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 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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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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0 Blog Log: Spirochetes Unwound On Persisters

Well, our favorite spirochete blogger has posted about the magic of antibiotic tolerance today - an apropos choice for this evening's post to follow on the heels of the discussion about Embers et al paper, "Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection".

While this entry on persisters in Spirochetes Unwound is not specifically about Borrelia, it is an interesting read for those who wish to learn more about the mechanisms related to antibiotic tolerance and how some bacteria can be dormant and survive its wrath.

Excerpt:

"Bactericidal antibiotics are effective at killing proliferating bacteria as long as they don't carry mutated or acquired genes that encode resistance to the antibiotics. Unfortunately even antibiotic-sensitive bacteria can tolerate antibiotics under some circumstances. Bacteria that are in a nondividing "dormant" state often survive antibiotic exposure. When the antibiotic is removed and growth resumes, the bacteria regain susceptibility to antibiotics.

At first glance antibiotic tolerance appears to be a passive process in which nondividing cells survive simply because the target of the antibiotic is inactive. However, this is not correct. Antibiotic tolerance requires an active response by the bacteria. The nondividing bacteria that survive antibiotic treatment are called persisters. Persisters may account for infections that are difficult to eradicate with antibiotics..."

Read more here: http://spirochetesunwound.blogspot.com/2012/02/magic-of-antibiotic-tolerance.html

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1 Patent: Viral Genetics Chronic Lyme Disease Treatment

This post is related to an earlier post about Viral Genetics application for a pre-IND to begin clinical trials on their VGV-L product for the treatment of Chronic Lyme disease.

See: http://campother.blogspot.com/2012/02/viral-genetics-issues-latest-report-on.html

Apparently, I found Viral Genetics' current patent. And as I had previously speculated, I was correct that the treatment is going to modify B cells. I knew it! Buy me a drink, eh?

Here is a link to the patent online: http://www.faqs.org/patents/app/20100166789

Patent application title: PROTEINS FOR USE IN DIAGNOSING AND TREATING INFECTION AND DISEASE

Inventors: Haig Keledjian (San Marino, CA, US) Michael Agadjanyan (Huntington Beach, CA, US) Martha Karen Newell (Colorado Springs, CO, US) Evan Newell (Menlo Park, CA, US)
Assignees: THE REGENTS OF THE UNIVERSITY OF COLORADO Viral Genetics, Inc.
IPC8 Class: AA61K3900FI
USPC Class: 4241851
Class name: Amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same
Publication date: 07/01/2010
Patent application number: 20100166789

And here are the excerpts on how the invention will be used to treat chronic Lyme disease (patent text slightly modified to correct spelling errors):

[0114] Lyme Disease is a tick-borne disease caused by bacteria belonging to the genus Borrelia. Borrelia burgdorferi is a predominant cause of Lyme disease in the US, whereas Borrelia afzelii and Borrelia garinii are implicated in some European countries. Early manifestations of infection may include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. Long-term the disease involves malfunctions of the joints, heart, and nervous system. Currently the disease is treated with antibiotics. The antibiotics generally used for the treatment of the disease are doxycycline (in adults), amoxicillin (in children), and ceftriaxone. Late, delayed, or inadequate treatment can lead to late manifestations of Lyme disease which can be disabling and difficult to treat.

[0115] A vaccine, called Lymerix, against a North American strain of the spirochetal bacteria was approved by the FDA and later removed from the market. It was based on the outer surface protein A (OspA) of B. burgdorferi. It was discovered that patients with the genetic allele HLA-DR4 were susceptible to T-cell cross-reactivity between epitopes of OspA and lymphocyte function-associated antigen in these patients causing an autoimmune reaction.

[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.

[0117] Chronic Lyme disease is sometimes treated with a combination of a macrolide antibiotic such as clarithromycin (biaxin) with hydrochloroquine (plaquenil). It is thought that the hydroxychloroquine raises the pH of intracellular acidic vacuoles in which B. burgdorferi may reside; raising the pH is thought to activate the macrolide antibiotic, allowing it to inhibit protein synthesis by the spirochete.

Read more: http://www.faqs.org/patents/app/20100166789

How will this theoretically stop inflammation present in chronic Lyme disease? What does the above all mean, in English? More details coming soon - for now I wanted to share this.

In the meantime, you might want to review this: http://en.wikipedia.org/wiki/Thymus

Image credit: Thymus by LearnAnatomy from Wikipedia under a CC 3.0 license.

