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.
"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?
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...
On June 26, 2012, WBUR, Boston's NPR (National Public Radio) station, 90.9, began publishing a series, "Living With Lyme", on their website.
The series of articles are accompanied by free downloadable podcasts and sometimes photos and slideshows. In addition to these articles, a live streaming video discussion on Lyme disease was broadcast on June 28 and is available online in its archives.
Additional Lyme disease related articles continue to be published on WBUR after the initial series was posted.
Here is a comprehensive list of all the articles published in the "Living With Lyme" series, from the oldest to the newest post:
For a series on Lyme disease, it is surprising how few patients have left comments on a number of these posts to date. It's been my observation that most of the time, patients participate in commenting on articles about Lyme disease and ticks far more frequently than this series has been responded to so far.
There are a few exceptions, such as the vaccine thread, which I commented on some days ago and which is still receiving more new comments. Sometimes the comments are more informative than the article itself, so they are worth a look. (Other times, they are educational only as a magnifying lens under which one can view other people's psychology... use your judgment, do your own research, and weigh the evidence linked to what people have to state.)
Infection with B. burgdorferi elicits robust, yet disparate antibody responses in infected individuals. A longitudinal assessment of antibody responses to multiple diagnostic antigens following experimental infection and treatment has not previously been reported.
Our goal was to identify a combination of antigens that could indicate infection at all phases of disease and response to antibiotic treatment. Because the rhesus macaque recapitulates the hallmark signs and disease course of human Lyme disease, we examined the specific antibody responses to multiple antigens of B. burgdorferi following infection of macaques.
Five macaques infected with strain B31 and 12 macaques infected with strain JD1 were included in the analysis. Approximately half of these animals were treated with antibiotics at 4-6 months post-inoculation.
Antibody responses to several B. burgdorferi recombinant antigens, including OspC, DbpA, BBK32, OspA and OppA-2 were measured at multiple points throughout infection. We have previously shown a decline in the response to the C6 peptide following antibiotic treatment.
Responses to OspA and OspC, however, were variable over time among individuals, irrespective of antibiotic treatment. Not every individual responded to BBK32, but anti-DbpA IgG levels were uniformly high and remained elevated for all animals. All responded to OppA-2, with a decline post-treatment that was slow and incomplete. This is the first demonstration of B. burgdorferi OppA-2 antigenicity in nonhuman primates. The combination of DbpA, OspC, OspA, and OppA-2 with the C6 diagnostic peptide has potential to detect infection throughout all disease phases.
Today Viral Genetics posted a letter to shareholders with the following information which relates to their experimental chronic Lyme disease candidate, VGV-L.
The letter stated:
"We are still finalizing scheduling a meeting with the FDA and our team to discuss our Lyme disease pre-IND submission from earlier this spring. Scheduling has been difficult with the number of people involved and the looming summer season. Following this meeting we expect to have a clear road map to follow towards clinical trials"
I am looking forward to hearing more about this meeting. It seems there have been a number of delays on the pre-IND road for VGV-L so far, though, and I can't predict when one is going to see progress on getting this treatment to trial stage.
Now might be the time to contact Viral Genetics and begin asking them questions about what sort of ideas they are coming up with in terms of clinical trial design. As a chronic Lyme disease patient reading along, your input may be informative and useful for researchers.
Stay tuned for more updates on VGV-L as I get them.
Last year someone who identified only as "anonx" wrote some interesting comments on last January's blog post on quorum sensing, "How bacteria "talk" and how to make them shut up". (There is a fascinating video there by researcher Bonnie Bassler. I highly recommend checking it out.)
To my knowledge, comments on Blogger do not get syndicated on post rss feeds - though one can request rss comments separately. I think that anonx's comments are worth taking another look, so I have decided to repost select excerpts of them here. (Refer back to the original comment thread for the CO side of the dialog, if needed.)
Anonymous January 21, 2011 5:06:00 PM HST
persister cells
quorum sensing
round bodies
blebs
biofilms made of borrelia
biofilms made of a mix of pathogens
symbiosis in all its forms and iterations
intracellularity
the cascade of genospecies and strains
quiescence and dormancy
transfections
borrelia stuck in B cells with CLIP attached
the role of toll-like receptors&inflammation
the growing list of possible co-infections
xmrv
new view of PANDAS that goes way beyond strep...
molecular mimicry
guess I'll stop here but I could go on --you just need a little proof: not just proof it is happening but proof that treating it will end the illness. that's the problem.
Anonymous January 21, 2011 11:03:00 PM HST
Don't you think that looking at persistence in isolation could be futile, given the large number of people treated for years without cure? Persistence could be the imperceptible spark that incites an immune tsunami. What if it is easier to treat the tsunami --by its nature outsized and blatant and in your face-- than the spark which, though present and incendiary, we cannot find? Is it possible that the yin and yang nature of the fight has prevented a view of the system overall? What if a tiny amount of infection has caused a huge inflammatory response? What if that infection is hiding in B cells because a glitch in the system has prevented T cells from recognizing the foreign invader, and thus, from finishing the job? What IF to get rid of the persistent infection, you must treat immune dysfunction first? So no ...I don't think it is as simple as you say, or that by conceiving the issue along the old paradigms of the fight, raging fruitlessly for 30 years, you will get what you want ... unless of course, it is really as simple as you suggest. I mean, what if you prove persistence, but it still won't bring a cure?
Anonymous January 22, 2011 11:57:00 AM HST
regarding the four trials cited, they do in fact show benefit to retreatment for fatigue and pain --and thus, in fact do not match with the wording of the conclusions rendered or (in the case of two of three authors) forced down those authors throats by the powers that be. There is actually ample evidence in these trials to suggest a benefit to retreatment and longer treatment --and in the case of the Klempner trial, serious issues with methodology. But as you say, the outcome of these trials must be detached from pathophysiology --what is the mechanism of the benefit? That question the trials do not address.
Still, to detach infection from immune response is just plain wrong-headed. We are 10% by weight bacteria, and most of those organisms are beneficial. The question is --which are the pathogens, and how do those pathogens do us in? In every case, inflammation and cellular immunity are going to play a role.
