2 Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of B. burgdorferi S.S.

This interesting abstract just got posted on PubMed and is in PLoSONE:

Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of Borrelia burgdorferi Sensu Stricto

Schramm F, Kern A, Barthel C, Nadaud S, Meyer N, Jaulhac B, Boulanger N.

Abstract

In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease.

Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans).

The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-κB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated.

In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts.

Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs.

Link: http://www.ncbi.nlm.nih.gov/pubmed/22768217

Comments:

Take note of that last paragraph:

"Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs."

Do you think the implications of the above fit in nicely with the mathematical modeling of Borrelia burgdorferi infection cycles mentioned in an earlier entry?

Why or why not?

See:

Abstract: Population Dynamics Of Borrelia burgdorferi In Lyme Disease
http://campother.blogspot.com/2012/04/abstract-population-dynamics-of.html

The implications - for me at least - seem to fit a model where the first wave of infection dies off but then a bigger, immune-resistant subpopulation explodes onto the scene (the site of infection).

Awaiting PLoSONE to publish the full text so I can give a more thorough analysis...

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1 Three Notable NIAID 2012 Research Projects On Lyme Disease

The National Institute of Allergy and Infectious Disease (NIAID) is conducting some Lyme disease related research which I think readers should know about. There are a number of projects to be found on the Project Reporter web site which may be fascinating, but I took the time to select and highlight a few projects which would be of greater interest to patients suffering with Lyme disease and/or its coinfections.

Project: AN INTRACELLULAR NICHE FOR BORRELIA BURGDORFERI
Institution: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
PI: Skare, Jonathan

Description (by applicant):

Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the leading arthropodborne infection in the United States and causes significant morbidity in endemic areas. If untreated B. burgdorferi can persistently infect individuals even though the host mounts a potent adaptive immune response such that antibodies obtained from infected patients or experimentally infected animals effectively kills in vitro cultivated B. burgdorferi. In addition, a robust cell-mediated proinflammatory response is observed that induces IL-6, IL-12 and IFN- and inhibits IL-10. Furthermore, the spirochete can resist complement killing demonstrating that this important component of the innate immune response is not sufficient to eliminate B. burgdorferi infection.

The observation that B. burgdorferi persists in such a hostile environment indicates that the spirochete is adept at evading the host immune response via mechanisms that have not been completely elucidated. One possibility is that B. burgdorferi invades host cells and survives at low levels. Recently we have determined that B. burgdorferi invade both immortalized and, more importantly, primary cells (both fibroblasts and endothelial cells) and persist as viable cells in o-culture. In addition we have preliminary data suggesting that the ability to invade host cells involves both integrin binding and Src kinase activity.

In this application we propose to further characterize the internalization of B. burgdorferi and track the fate of B. burgdorferi within thes infected cells to determine how they affect the localized host response following infection. To accomplish this we will use both in vitro correlates of invasion and intracellular survival as well as in vivo imaging of experimentally infected mice as readouts for our studies.

Specifically, we propose to:

(1) Characterize the invasion of Borrelia burgdorferi into primary fibroblasts. The working hypothesis here is that B. burgdorferi exploits invasion as an additional mechanism to avoid host clearance. Our preliminary studies demonstrate that B. burgdorferi invasion is not dependent on host fibronectin, but does involve B1 integrins other than a5B1. In this Aim we will identify the subunit that pairs with B1 to promote invasion and will also evaluate how B. burgdorferi traffics within these cells; and

(2) Determine if invasion is required for B. burgdorferi persistence in vivo. Our working hypothesis is that invasion contributes to persistence by providing an immunoprotected niche for B. burgdorferi. Since Src kinases are required for borrelial internalization in vitro, we will determine whether Src kinase inhibitors alter the infectivity potential of B. burgdorferi in vivo. In addition to standard cultivation and molecuar approaches, novel in vivo imaging will be employed to assess how the inhibitor affects colonization.

The overall goal of these studies is to determine the extent in which an intracellular locale contributes to borrelial persistence.

PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and, as such, represents an important Public Health issue. The studies described in this application are designed to address how B. burgdorferi is able to persist effectively in infected mammals despite effective innate immune killing mechanisms and a potent adaptive immune response directed against this pathogen. The hypothesis being tested herein is that B. burgdorferi is capable of low-level intracellular survival in non-immune cells as an additional strategy to prevent borrelial host clearance.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8300386&icde=12284856

Comment: This really begins fulfilling my wishlist, and I look forward to the imaging study videos that I hope will be made and posted online. If there is some sort of confirmation of intracellular Bb in vivo this may explain why some patients need additional antibiotics and why existing treatments may be inadequate as a matter of timing.

This next project is bound to generate discussion, as it involves the potential role of toxins in Borrelia burgdorferi. In this case, the researcher is looking for gene clusters in Borrelia burgdorferi which may create cytolysins similar to the toxins which are found in Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum.

Project: A COMMON DENOMINATOR OF PATHOGENESIS; A RARE OPPORTUNITY FOR NOVEL THERAPEUTIC DE(VELOPMENT)
Institution: UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN
PI: Mitchell, Douglas

Description (by applicant):

Abstract: The 20th century witnessed several major advances in medicine. Perhaps most important were the discovery of antibiotics for bacterial infections and effective vaccines for several major viruses. Unfortunately, the creation of effective vaccines for bacteria has lagged behind analogous anti-viral strategies. Compounded with the rise in antibiotic resistance and a lack of interest from the pharmaceutical industry in pursuing novel antibiotics, we risk losing the fight against bacterial pathogens.

Described herein is an unconventional strategy to exploit bacterial toxins as both novel targets for antibacterial agents and antigens for vaccine development. To intelligently address the increasing threat posed by bacterial pathogens, more effort is needed to uncover the molecular underpinnings of virulence. Our group specializes in the use of bioinformatics, in vitro reconstitution, and genetic manipulation to identify and characterize gene clusters that are responsible for the biosynthesis of virulence-promoting cytolysins. The best-known toxin in this family is the highly modified peptide, streptolysin S (SLS, produced by Streptococcus pyogenes).

SLS production is required for the infective process, but not essential life processes. Our work has uncovered SLS-like toxins are synthesized by at least three other notorious human pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. We aim to study the potential role of the SLS-like toxin in an additional organism, Borrelia burgdorferi (Bb), which causes Lyme disease.

Although widely known, the Bb molecular mechanism of pathogenesis is inadequately defined. If the SLS-like toxin was indeed employed during Bb infections, this would represent the first demonstration of toxin utilization in this family of organisms and would prompt a major revision of borrelioses.

Because bacteria typically employ disparate pathogenic mechanisms, the conserved, SLS-like pathway provides a rare opportunity to develop more broadly applicable, yet targeted countermeasures. From our perspective, new antimicrobial strategies should directly target the pathogenic mechanism, rather than DNA replication, protein synthesis, or the cell wall. This approach holds enormous potential, as these drugs will theoretically be resistant to resistance.

This project will identify inhibitors of SLS toxin biosynthesis for the specific purpose of developing novel antibacterials. Moreover, SLS is non-immunogenic, rendering it an unfeasible candidate for vaccine development.

We have succeeded in generating attenuated variants with the anticipation that these can be used for raising toxin-neutralizing antibodies. The notion of immunizing against a bacterial toxin represents a potentially general strategy for future vaccine development.

With this proposal, we aim to not only fundamentally shift the accepted view of Bb pathogenesis, but also to challenge the paradigm that antibiotics must kill bacteria and non-immunogenic toxins are intractable vaccine candidates. These seemingly unrelated goals are actually quite intertwined. Our approach rests on the philosophy that a more complete understanding of toxin biosynthetic pathways and chemical structure can be rationally exploited to design novel therapeutics.

Public Health Relevance: Bacterial pathogens employ numerous mechanisms to evade the human immune system. We have discovered a novel strategy within the organism that causes Lyme Disease, who's pathogenesis remains largely enigmatic. A greater understanding of these processes will lay the foundation for developing the next generation of antimicrobial drugs.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8145943&icde=12284856

Comment:

Wait... I thought Radolf & co. said Borrelia burgdorferi does not produce a toxin? I know Donta patented some genes in Bb he saw as being analogous to a toxin.