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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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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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0 News: New Molecular Test Could Detect Early Lyme Disease

The Guardian has written about a new molecular test using nanoparticles which was developed by Alessandra Luchini, of George Mason University, to initially detect cancers. The new test is being used in clinical trials to detect early Lyme disease - even if there is no rash present in the infected patient.

Excerpt that will be of interest to readers:

"Q: In years to come, is this something that could be available in hospitals? 

That's the hope we have. The first clinical trial is on the detection of Lyme disease. A fraction of patients get a skin rash but for those without the rash it is very difficult to diagnose. So with the particles we are able to capture the antigens that come from the spirochaete that is the causative agent of Lyme disease. If we see in the urine a piece of the bacteria of the spirochaete, we are sure that the patient has Lyme disease. We are gathering all the evidence and then we will need to go first for FDA approval before it is available in clinics. 

Q: How much earlier will you be able to detect Lyme disease? 

Lyme disease has a window of two to three weeks before seroconversion [production of antibodies in the host blood, indicating infection]. With our tests, we're able to detect it before seroconversion, because we're not looking for the antibodies, we just look for the spirochaete. I would say here, yes, by weeks, and earlier diagnosis would be beneficial for the prognosis."

Comment: One thing this does make me wonder about is how similar this test is to Temple Douglas' hydrogel nanoparticle test for early detection of Lyme disease. Maybe it's time to do a compare and contrast of patent application content?

MORE here at the link: http://www.guardian.co.uk/technology/2011/oct/23/bright-idea-nanoparticle-trap-cancer

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1 The Curious Case Of Cholesterol

This article got passed on to me:

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

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

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

Excerpt:

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

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

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

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0 How To Avoid Sample Contamination

A quick and dirty primer for those who want to ensure that their samples do not get cross-contaminated with DNA from other bacteria, viruses, etc.:

1. Use aerosol barrier pipette tips.
   
2. UV-irradiate all workstations used for the setup of master mix preps and PCRs.
   
3. Treat all surfaces and tube racks with a 10% bleach solution.
   
4. Use frequent and careful glove changes.
   
5. Perform DNA extraction, PCR setup, and PCR product analysis in different rooms.
   
6. Use clean systems.
7. Use a negative control such as UV-treated, deionized water.
8. Do not do bacterial work, etc. during any human DNA extraction.

Follow these simple tips, and then when others look at your research and claim that Borrelia burgdorferi could only be found where it was due to  sample contamination, point out that a number of steps were taken to prevent it.

At what point when an experiment is repeatedly reproducible does one stop saying the end result was due to sample contamination and begin saying the end result was genuine?

What other steps can you take to prevent and eliminate sample contamination?

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34 News: Institute of Medicine Releases Lyme Disease Workshop Summaries

Source link: http://www.iom.edu/Reports/2011/Critical-Needs-and-Gaps-in-Understanding-Prevention-Amelioration-and-Resolution-of-Lyme-and-Other-Tick-Borne-Diseases.aspx?utm_medium=etmail&utm_source=Institute+of+Medicine&utm_campaign=04.20.11+Report+-+Lyme+Disease+%26+Other+Tick-Borne+Diseases&utm_content=New+Reports&utm_term=Media

To read the Workshop Summaries, click on the link at the top and look in the righthand column - you'll see gray buttons that say "Download Report" or "Read Report Online For Free". If you want to download a pdf of the report, you'll need to supply your email address and other info. If you just want to view it online, click "Read Report Online For Free" and there will be no request for personal information.

Once you select "Read Report Online For Free", by the way, you have an opportunity to download a 21 page summary of the report (direct download) by clicking on a link for it on the lefthand column. Otherwise, the full report is 468 pages long.

[CO update: The 21 page summary gives the barest of outlines of what was discussed, along with a list of those who attended - you are much better off reading the 468 page report.]

One thing to note here is that this appears to be a report on what was discussed at the Institute of Medicine (IOM) workshop on tickborne diseases in October 2010 and not a final position piece stating the IOM's consensus on how Lyme disease and tickborne infections should be approached through research, prevention, or treatment.

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5 One way to treat Borrelia naturally?

Many Lyme disease patients have used antibiotics for treating Lyme disease and other tickborne coinfections. They have years of scientific study behind them and many reports of patient improvement come from doctors and specialists - patients have had a lot of success with them.

But sometimes antibiotic use leads to various side effects, digestive problems, and potentially, undesirable secondary infection with C. difficile. Using probiotics can often help with digestive problems and prevent C. difficile, but it is not guaranteed.

In some cases - due to allergies or intolerance of the side effects - patients have to stop antibiotic treatment. Because of this, patients have opted at some point in their treatment to stop taking antibiotics after a while and switch to alternative treatments such as herbs.