Anonymous January 22, 2011 11:15:00 PM HST
It is a very complicated problem, as you say.
It is TRUE that if you suppress inflammation infection would spiral out of control: The murine studies on borrelia and toll-like receptors show that to be right.
But here's the thing: Most researchers, even most of the IDSA researchers, don't actually deny that organisms can persist. They just deny that those quiescent remaining organisms are driving the continued symptoms --they say these persisting borrelia are too few, and too dormant. And they would cite the research on quorum sensing to support their case.
As I see it, it is not really persistence you need to prove, but rather, the mechanism by which persistence at the low level research suggests drives the disease. Inflammation is not the only immune mechanism --cellular immunity or dysfunction thereof can play havoc, too, and persistent infection could drive it in an endless loop.
Also: Treatment studies without knowing more about pathophysiology can work against you, because --hell-- they are just empiricism on top of empiricism, more wandering in the dark.
I contend you need more basic biology to target such studies, strategically. If you were to move forward without that, you would need an elaborate methodology with many variables and large enough numbers of patients to test for many possibilities and separate the data from the noise. And with Lyme patients still so ill-defined, with no test extant for active infection ...
Makes my head spin. A hundred million dollars would help.
Anonymous January 23, 2011 1:34:00 PM HST
Other,
My comments refer specifically to the Barthold work, with which I am extremely familiar. Barthold's findings of small numbers of dormant, quiescent spirochetes within collagen across the range of mammalian species following treatment have been well-known inside the mainstream (though published only recently) for decades. There is no great rush to debunk Barthold, whose research really is beyond dispute --Barthold himself being an especially meticulous and careful scientist. What his critics say, however, is that these spirochetes are not active enough and not numerous enough to cause disease. (to wit: issue of quorum sensing.) Barthold theorizes otherwise, contending that the small numbers of chetes may provoke an outsized --but heretofore undetected-- cytokine cascade that causes the disease. Barthold would classify this cascade under the heading of INFLAMMATORY response. This is his theory --and a powerful one that should be explored.
You may have noticed that NIH has begun to test the Barthold work with a study of xenodiagnosis, but patients are protesting that study for fear that the ticks used might not be as naive as claimed: And really, given the confusion over pathogens involved, who knows?
Other theories of persistence to pursue include the impact of round bodies AND the work of Newell, who finds Borrelia stuck inside B cells because of a dysfunction in MHC.
In the case of Newell, especially, the notion is that persistent infection can never be cured without correcting the recognition dysfunction of MHC. In other words, even though the disease is driven by persistence, Newell says you have to correct the immune problem first.
And by the way, both she and Barthold insist you can never entirely clear borrelia infection with antibiotics alone --you need the immune system to do the final kill, and so you must have an immune correct FIRST, even if the driver is persistence.
Or it could be the round bodies... but whatever it is, it is complicated --and simply fueling the fight of persistence versus immunity isn't helpful.
There is a difference between what patients use to get well right now --the ax in the form of huge quantities of endless suppressive antibiotics-- and what they should want for the future --the chisel, which could well be an immune correction that allows infection finally to be resolved. There should be a divide between the effort to protect a Lyme doc in the here and now and the direction of research for the future --but it is hard for a lot of people to understand this.
I agree with you that if we knew the mechanism we would have a target --and that is why I am so equivocal if not outright squeamish about the continued treatment trials some patients are calling for.
What are they treating? --and if they don't really know, there is a big risk that study could bury them deeper and darker than ever before.
anonx
Anonymous January 23, 2011 7:27:00 PM HST
Given current state of knowledge immune treatments could backfire, big time --as the literature shows. If you look at the work on toll-like receptors you find a genetic curve for inflammatory response to borrelia ranging from almost nothing to off-the-charts and everything in between. That is just one immune parameter, and there are many others. These parameters could vary for every infection or strain and every person. Therefore it is possible with current state of knowledge that suppressive abx are really the best we have... there needs to be a crunching of data to understand what we are looking at --otherwise, it is just stumbling in the dark. The amazing thing is that the IDSA crew has gotten away with such a grotesquely oversimplified story of this disease for so long --and that to explain it, they perpetuate the explanation that it is a psychosis instead of a complex spectrum of infection and immune response. But by giving an oversimplified rejoinder, patients have hurt their cause, too.
In a gross way one could do a study treating infection, treating immune issues or treating both: but this would be very crude without more data up front.
anonx
Comments:
So I do have a few comments on this, now that it's nearly a year and half since these comments were posted. My thoughts on the matter have shifted over time, and after exposure to more research.
I'd like to see evidence that Borrelia burgdorferi is hiding in B cells in vivo. That would be informative. There has been some mention of Bb being intracellular in a few in vitro studies and one in vivo study; we need more.
This anonymous author may be on to something, and it may be that the host immune response may need to be addressed in order to manage the remaining infection if it is still present. Not enough is known, but when I read about filgrastim and rixtuximab and how they have some positive effect on a patients with persisting symptoms of Lyme disease or CFS/ME, I think that adjusting the host immune response is an avenue worth exploring. It should have been explored more years ago.
I still think the trials must be detached from pathophysiology. My position on this has not changed. Treatment trials have done nothing to provide evidence of persistence of Borrelia burgdorferi one way or the other.
I strongly agree we need more basic biology research. The anonymous author's comments on the role the immune system plays in infection are noteworthy. But we also need to know what is going on if the infection does persist in some form. Is there a persister phenotype? Ongoing research into persister phenotypes should not be neglected. But I wouldn't leave all research at that, because there are other hypotheses to consider.
One thing I wonder about is the issue of quorum sensing and efficiency sensing, and if blebs or vesicles play any role in the dissemination and pathogenesis of Borrelia burgdorferi. Blebs as a form of communication are observed in other bacterial species, and perhaps Bb uses blebs and vesicles in a different manner and they are not part of cells undergoing apoptosis. Plasmid DNA and outer surface lipoproteins have been found within blebs; there is some suggestion of blebs containing adhesins... I think there's more to blebs than meets the eye.