Is there now evidence of newly researched genes which create a toxin in Bb? Or is this an old hypothesis which is being revisited?

Project: ASSESSMENT OF PATIENTS WITH BORRELIA INFECTION
Institution: NIAID
PI: Marques, Adriana

Description (by applicant):

Lyme disease is a multisystem illness caused by infection with the spirochete Borrelia burgdorferi and it is the leading vector-borne disease in the United States. Our current work addresses the following areas in Lyme disease: development of new tests and biomarkers for infection, investigation of persistence of infection with B. burgdorferi in humans, search for the cause of Southern Tick-associated Rash Illness (STARI), and investigation of the role of immune response in Lyme disease and PLDS.

One of the main problems in Lyme diagnosis has been the lack of highly specific and sensitive assays for B. burgdorferi and the lack of a test that could be used to assess response to therapy. Such assays should greatly facilitate the accurate diagnosis of Lyme disease and assessment of response to therapy in individual patients. Currently, no such test is available.

We have developed a new test using the luciferase immunoprecipitation systems (LIPSs) for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. A synthetic protein consisting of a repeated antigenic peptide sequence, named VOVO, had the best diagnostic performance, similar to the C6 test (a diagnostic test using a peptide ELISA that we have helped develop and is highly sensitive and specific). The VOVO LIPS test displays a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution; and offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

Recent studies have shown that B. burgdorferi may persist in animals after antibiotic therapy and can be detected by using the natural tick vector (Ixodes scapularis) to acquire the organism through feeding. Whether this occurs in humans is unknown.

We have implemented a new clinical protocol to investigate the utility of this approach for identifying persistence of B. burgdorferi in treated human Lyme disease.

STARI is a rash similar to the rash of Lyme disease that occurs in persons residing in southeastern and south-central states and is associated with the bite of the lone star tick, Amblyomma americanum. The cause of the rash is unknown, as it is the natural course of the disease.

We have a clinical protocol to investigate the cause of STARI, and we are applying new genomic tools that identify bacteria based on species-specific sequences in the 16S rRNA ribosomal genes to the skin biopsies from patients with STARI.

Inflammatory innate immune responses are critical in the control of early disseminated infection, while adaptive immune responses are vitally important, particularly the humoral immune response, in controlling spirochete levels in tissues and resolution of Lyme arthritis in animal models. We are examining the antibody response to immunogenically dominant antigens of B. burgdorferi in PLDS patients and controls.

Further investigation of the anti-borrelia immune response may help in elucidating the pathogenic mechanism of PLDS and yield important information for future approaches to diagnosis and treatment. We have a clinical protocol in which we use DNA microarrays to characterize gene expression patterns in skin biopsies from individuals with EM, with the aim of capturing the human host response to pathogen exposure.

We are also investigating the differences in immunological response between predominantly lymphocytic meningitis and predominantly neutrophilic meningitis. Results from these studies will serve as a window into the fundamental biology of the infection.

Link: http://projectreporter.nih.gov/project_info_description.cfm?aid=8336099&icde=12284856

Comment:

The existence of the VOVO LIPS test is nothing new - reports on the development of this test have been around since 2010. Also, there is already information about a chronic Lyme disease xenodiagnosis study out there.

It seems like this project has a large scope - or consists of more than one project under the same umbrella. So far, no project end date has been posted for this entry.

What would be of most interest to me would be finding differences in immunological response between patients with acute Lyme disease and those with assumed PLDS - something Alaedini has already been studying.

(Side note: I thought that it was already determined that Borrelia lonestari, a relapsing fever spirochete, was the cause of STARI or Masters disease - did I miss something?)

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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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0 Lyme Disease Presents Differently In Women Compared To Men

Recently, Lauren A. Crowder, M.P.H. reported observations on some differences between women and men in response to Lyme disease in a poster at the International Conference on Emerging Infectious Diseases.

The short story: Women with Lyme disease display more clinical symptoms than do men with the disease and also are less likely to seroconvert following treatment, according to findings from a prospective cohort study involving 77 patients.


