. I've also stated that I'm not sure chronic infection is always the cause of persisting symptoms. I simply don't know.

Because of my background and who I have been, I have found it difficult to stand in the middle of the Lyme disease controversy and ask the basic question of, "What is this all about, anyway?". But that is the question I am asking, and while I have a lot of readers who are supporters - because I haven't firmly fallen into one camp, I have detractors, too.

On one hand, I can potentially be viewed suspiciously by some Lyme disease patients because I do write about autoimmunity and Lyme arthritis, and I do wonder if the disease itself leads to changes in the immune system which lead to chronic symptoms. The latest research on B cells in lymph nodes certainly align with my musings about this... On the other hand, I can potentially be viewed suspiciously by those who align with the IDSA because I think Lyme disease may be able to persist as an infection... Barthold's research on surviving spirochetes after antibiotic treatment in a mouse model can point to the possibility of persisting infection.

In a way, I really can't win. Someone is going to take issue with something I write regardless of what it is. And that's fine - I give them a venue to express their opinion even though I may disagree. Or perhaps I don't even have an opinion on a particular topic yet, and whatever information they are sharing isn't something I was aware of before they mentioned it.

My goal here isn't to always find consensus - though I appreciate and welcome it when it is found; agreeing on some ideas can be a form of progress provided the evidence is sound. My goal here is to learn about facets of this disease and tickborne illnesses in general, and how they impact society, individuals, and research, and what is the relationship between each of those aspects.

I actually wish more skeptics would drop by for a visit, have some scotch or a coffee, and just share what research they have under their belt which supports their view and knowledge which reflects their understanding of it. It is easy to say, "You're making a mistake in supporting the chronic Lyme disease model". Criticism is easy to come by, and attaching that criticism to the letter which was posted to the Lancet recently in no way supports the science underlying your position.

If you don't support the concept of chronic Lyme disease as possible, okay, but at least explain why you individually do not and give reasons about it from a biological, microbiological, and molecular biological point of view. That would be educational for everyone reading along.

That gets the focus back on the reason for controversy to begin with: A dispute over what is happening in patients with persisting symptoms.

We may actually agree on a number of points, even if I don't agree with you on all of them. But whether we agree or not, discussing conflicts of interest from either the ILADS camp or IDSA camp sidesteps the issue of what the research out there reflects about what is known and unknown about Borrelia. Where does the definitive knowledge about Borrelia end and where does the speculation begin? What is inconclusive? What requires more research at this point in time?

I am looking for common ground at times because I am tired of the controversy. And I am also looking for any tie breakers: Is evidence in category A stronger in this instance than it is in category B? What is the weight of the evidence? What if there isn't a preponderance of evidence yet?

So, this is the basis on which I want to engage in discussion with people on this blog. I can talk about the social aspects of it on a social post, and the political aspects of it on a political post - but mostly, I'd like to steer things back to what the data reflects and what results are. (And also what isn't reflected in the data and results but has yet to be known.)

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