Anonymous said, "There is a difference between what patients use to get well right now --the ax in the form of huge quantities of endless suppressive antibiotics-- and what they should want for the future --the chisel, which could well be an immune correction that allows infection finally to be resolved. There should be a divide between the effort to protect a Lyme doc in the here and now and the direction of research for the future --but it is hard for a lot of people to understand this." I keep reflecting on what they wrote, and its implications. Is there a more targeted approach which could be designed to help treat patients?" I think they are right - there is no reason why patients cannot ask for protection for the treatment they are receiving now while promoting research on new and different treatments. I think VGV-L is one effort in this direction, but I am not sure it will work. There is a lot of complexity involved.
Last but not least, my anonymous commenter said this: "The amazing thing is that the IDSA crew has gotten away with such a grotesquely oversimplified story of this disease for so long --and that to explain it, they perpetuate the explanation that it is a psychosis instead of a complex spectrum of infection and immune response. But by giving an oversimplified rejoinder, patients have hurt their cause, too."
I think there's a lot of truth in that last statement. Now, what does one do about it?
Just recently, the Lyme disease advocacy group, Time For Lyme - which is credited with creating a partnership with Columbia University Medical Center and the national Lyme Disease Association to establish and endow a Lyme and Tick-borne Disease Research Center in New York City at Columbia - changed its name to the "Lyme Research Alliance".
The name change came about as an effort to reflect the organization's growing emphasis on supporting more research into chronic Lyme disease and how to improve serological testing and treatment.
See this page to view a list of their currently funded research projects:
I've checked out the Lyme Disease Alliance's new web site, and I approve of their overall mission and think it was fantastic they gave $3 million to fund the Columbia Lyme and Tick-borne Disease Research Center.
One thing I'm hoping to see with the change in name and greater commitment to research is a shift in the kind of accomplishments they had in the past, where videos, education packets, and legislation has been a focus - to a focus in assessing what kind of research projects would settle the Lyme disease controversy as well as focus on backing research on more novel treatment methods, such as they have done with VGV-L.
It is my dream that someday all the different Lyme disease organizations will unite for a single effort and come up with one large scale, fully funded Lyme disease research project that will help thousands of patients. I'd like to see focus on that project alone for a solid two or three years and see how far we can get in our understanding of Lyme disease.
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:
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.).
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:
"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
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.
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.
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.
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?)
Credit goes to Joanne, of the Looking at Lyme blog, for mentioning this abstract: It describes a response to Borrelia burgdorferi infection in mice where the first immune response almost clears the infection - but approximately 1 week post infection, the bacterial population recovers and reaches an even larger size before entering the chronic phase.
Front Microbiol. 2012;3:104. Epub 2012 Mar 22.
Population Dynamics of Borrelia burgdorferi in Lyme Disease.
Binder SC, Telschow A, Meyer-Hermann M.
Source
Department of Systems Immunology, Helmholtz Centre for Infection Research Braunschweig, Germany.
Abstract
Many chronic inflammatory diseases are known to be caused by persistent bacterial or viral infections. A well-studied example is the tick-borne infection by the gram-negative spirochaetes of the genus Borrelia in humans and other mammals, causing severe symptoms of chronic inflammation and subsequent tissue damage (Lyme Disease), particularly in large joints and the central nervous system, but also in the heart and other tissues of untreated patients.
Although killed efficiently by human phagocytic cells in vitro, Borrelia exhibits a remarkably high infectivity in mice and men. In experimentally infected mice, the first immune response almost clears the infection. However, approximately 1 week post infection, the bacterial population recovers and reaches an even larger size before entering the chronic phase.
We developed a mathematical model describing the bacterial growth and the immune response against Borrelia burgdorferi in the C3H mouse strain that has been established as an experimental model for Lyme disease.
The peculiar dynamics of the infection exclude two possible mechanistic explanations for the regrowth of the almost cleared bacteria.
Neither the hypothesis of bacterial dissemination to different tissues nor a limitation of phagocytic capacity were compatible with experiment.
The mathematical model predicts that Borrelia recovers from the strong initial immune response by the regrowth of an immune-resistant sub-population of the bacteria. The chronic phase appears as an equilibration of bacterial growth and adaptive immunity.
This result has major implications for the development of the chronic phase of Borrelia infections as well as on potential protective clinical interventions.
(Special thanks to Frontiers in Microbiology journal for having what appears to be a solid peer review process and Creative Commons license.)
Comments:
I can't wait to read the full text of this paper. This is an intriguing abstract and it leads to more questions.
How did these researchers come up with their mathematical model? Have other researchers previously observed this second wave of bacteria during infection? What about evidence of peaks in immune response in Borrelia burgdorferi infected animal models which have been documented?
In which way, precisely, is the regrowing immune-resistant sub-population actually immune-resistant? What is happening to B cells and T cells in relationship to this second phase of Borrelia?
From Fig. 3 of Tunev et al., 2011. Day 8 of infection. The arrows point to intactextracellular B. burgdorferi in the subcapsular sinus of the lymph node, which was culture positive beginning on day 1 of infection. Source
And does this model have any implication for antibiotic treatment? As in: Does this second immune-resistant sub-population also have a different response to antibiotics than the initial wave? This model is about infection without treatment, and it is not discussed what the implications could be.
I posted some of my notes on VGV-L on LNE, related to the previous entry on VGV-L which I posted on Monday. While there is a lot I posted about VGV-L based on interviews, press releases, and patents - and while I outlined how the immune system normally should work - I did not give a concise explanation of how the immune system doesn't work relative to polyclonal b cell activation.
I think another entry on this topic is in order. But in the meantime, I have decided to share the notes I posted on LNE about VGV-L here before I write a more concise post on the immune system and polyclonal b cell activation...
I did as much poking around about Viral Genetics' new chronic Lyme disease candidate drug, VGV-L, as I could and it wasn't easy. One CV, several patents, and a few Dr. Karen Newell Roger interviews later, and the best I could get out of it is that it is not just a synthetic thymus peptide that they patented but a method of replacing a peptide, CLIP, on the surface of B cells with this synthetic thymus peptide (which somehow maps to the MHC genetic type (HLA) of the patient) so that it activates Treg cells. The activation of these Treg cells is supposed to lead to reduction of non-specific B cells (polyclonal B cell activation).