The study revealed the following observations:

• Significantly more women than men reported joint pain, muscle pain, headache, back pain, heart palpitations, nausea, vomiting, anxiety, numbness and tingling, and changes in vision during at least one of six preplanned study visits with a physician.
• At the initial study visit, a similar proportion of men and women (about 60% of each) tested negative for Lyme disease using the Centers for Disease Control and Prevention’s recommended two-tier testing criteria for serodiagnosis. At the first post-treatment interview, 70% of women who tested negative at the first pre-treatment visit remained negative, compared with only 35% of the men who initially tested negative.

Read more about this Lyme Disease Foundation funded study here:
http://www.internalmedicinenews.com/news/infectious-diseases/single-article/lyme-disease-presents-differently-in-men-and-women/1bf48578d5.html

And see the original source with study here:

SEE page 151 of ICEID 2012 Abstracts
March 11-14, 2012 | Hyatt Regency Atlanta | Atlanta, Georgia
(PDF) http://www.iceid.org/images/iceid_2012_finalprogram_final.pdf

Board 264. Another Difference between Boys and Girls: Sex-Based Differences in Lyme Disease.
L.A. Crowder, A. Rebman, V. Yedlin, M. Soloski, J.N. Aucott; Lyme Disease Research Foundation of Maryland, Lutherville, MD, USA, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.

This isn't the first time, however, that someone observed a difference between men and women's immune responses in relation to Borrelia burgdorferi.

Let's take the time machine back to Sweden, in 2004...

Lyme borreliosis reinfection: might it be explained by a gender difference in immune response?
Sara Jarefors, Louise Bennet, Elin You, Pia Forsberg, Christina Ekerfelt, Johan Berglund, and Jan Ernerudh
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1782288/

This study had a different goal than Ms. Crowder's in that it was intended to measure the difference in immunological response between people of both genders who had only been infected once and those who had been reinfected with Lyme disease within a five year period.

The findings relevant to women in this case:

"...for the immunological response there were major differences between men and women. The women displayed higher spontaneous secretion of all cytokines measured, i.e. IL-4, IL-6, IL-10, IFN-γ and TNF-α. Spontaneous secretion, at an infection-free time-point, reflects the habitual immune status and may suggest what type of immunological defence an individual generally displays. For instance, allergy has been considered a Th2-type related condition and, accordingly, atopic individuals have higher spontaneous IL-4 expression than non-atopic controls.

Women of reproductive age are believed to handle infections better than men, having a stronger tendency to show Th1-type responses and expression of higher levels of pro-inflammatory cytokines, and they also develop higher antibody titres than men when vaccinated. However, the female immune response fluctuates with the menstrual cycle. In general, oestrogen has a stimulatory effect on the immune system whereas testosterone acts as a suppressor. When women enter the menopause their levels of oestrogen decrease and thereby the stimulatory effect diminishes, leading to an altered immune status. All except one of the women in our study were postmenopausal, and this could be a factor explaining why more women than men became reinfected with B. burgdorferi."

And...

"Serology was not performed on the individuals in this study because, at the time of EM diagnosis, only 30–40% of patients displayed antibodies to Borrelia. Studies following patients with culture-confirmed EM have shown that, although antibodies can be detected 10–20 years after initial infection, titres decline gradually during the first year."

A paper which cited the previous one discusses the functions of IL-10 in relationship to Borrelia burgdorferi:

Interleukin-10 alters effector functions of multiple genes induced by Borrelia burgdorferi in macrophages to regulate Lyme disease inflammation.
Gautam A, Dixit S, Philipp MT, Singh SR, Morici LA, Kaushal D, Dennis VA.

Source: http://www.ncbi.nlm.nih.gov/pubmed/21947773

To sum it up: IL-10 (an interleukin) which is produced in higher amounts in women than it is in men, is responsible for inhibiting the actions of some genes in Borrelia burgdorferi - but it is also responsible for empowering the actions of some genes, too.

What implication this has on infection in different genders remains to be seen and requires more study.

But what is already known about the role of inflammation in the presence of Borrelia burgdorferi is important to take note of here: Inflammation facilitates Borrelia burgdorferi's adaptation to its host; it stimulates antigenic variation and it leads to increased spirochetal burden in mice. So if this applies to humans: All that pain, swelling, and inflammation patients feel? It is good for the spirochetes, and it is bad for you.