Whether a patient decides to use antibiotics or herbs, one thing on the horizon seems certain: Eventually antibiotic resistance will lead to more restrictive use of antibiotics, and antibiotic resistance may challenge patients' ability to treat some of their own infections.

However, there is one completely natural possibility that might treat Borrelia and some other tickborne infections in the future which is rarely mentioned in the west other than as a curiosity - yet everyone in the world is surrounded by this abundant and prosperous source of healing from nature all the time.

Much as there are different probiotic bacteria are found in yogurt and probiotic supplements that Lyme disease patients take -- there are viruses in our environment that are helpful to us.

A lot people think of a few things when they hear the word "virus": they think of H1N1 or the swine flu, colds, herpes, HIV, and meningitis, for a start. Not good things. But like the probiotic bacteria that we consume in yogurt all the time, viruses are also present in our environment - in our food, our soil, our drinking water, and our own digestive systems.

Like adding probiotic mixes to your yogurt, these helper viruses have been approved by the FDA to be sprayed on the surface of cheese across the US in order to prevent the development of the bacteria, Listeria monocytogenes, from causing serious disease in pregnant women,  immunocompromised people such as cancer patients, and those with immuno-deficiences. Thousands of people can be severely sickened by Listeria and in some cases even die. So the use of these viruses in food such as cheese is beneficial.

In addition to providing protection from harmful bacteria in food, these helpful viruses have also been used to help save baby calves from dying of diseases which cause severe diarrhea and prevent salmonella from colonizing chickens.

The method for treating these cases was find out which bacterial strains the animals were infected with in order to find the viruses which would eat them. Then use these viruses just as they are found in nature, with no genetic engineering required - put the viral material in pills, injections, or lotions in order to treat the infection.

So this leads one to wonder if this all-natural, non-GMO treatment which is low-cost compared to antibiotics and so abundant in nature can kill off bacterial infections in animals - why can't they kill off infections in people too?

Well, they can.

Watch the next two videos, paying special attention to the first video.


The first video is a 48 minute BBC documentary on the use of viruses to kill bacteria, also known as "bacteriophage therapy" in the former Soviet republic of Georgia, in the Eliava Institute of Tblisi.

Note that if the institute seems run down, filming was done after the collapse of the Soviet Union and the hospital just came out of a civil war - thus buildings had poor maintenance, but the technology to use bacteriophage therapy was in place and used. (After a period of economic instability and social problems - followed by the Rose Revolution - Georgia and Tblisi have been doing much better in the past several years.)

So this documentary is a little dated but general principles remain the same - it explains very well what bacteriophage therapy is and how it has been used in Europe for over 60 years through the 1990's (it continues to be used today - more on recent research using phages will be posted this week).

Youtube (3 parts)

BBC Horizon - 1997 - The Virus That Cures

This second video is from Canadian television as well as CBS news and is more recent - it contains two clips back to back about two people who were treated with phage therapy and their results. Don't miss it - the results are amazing when you realize the initial prognosis each patient was given.

Case studies on phage treatment plus Evergreen College, 

Washington State phage research - [Time: 9:26 minutes]

Is bacteriophage therapy this effective? Does it have any pitfalls? Why don't we hear more about it here yet, given the rising number of cases of antibiotic resistance to deadly bacteria such as MRSA? What can it treat so far? How can this treatment help Lyme disease patients in the future? Here's just one more video just to get a different angle on it from Australian news (Channel 7 and Channel 9). It talks more about history, plus business investments and projections for human trials...

The Forgotten Cure - on Sunday Sunrise, Channel 7 - 

plus a short clip on phages from Channel 9

More on this later this week - for now, check out the videos and let me know what you think, including your own questions and concerns about this kind of medical treatment. [CO note: Continue reading part two of this series, "Phage Therapy and Borrelia burgdorferi".]

This work by Camp Other is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

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13 Books: Borrelia. Plus a lesson in terminology.

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

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


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

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

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

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

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

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

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

Translation for biology/genetics beginners:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Its significance in Borrelia burgdorferi:

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

phage particles in Borrelia burgdorferi
from Eggers et al

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

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

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

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

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

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

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

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

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

Translation for biology/genetics beginners:

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

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

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

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

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

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

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

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

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

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

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

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

Translation for biology/genetics beginners:

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

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

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


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

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

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

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

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

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

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

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

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

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

Translation for biology/immunology/genetics beginners:

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

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

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

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

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

protease = an enzyme that breaks down proteins.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Onward...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Not.

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

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

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