In the patent, there is also mention of using bacterial antigens and antibiotics as adjunct treatments which are optional. The impression I'm left with is the bacterial antigens are used to prime new B cells and if there is any existing infection, antibiotics are used.
So the entire method of treating patients may be: a) VGV-L alone, b) VGV-L and antigen exposure, or c) VGV-L, antigen exposure, and antibiotics are used. (antivirals and antiparasitics may also be used, depending on the patient's diagnosis)
[...] I don't get the impression after reading everything I've read thus far that chronic Lyme disease is a pure B-cell disorder. But maybe it's a variant on that? Maybe cell-mediated immunity is somehow affected by B. burgdorferi?
I've made a major edit on my blog entry. I still think I need a revised version of the post to be made in the future, but I am so exhausted at the moment that I think it won't be for a few days at least. Maybe call it the revised executive summary version, because the current post is rambling even relative to rambling for me.
Why do I mention this edit here? Because it is very important to note, relative to how I infer the way VGV-L has been hypothesized to work:
The edit was about Tunev and Barthold's research on lymphadenopathy in Borrelia burgdorferi infection in mice. In their research, they noticed an outsized B-cell response to the presence of spirochetes. However, what they found differed from what has been found in polyclonal B-cell activation in other infections - where it's clear runaway non-specific polyclonal B-cell activation leads to autoimmune disorders. In Tunev and Barthold's research, the outsized immune response had B-cells which were specific for Borrelia burgdorferi yet were of low quality. This is notably different from typical polyclonal B-cell activation.
"From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality. By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed. The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3. Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally. Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies. If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host."
I don't know how this difference in response would work with VGV-L. It isn't clear to me, and I have to read through more research to understand it - at least hypothetically. I also want to know if anyone else has used similar technology to treat pure B-cell disorders and if so, what the pros and cons were. If this is a completely novel invention, then it's harder to evaluate and all one can do is look at the animal trials if one has access to them and examine the underlying hypothesis for its application.
The paper that this technology primarily appears to be based on is this one:
M. K. Newell, R. P. Tobin, J. H. Cabrera, M. B. Sorensen, A. Huckstep, E. M. VillalobosMenuey, M. Burnett, E. McCrea, C. P. Harvey, A. Buddiga, A. Bar-Or, M. S. Freedman, J. Nalbantoglu, N. Arbour, S. S. Zamvil, and J. P. Antel. 2010. TLR-Mediated B Cell Activation Results in Ectopic CLIP Expression that Promotes B Cell-Dependent Inflammation. Journal of Leukocyte Biology.
Online e-Pub. July 14, 2010.
I'll just post the abstract here, and you can read the entire text at the link above:
Abstract
Infectious pathogens produce compounds called Toll ligands that activate TLRs on lymphocytes. Acute activation triggered by certain TLRs appears to "jump start" the innate immune response, characterized by the release of inflammatory cytokines and cellular expansion.
In some individuals, there is a failure to control acute inflammation, resulting in postinfectious, chronic inflammation. Susceptibility to chronic inflammation is strongly associated with an individual's MHC genes. Recent clinical trials for several autoimmune diseases characterized by chronic inflammation suggest that B lymphocyte depletion therapies dampen chronic immune activation. However, currently, there is no known mechanism that accounts for the correlation among TLR activation, MHC genetics, and a pathological role for B-lymphocytes.
Our hypothesis is that TLR-activated B cells (B cells that have been polyclonally activated in the absence of antigen-specific signals) are not controlled properly by T cell-dependent B cell death, thereby causing B cell-dependent chronic inflammation.
Here, we show that treatment with Toll ligands results in polyclonal B cell activation accompanied by ectopic expression of CLIP. Furthermore, by adoptively transferring purified CLIP+ B cells in syngeneic animals, we find that CLIP+ B cells induce production of TNF-α by host T cells. Finally, we demonstrate that CLIP-targeted peptide competition results in the death of polyclonally activated CLIP+ B cells.
I think I know at this point what my missing pieces are now:
- describe the role of Treg cells (regulatory T cells) and how they relate to killing off B cells and generating more targeted immune responses.
- describe the hypothesis that Foxp3+ Treg generation in the thymus is somehow dysfunctional in those with persisting symptoms (I think this has some relationship to the core hypothesis behind VGV-L's use).
- And I have to more clearly state that even if the Foxp3+ Treg generation is what it is happening, there may be some risk involved in proceeding with this kind of treatment. Wikipedia managed to explain some of it, and while Wikipedia is not the most reliable resource on everything, it is relatively easy to follow and this particular entry (so far) jives with what I've seen in other sources:
Plus, of course, there are the papers I link to on the blog.
The more I delve into this, the more questions I have... Tunev and Barthold's paper, in particular, lead me to wonder if the hypothesis underlying VGV-L's design developed with the scenario described in their research being taken into account. Between T & B's research plus other publications, I get the impression that in at least the animal model of immune response to Bb, that there is a mixed state response to it - It is both immune suppressing and immune stimulating. Some research has even indicated a certain amount of tolerizing is involved. (http://en.wikipedia.org/wiki/Peripheral_tolerance - anergy plus inflammation; see also http://users.ox.ac.uk/~path0116/tig/tolg2.html for a more detailed explanation)
Adding a bacterial antigen/adjuvant (and possibly antimicrobial medicine) to the treatment may be a way of working around this combination, in order to produce new B cells which are stimulated and respond specifically to Bb and not be nonspecific - and in order to eliminate any remaining infection if there is one present.
This is complicated - and while more and more is being learned about immunology every day, there are still an enormous number of unknowns...
On Friday I posted about the use of Filgrastim and Ceftriaxone for treating persisting symptoms in a Lyme disease case study as well as the use of Rituximab for treating CFS/ME. I also touched upon Viral Genetics' VGV-L or targeted peptide therapy for treating chronic Lyme disease, and wanted to write an entry about this treatment on its own.