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6 Viral Genetics Issues Latest Report On Chronic Lyme Clinical Trials Status

Recently, Viral Genetics posted an update on its move towards testing its product, VGV-L, for treating chronic Lyme disease.

Research for this product was funded by two Lyme disease advocacy organizations, Time For Lyme, Inc. and the Turn The Corner Foundation.

You might be familiar with the project lead, Dr. Karen Newell Rogers, whose research was mentioned at the LDA-Columbia University Lyme 2011 Lyme and TBD Conference.

I previously wrote about Dr. Newell Rogers research, and highlighted the following excerpt from a summary report on her conference presentation:

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

She pointed out: 

"[...]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."

So what is VGV-L, and what does this soon-to-be-tested product supposedly do? It would seem to fall more in line with Dr. Newell Rogers' research addressing chronic inflammation than addressing chronic infection, based on what I've read thus far.

According to Viral Genetics' web site, VGV-L seems to be related to Targeted Peptide Therapy (TPT), a therapy which uses synthetic peptides to "trick" cells that may be responsible for harmful symptoms and makes these cells vulnerable to the body's natural immune response mechanism.

What exactly are targeted peptides? According to Viral Genetics (VG), targeted peptides are custom-designed protein fragments that work to modify certain immune system reactions that they believe cause or worsen some inflammatory diseases.

More about Targeted Peptide Therapy is found on its own VG page:

 "Autoimmune diseases occur when the body reacts to itself or self-tissues. In some cases, an external threat from disease-causing organisms activates too many of certain types of immune cells which in turn cause damage the body. A physical trait of those cells also makes them impervious to the body's natural defense system that would ordinarily limit their numbers.  

TPT works by tricking those impervious cells into dropping their defenses. They can be fooled into releasing their protective shields, swapping the shield for a synthetic TPT-polypeptide instead. Those peptides, created by our research team, have been engineered to make the cell susceptible to the body's natural defenses. We expect our TPT drug compounds to enable the body to destroy the cells that help trigger the symptoms of autoimmune diseases." 

 So what this sounds like is it a treatment for chronic Lyme disease which lessens inflammation by modifying the immune system in some way.

And judging from the content above, I am wondering if the "certain types of immune cells" of which there are too many could be referring to the overabundant low quality plasma cells that were found in lymph nodes in Tunev and Barthold's study, "Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation."?

So what is the status of getting VGV-L's TPT to the clinical trial stage?

Excerpts from its letter to shareholders indicated that due to the time required for coordinating schedules, screening clinicians, and presenting the data, that it was only "recently that we neared finalization of securing a clinician to act as lead on this program. We expect to be able to discuss this in much more detail very soon, but I am very confident that we will be filing our pre-IND for our Lyme disease candidate this year."

 For those who are reading along who do not know what a pre-IND is, "IND" stands for Investigational New Drug, and any clinician who wishes to begin clinical trials to test a new drug must file for approval from the FDA.

From the FDA's own site:

"During a new drug's early preclinical development, the sponsor's primary goal is to determine if the product is reasonably safe for initial use in humans, and if the compound exhibits pharmacological activity that justifies commercial development. When a product is identified as a viable candidate for further development, the sponsor then focuses on collecting the data and information necessary to establish that the product will not expose humans to unreasonable risks when used in limited, early-stage clinical studies.

FDA's role in the development of a new drug begins when the drug's sponsor (usually the manufacturer or potential marketer) having screened the new molecule for pharmacological activity and acute toxicity potential in animals, wants to test its diagnostic or therapeutic potential in humans. At that point, the molecule changes in legal status under the Federal Food, Drug, and Cosmetic Act and becomes a new drug subject to specific requirements of the drug regulatory system."

(For more specifics, see: http://www.fda.gov/drugs/developmentapprovalprocess/howdrugsaredevelopedandapproved/approvalapplications/investigationalnewdrugindapplication/default.htm)

I am finding this all very interesting and will continue to watch Viral Genetics as they move towards the trial phase.

I wonder what requirements will need to be met for study participants - and how many chronic Lyme disease patients would be willing to sign up?

Would you?

Additional Information: http://www.viralgenetics.com/investors/2012-Letter-to-Shareholders.pdf

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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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