What I can tell you is to some degree limited by the fact that VGV-L's exact design and mechanism is proprietary in nature, so I can only report based on what the researchers and Viral Genetics choose to disclose. But hopefully, what I post here and future publications by Dr. Karen Newell Rogers will shed some light on the matter.
Dr. Karen Newell Rogers from Texas A & M is in the middle of contributing to the following three papers which seem to have a relationship between VGV-L and chronic Lyme disease:
S. Harris, E. W. Newell, R. P. Tobin, C. P. Harvey, N. Kurzman, E. M. Hechinger, P.
Cipriani, and M. K. Newell. 2010. Comparative Analysis of Peptide Binding, MHC alleles,
and B cell activation in patients meeting CDC criterion for Chronic Lyme Disease.
(manuscript in preparation).
E. Connick, R. Schlichtemeier, J. Folkvord, R. Tobin, C. P. Harvey, and M. K. Newell.
2010. TLR activation of human peripheral blood B cells can be reversed by peptide
treatment. 2010. Manuscript in preparation.
Cabrera, J. and M. K. Newell. 2010. Polyclonal TLR-induced B cell activation is
controlled by Peptide-dependent B cell death (manuscript in preparation).
All three in preparation, but I think they are tightly related to the same research and stem from this previous publication:
M. K. Newell, R. P. Tobin, J. H. Cabrera, M. B. Sorensen, A. Huckstep, E. M. VillalobosMenuey, M. Burnett, E. McCrea, C. P. Harvey, A. Buddiga, A. Bar-Or, M. S. Freedman, J. Nalbantoglu, N. Arbour, S. S. Zamvil, and J. P. Antel. 2010. TLR-Mediated B Cell Activation Results in Ectopic CLIP Expression that Promotes B Cell-Dependent Inflammation.Journal of Leukocyte Biology.
Online e-Pub. July 14, 2010.
In this patent, the portion attributed to Lyme disease states:
"[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."
However, I just found out that there are additional patents on this technology of which I was previously unaware. These patents contain a great deal of detail about what these targeted peptides can do and their effect on polyclonal B cells:
In addition to the above published paper on CLIP expression, Viral Genetics published the following excerpt in its research newsletter which explains what VGV-L does for HIV in easy-to-understand terms - substitute "Lyme disease" for "HIV" here:
"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 havecreated 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."
One thing which I have thought of (and heard a few patients mention in passing) is that this candidate drug is only for treating inflammation and would only address an autoimmune angle relating to chronic Lyme disease.
However, this is not the case:
If you read the full patents, VGV-L's technology works not only to reduce inflammation, it also works to rebalance the immune system so that it is focused on fighting infection in a targeted manner. And in terms of treatment with VGV-L, patients may not just receive VGV-L alone - but also receive a bacterial antigen and antibacterial (possibly also antiparasitic and/or antiviral) therapy concurrently to treat their condition.
Here is the excerpt from the patent concerning the treatment of infections using this technology - including Lyme disease:
[0169] Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, mycobacteria, rickettsia, mycoplasma, neisseria, Borrelia and legionella.
[0170] Although Applicant is not bound by a specific mechanism of action it is believed that the CLIP inhibitors of the invention displace CLIP from MHC class I and cause down regulation of Treg activity and/or activation of effector T cells such as γδT cells.Downregulation of regulatory function of Treg activity prevents suppression of the immune response and enables the subject to mount an effective or enhanced immune response against the bacteria.At the same time the Treg cell may shift to an effector function, producing an antigen specific immune response. Thus, replacement of CLIP with a peptide of the invention results in the promotion of an antigen specific CD8+ response against the bacteria, particularly when the peptide is administered in conjunction with a tumor specific antigen. Activation of effector T cells also enhances the immune response against the bacteria, leading to a more effective treatment.
[0171] One component of the invention involves promoting an enhanced immune response against the bacteria by administering the compounds of the invention. The compounds may be administered in conjunction with an antigen to further promote a bacterial specific immune response. A "bacterial antigen" as used herein is a compound, such as a peptide or carbohydrate, associated with a bacteria surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Preferably, the antigen is expressed at the cell surface of the bacteria.
[0172] The compounds of the invention may be used in combination with anti-bacterial agents. Examples of such agents to treat bacterial infections include, but are not limited to, folate antagonists (e.g., mafenide, silver sulfadiazine, succinylsulfathiazole, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, pyrimethoamine, trimethoprim, co-trimoxazole), inhibitors of cell wall synthesis (e.g., penicillins, cephalosporins, carbapenems, monobactams, vacomycin, bacitracin, clavulanic acid, sulbactam, tazobactam), protein synthesis inhibitors (e.g., tetracyclines, aminoglycosides, macrolides, chloramphenicol, clindamycin), fluoroquinolones (e.g., ciproloxacin, enoxacin, lomefloxacin, norfloxacin, ofloxacin), nalidixic acid, methenamine, nitrofurantoin, aminosalicylic acid, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampin, clofazimine, and dapsone.
I don't know entirely what the researchers intend to use as a bacterial antigen... An Osp? They are suggesting a peptide or carbohydrate, though, and not a highly immunogenic lipoprotein from the cell's outer membrane - even though that's what I think they would have to use if they were to use an antigen. Reading ahead, though, there is the potential that any of a number of Borrelia burgdorferi antigenic products may be used.
Both items #0171 and #0172 have wording which implies that they are optional treatments, as they use the word, "may be administered" or "may be used" rather than "will be administered" or "will be used", respectively. I would assume that whether or not these individual treatments are applied depends entirely on the individual patient and their needs and clinical diagnosis.
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 a more targeted immune response towards bacteria rather than the overload that polyclonally expanded B cells can be.
One of the more fascinating sections of the patent is towards the end. The researchers give a number of examples of how their technology was applied and what the results were. Example 13 of this patent appears relevant to demonstrating how Borrelia burgdorferi activators affect tissue and about eliminating excessive B cells which cause inflammation in tissues. They did an in vitro post-mortem study of these actions in mice:
Example 13 - TLR Activators Promote CLIP-MHC HLA Association and CLIP Inhibitor Peptides Reduce an TLR Activator Promoted CLIP-MHC HLA Association
[0480] Methods
[0481] Preparation of Cells: Mice were Sacrificed by Cervical Dislocation. Spleens and lymph nodes were removed; the tissues were minced through cell strainers to create single cell suspensions; red cells were lysed using buffered ammonium chloride followed by addition of phosphate buffered saline and centrifugation to wash out the ammonium chloride; and the cells were counted using trypan blue exclusion to determine live versus dead cell discrimination and to determine the number of cells per tissue.
[0482] Treatments: The spleen or lymph node cells were treated in vitro with various stimuli (TLR activators: CpG ODN (Alexis), LPS (Sigma), Polyl:C (BD Pharmagen), Pam3Cys (Genway); IL-4 (BD Pharmagen), anti-CD40 monoclonal antibody (BD Pharmagen), both IL-4 and anti-CD40 antibody and OspA and Osp C (Genway) and the cells were cultured for the indicated time periods. The cells were grown in RPMI 1640 medium supplemented with standard supplements, including 10% fetal calf serum, gentamycin, penicillin, streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and 2-ME as well as (where indicated) the stimuli listed above. The cells were incubated at 37° C. in an atmosphere containing 5% CO2 and approximately 92% humidity. The cells were incubated for 3, 24, and 48 hours. At each time point, the cells from that experimental time were harvested and stained for flow cytometric analysis of cell surface expression of CLIP (MHC Class II invariant peptide/IAb, Santa Cruz) by using the commercially available anti-mouse CLIP/IAb peptide, anti-mouse B220, anti-mouse CD4, anti-mouse CD8, and anti-mouse FoxP3 (all commercially available from Becton Dickinson/Pharmingen). Harvested cells were stained using standard staining procedure that called for a 1:100 dilution of Fitc-anti-mouse CLIP/IAb or isotype control. Following staining on ice for 25 minutes, cells were washed with PBS/FCS and resuspended in 100 microliters and added to staining tubes containing 400 microliters of PBS. Samples were acquired and analyzed on a Coulter Excel Flow Cytometer. The data were analyzed using FloJo software.
[0483] Results
[0484] B cell death, including total B cell death and % CLIP positive B cell death in cells treated with a TLR activator (CpG ODN) alone or in combination with MKN3 in the presence or absence of CLIP was assessed. The results are shown in FIG. 12. FIG. 12 is a line graph having a double Y axis, on one side depicting % total B cell death (diamonds, representing CpG ODN alone and squares representing CpG ODN+MKN3) and on the other side depicting % CLIP+ B cell death (triangles, representing CpG ODN and CLIP alone and Xs representing CpG ODN+MKN3 and CLIP). The data reveal that CpG ODN cause an initial increase in B cell death which after 72 hours appears to level off. The CpG ODN+MKN3 data demonstrate that MKN3 is capable of preventing the increase in B cell death.
[0485] Changes in CLIP positive B cells in spleen versus lymph nodes were also assessed. FIG. 13 is a line graph having a double Y axis, on one side depicting % CLIP+ B cell numbers in spleen (light gray square with solid lines representing CpG ODN alone and dark gray square with solid lines representing CpG ODN+MKN3) and on the other side depicting % CLIP+ B cell numbers in lymph nodes (diamonds with dashed lines representing CpG ODN alone and light gray square with dashed lines representing CpG ODN+MKN3). In both spleen and lymph nodes the addition of the peptide to the cells with CpG ODN resulted in less CLIP positive B cells.
[0486] CLIP positive B6.129 cultured B cells (H-2b haplotype) and H2M-/- (from C3H HeJ mice) cultured B cells were also examined in the presence or absence of treatment with a number of different TLR activators. The data is shown in FIGS. 14A and 14B. As shown in the Figures, several TLR activators were able to induce levels of CLIP+ B cells.
Just so it's clear, this isn't the treatment a patient would receive - Dr. Newell Rogers and her colleagues won't be breaking your neck and removing your tissues if you sign up for a clinical trial, okay?
This is an example of an experiment they did to show that VGV-L technology is effective in reducing the number of ineffective B cells which cause inflammation. The end result measured this change, and also measured the end of the sordid relationship between TLR-promoted CLIP MHC-HLA association in the immune system.
[Edited Apr. 3, 2012: Removed mention of CLIP positive cells - these cells need to be removed not added. ]
Now time for a brief lesson in immunology, based on what normally happens in immune response:
Terminology: MHC = major histocompatibility complex; key components of T cell immunity. Think of them as immune response genes. HLA = human leukocyte antigen (think of earlier discussions on this blog about HLA-DR4 and HLA-DR11, and different alleles which respond to infection differently)
So the story goes, B cells express MHC class II. Once antigen has been bound on the antigen receptor on the B cell, the antigen and its receptor are sucked into an endosomal compartment inside the B cell. Then the endsomal compartment fuses with another compartment, the lysosome.
Antigens are broken down into smaller pieces inside the lysosome and then loaded onto the MHC class II component, then the MHC is transported to the B cell surface where the B cell displays the antigen to a CD4+ T cell. This T cell is also known as a helper cell, of which there are two types - Th1 and Th2.
Susceptibility or resistance to many diseases appears to be determined by the genes encoding Major Histocompatibilty Complex (MHC) molecules. Often referred to as immune response genes (or IR genes), these molecules are the key players in restricting T cell activation.
T cells, both CD8 and CD4 positive T cells, recognize antigens only when the antigen is presented to the T cell in association with MHC class I (expressed on all nucleated cells) or MHC class II molecules (expressed on cells that present antigens to CD4+ T cells), respectively.
To sum up:
B cells express MHC class II.
Different people produce different levels of allele variation in MHC locus.
Because of this genetic difference, some people are more or less vulnerable to certain diseases.
The B cell's expression of MHC class II is noticed by CD4+ T cells.
These CD4+ T cells are known as helper cells - of which there are two types, Th1 and Th2.
CD4+ T cells are a major player in our immune systems for fighting infection.
These helper cells do not kill - they activate and direct other immune cells. They are essential in B cell antibody class switching, in the activation and growth of cytotoxic T cells, and in maximizing bactericidal activity of phagocytes such as macrophages.
Look at these handy diagrams as an overview to what happens with B cells, MHC molecules, and T-cells...
Or, if your learning style is better geared towards watching videos, watch this one (warning: might be preceded by an ad, which you can mostly skip):
A simple overview of the immune system [Time: 5:56]
If you've gotten this far, right about now you might be asking yourself, "So what's the big deal? Why is it an issue that there are excess B cells, and how getting rid of them going to make a difference in fighting off Lyme disease if I have a chronic infection?"
Good questions.
Obviously, you can see so far that one reason to not have certain B cells around is that they trigger autoimmune responses. No one wants that. But there are other reasons to avoid an overzealous non-specific B cell response.
Let me unwind the answer, step by step.
Google "polyclonally expanded B cells Borrelia" and tell me what you find. Or, read on and I'll tell you what I found:
Remember that study on Borrelia burgdoferi that Tunev and Barthold did, where it was noted that there was an outsized yet seemingly inadequate immune response to Borrelia burgdorferi found in lymph nodes? One with ill-formed B cells? This one: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002066 ?
Well, the phenomenon that's happening there is somewhat different from what is happening in polyclonal B cell activation in general. In polyclonal B cell activation, there can be lots of B cells which are produced which are non-specific. In this case, in Tunev and Barthold's research, a notable percentage of the B cells were Borrelia burgdorferi antigen specific - yet the cells were of low quality and inadequate to the task.
That's different than what is generally understood to be the typical polyclonal B cell activation, which is what I think is the hypothesis behind this treatment, VGV-L. In both cases, what one observes is an overwhelming number of B cells being produced.
[Edit Apr. 3. 2012: Updated correction to describe Tunev and Barthold difference in outcome of B cell activation.]
Polyclonal b cell activation has been thought to be a useful immune defense mechanism early in acute infection. What has not been investigated as much is how it might be a damaging process in chronic infection and immune dysregulation.
It's important to settle this argument because dysregulated and mis-targeted B cell antibody responses could result in autoimmunity, whereas impaired antibody responses during an actual infection could result in an immune deficiency.
Either way, over time it has become clearer that the production of these B cells relates to the appearance of an IgM response, and the relevance of the presence of an IgM response - particularly a prolonged IgM response - has also become a subject of heated discussion. See: "IgM in microbial infections: Taken for granted?"
"We have used the murine model for Lyme disease described by Barthold et al. (S. W. Barthold, D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody, J. Infect. Dis. 162:133-138, 1990) to determine whether the B. burgdorferi B-cell mitogen is expressed during active infection.
To correlate arthritic changes with immune events, we have studied two strains of mice injected with B.
burgdorferi; one of them, C3H/HeJ, developed severe disease, and the other, BALB/c, developed only mild
disease.C3H/HeJ mice displayed a persistent 10-fold increase in circulating immunoglobulin G (IgG) levels, a
2-fold increase in IgM levels, and a 15-fold increase in peripheral lymph node B-cell numbers, providing
evidence of mitogenic activity. Infected BALB/c mice also had evidence for mitogen activity, since the IgG level
in serum increased three- to fourfold.
The bulk of the increase in circulating IgG levels was not directed against B. burgdorferi antigens, supporting the occurrence of polyclonal B-cell activation. Analysis of IgG isotpes pointed out a contrast between C3H/HeJ and BALB/c mice in that levels of all isotypes were elevated somewhat
in both strains of infected mice but IgG2a levels were much more dramatically increased in the C3H/HeJ mice
(28-fold) than in the BALB/c mice (4-fold). In this study, interleukin-6 levels were found to be persistently
elevated in the serum of infected C3H/IHeJ mice. Interestingly, interieukin-6 levels in serum were much lower
in the infected BALB/c mice. These findings indicate that the B. burgdorferi mitogen is active in infected
animals and may contribute to the inflammatory and immune response to infection."
Right from the start, you get the idea that this paper is going to tell you that the presence of these polyclonally activated B cells have a relationship to IgM and IgG levels.
This is relevant, very relevant - because it can reflect how antibodies to Borrelia burgdorferi are present and how they are picked up in serological testing like ELISA and Western Blots.
Meaningful excerpts from this paper include:
"Immunological abnormalities, including hyperactive B cells, elevated IgM levels in serum, lymphadenopathy, impaired natural killer function, and delayed development of humoral immunity, have been documented in patients with Lyme disease (11, 16, 29, 30, 32). This has suggested a possible involvement of the specific or innate host responses
in the pathogenesis associated with stage 2 and 3 disease(32)."
"Because of the persistent nature of infection and the ability of the organism to gain access to the joint and other tissues (5, 15, 29), a B-cell mitogen present during infection could play a role in the pathology of Lyme disease. To support this possibility, it was important to determine whether the mitogen functioned in vivo. This paper provides evidence that a B. burgdorferi mitogen is active in vivo in infected animals.
Three lines of
evidence support the conclusion that B-cell activation in vivo is polyclonal or oligoclonal in addition to being antigen specific.
First, the level of IgG in serum in infected mice was elevated about 10- to 15-fold, with the value ranging from 10
to 15 mg/ml (Fig. 1C). In comparison, the amount of IgG specific for B. burgdorferi antigens was approximated at 0.6
mg/ml (Fig. 2).
Second, the number of B lymphocytes in peripheral lymph nodes of infected animals was increased
10- to 15-fold, with a 5-fold rise in the ratio of B to T cells (Fig. 4). The number of B cells also increased about twofold relative to the number of T cells in spleens from C3I/HeJinfected mice.
Third, the IgG titer in the serum of infected animals to an unrelated antigen, ovalbumin, was increased 10- to 15-fold, which resembles the increase in the total IgG level (Fig. 3).
These findings suggest that levels of autoreactive antibodies might be also expanded in infected animals, although anti-collagen antibodies were not identified. Because CD5+ B cells have been shown to produce autoreactive antibodies and are selectively increased in patients with rheumatoid arthritis (7), we determined whether they were expanded in B. burgdorferi-infected animals.No selective increase in the number of B cells of this lineage were found in C3H/HeJ animals at any stage of infection. Further studies are required to determine whether autoreactive antibodies are generated during infection."
So their initial experiment to see if there was an overwhelming B cell response provides us with evidence that yes, there is, and also - while there is a high IgG response, only a small percentage of IgG produced is B. burgdorferi specific. There was at the time no indication that autoreactive antibodies were involved.
(This process can be a precursor to autoimmunity developing - but that's later on.)
A later paper, from 1997, "Why is chronic Lyme Borreliosis chronic?"(full text), also brings up a host of issues related to TLRs, MHC class II, and the relationship between B and T cells in lymph nodes.
Doesn't it seem a little prescient?
"The question remains whether downregulation or even loss of MHC class II molecules on LCs might influence a patient's disease susceptibility. It is MHC class II molecules that bind antigenic peptide fragments, present them to CD4+ Th cells, and induce cytokine secretion and IgG secretion by B cells [55]. In vitro investingations have shown that MHC II class molecules are downregulated on antigen-presenting cells after coculture with Th cell clones in the presence of antigenic peptides of tetanus toxoid or staphylococcal superantigen, which elicit a strong HLA-DR-restricted T cell response.
Several hypotheses were suggested as the cause of this down-regulation.
(1) Downregulation occurs when antigenic peptides catabolized in macrophages are recognized by CD+4 helper T cells, in order to control the size of a T cell clone and provide a homeostatic mechanism [55]. (2) Downregulation occurs for completion of T-B cell collaboration after antigen presentation, limiting excessive T cell help to the triggered B cells, or (3) it occurs for focusing the T cell repines to one or a few immunodominant peptides.
(4) LCs of patients with AIDS express decreased amounts of MHC class II molecules. Polyclonal B-cell activation, as seen in these patients and in patients with ACA, could cause the appearance of autoantibodies or immunocomplexes that interact with LCs and block their surface-staining characteristics [45]. (5) IL-10, originally identified as a product of Th2 cells, has a significant inhibitory influence on the antigen-presenting functions of macrophages and LCs by downregulation of MHC class II molecules. In fact, LCs pretreated with IL-10 were converted from specifically sensitizing to specifically tolerogenic antigen-presenting cells in vitro and in vivo [56]. In other studies treatment of LC cultures with IL-10 inhibited to upregulation of HLA-DR [57].
(6) Downregulation is initiated for establishment of self-tolerance. This downregulation can protect the antigen-presenting cell by inhibiting the presentation of self-antigens [58]. On the other hand, the downregulation of MHC class II antigens on LCs could result in inadequate presentation of antigens in lymph nodes, which in turn may reduce activation and proliferation of both B and T cells and the secretion of relevant cytokines. This may be what happens in CLB."
Dr. Karen Newell Rogers et al recent patent has this to say about TLRs (Toll Like Receptors) and B cells:
"Many bacteria and viruses produce substances, collectively called Toll ligands, that elicit an immediate response from an individual's immune system. These Toll ligands appear to promote inflammation by activating a wide variety of immune cells to bring them rapidly into battle against the invading pathogen.
In most cases, these events correlate with a healthy and productive immune response to the pathogen. However, in some cases the Toll ligand binds to a Toll-like Receptor (TLR) on lymphocytes and non-specifically activates immune cells called B and T lymphocytes that would normally to respond to infectious pathogens with an exquisitely specific response. When Toll ligands activate B cells in a non-specific way, the non-specific activation is a pro-inflammatory event that may result in uncontrolled, or even auto-reactive, production of antibodies. When a B cell is activated non-specifically, we have discovered that the B cell expresses an important, small self-peptide called MHC class II invariant peptide, CLIP. In most individuals, a control cell, known as a T regulatory cell (Treg for short), has been shown, to kill the activated B cell.
During a viral or bacterial infection, non-antigen specific B cells in close proximity to an inflammatory or inciting lesion could manage to become activated in a bystander fashion. In those cases, CLIP would remain in the groove and get transported to the cell surface of the B cell. Its presence on the cell surface can be undesirable because if CLIP gets removed from the groove by a self antigen, the B cell would be in a position to present self antigens to self-reactive T cells, a process that could lead to autoreactivity and autoimmune disease.
For some B cells this may result in death to the B cell by a nearby killer cell, perhaps a natural killer (NK) cell, unless the antigen receptor on the B cell has engaged antigen. Antigen recognition would thereby provide a survival signal for the B cell. However, if a killer cell doesn't remove the potentially autoreactive B cell and it encounters a CD4+ T cell that can recognize that antigen (most likely one that was not in the thymus) the B cell might receive additional help from a T cell specific for the antigen that now occupies the groove (antigen binding location in the MHC molecule). Alternatively, a nearby cell whose job it is to detect damaged self cells, may become activated by the self antigen-presenting B cell. Such a damage detecting cell is, for example, an effector T cell (Teff) such as a gamma delta T cell, also referred to as a γδT cell (γδ refers to the chains of its receptor). The γδT cell can then seek out other sites of inflammation (for example in the brain in MS, in the heart for autoimmune myocarditis, in the pancreas in the case of Type I Diabetes). Alternatively, the γδT cell might attempt to kill the CD4+ T cell that may respond to self antigens."
So based on all this, I think one has to consider that the complex interactions within the immune system related to B. burgdorferi infection have to be paid close attention to - and not just any persisting spirochetes themselves.
I am really interested in seeing what VGV-L - along with supportive and antibacterial treatment together - can do for chronic Lyme disease. It appears it not only prevents autoimmune responses to infection, but redirects the immune response so it can better target infection.
I do wonder, though, how VGV-L would handle a situation where many B cells are being created and a good percentage of them are antigen specific but are of low quality - such as those mentioned in Tunev and Barthold's research.
* It may be that Lyme disease is more like HIV in that inflammation may allow or facilitate the spread of spirochetes as it encourages vlsE recombination. See: http://www.jimmunol.org/content/167/6/3383.long for one example.
