Wednesday, July 6, 2011

5 Fibroblasts And Lyme Disease: Sample Studies

One of the indications that Lyme disease may cause a persistent infection would be if it were intracellular and not just an extracellular obligate parasite. There are studies which show that Lyme disease spirochetes can be intracellular - at least in passing - in fibroblasts.

Fibroblasts are important cells to study in relation to Lyme disease and other diseases.

Mouse fibroblasts in cell culture
Fibroblasts are the most common cells of connective tissue in animals as well as humans. Tendons, blood, cartilage, bone, adipose tissue, and lymphatic tissue are all places where one will find fibroblasts and in many cases are  tissues where Borrelia burgdorferi likes to hang out.

Fibroblasts make collagens, glycosaminoglycans, reticular and elastic fibers, glycoproteins found in the extracellular matrix and the cytokine TSLP. On a more general note, they synthesize the extracellular matrix and collagen, the structural framework (stroma) for animal tissues, and play a critical role in wound healing.

To get a good idea of where fibroblasts are found in the human body, refer to this diagram:


In the above diagram:

epithelial cells = tissues which line the cavities and surfaces of structures throughout the body, and also form many glands. Functions of epithelial cells include secretion, selective absorption, protection, transcellular transport and detection of sensation.
basement membrane = a thin sheet of fibers that underlies the epithelium, which lines the cavities and surfaces of organs including skin, or the endothelium, which lines the interior surface of blood vessels.The primary function of the basement membrane is to anchor down the epithelium to its loose connective tissue underneath.
endothelium = the thin layer of cells that lines the interior surface of blood vessels.
interstitial matrix = a type of extracellular matrix found in interstitial connective tissue, characterized by the presence of fibronectins, proteoglycans, and different types of collagen.

A study from 2007, Fibroblasts as novel therapeutic targets in chronic inflammation,  has this to say about fibroblasts in its abstract:
"A characteristic feature of many chronic inflammatory diseases is their persistence and predilection for certain sites. The molecular basis for such tissue tropism and failure of the inflammatory response to resolve has until relative recently remained obscure. Recent studies have strongly implicated fibroblasts as cells which contribute to disease persistence and which help define anatomical location. Therefore fibroblasts make an attractive therapeutic target as they help orchestrate the inflammatory infiltrate. Current anti-inflammatory therapies target immune cells in an attempt to inhibit the production of pro-inflammatory mediators. However an equally important target is the active induction of pro-resolution programmes responsible for the resolution of inflammation. Fibroblasts are likely to be an important source of these anti-inflammatory mediators. Therapeutic manipulation of fibroblasts and their biologically active products is an emerging concept in treating cancer and is likely to provide a novel method to achieve improved control of chronic inflammatory disease." [1]
Fibroblasts are likely to be a source of anti-inflammatory mediators, but they could also be a home for invading pathogens.

In yesterday's paper outline, Interaction of of Borrelia burgdorferi in coculture with human fibroblasts, the results stated:
"Electron micrographs showed borreliae which were able to attach to the fibroblast membrane through protein bridges. Single spirochetes seemed to pervade fibroblast cytoplasm by invagination surrounded by an intact fibroblast membrane."
This recent research indicates that Borrelia burgdorferi (and afzelii) spirochetes attach to fibroblasts and individual spirochetes enter the cytoplasm by invagination while the fibroblast membrane remains intact.

Here, invagination means just what you imagine it would be: A single spirochete can enter the wall of the fibroblast without disrupting its integrity and happily live within its cytoplasm.

The conclusion reflects this statement:
"The interaction of B.b.s.s. and B. afzelii with human fibroblasts was verified by electron microscopy. Fibroblast integrity was not disturbed by borreliae. Intracellular accumulation of spirochetes was not detectable." [2]
So according to this research, single spirochetes enter fibroblasts without disturbing the fibroblasts, and there was no evidence that more than one spirochete was found within the fibroblasts. I'm taking this to mean that spirochetes neither entered fibroblasts en masse nor did they find any reproducing spirochetes inside fibroblasts. It doesn't mean that isn't what might happen, but such behavior was not observed.

Another study was conducted in the same year, in Poland, in 2010, Interactions between Borrelia burgdorferi and Mouse Fibroblasts, and this study states more happens within those fibroblasts:
"Electron microscopic studies reveal consecutive stages of B. burgdorferi spirochetes penetration to mouse fibroblasts in vitro. It has been observed, as a first step attachment and engulfment of spirochetes followed by formation of vacuoles. After 48 hours of infection, vacuoles of fibroblastic cells have been seen full of B. burgdorferi bacteria and latter they have been released from infected cells to extracellular space. It can be the evidence that B. burgdorferi multiply intracellulary."[3]
Here the authors have stated that vacuoles within fibroblasts were full of Borrelia burgdorferi, and that was evidence that Borrelia burgdorferi multiplies within fibroblasts.

What portion of their lives Borrelia burgdorferi spends within fibroblasts remains to be seen, but if they spend any amount of time as intracellular obligate parasites and not just extracellular ones, this could explain why infection may be persistent.

Earlier studies on Borrelia burgdorferi in relation to fibroblasts have been completed, some of which indicate that perhaps Borrelia burgdorferi does not survive inside fibroblasts very long and instead destroys them. A 2001 study,  Insights from a novel three-dimensional in vitro model of lyme arthritis - Standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts, states the following in its paper:
"Results: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for > 3 weeks, Bb invaded the synovial tissue and the artificial matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1 beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration.

Conclusion: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis."
Is this a reasonable conclusion to draw, based on this in vitro study? What other studies challenge the concept of intracellular persistence of Bb?  What about findings from other studies including Klempner's earlier study on not only fibroblasts, but Borrelia burgdorferi's intracellular relationship to other cells?

In a 1992 study, Fibroblasts Protect the Lyme Disease Spirochete, Borrelia burgdorferi, from Ceftriaxone In Vitro, stated this in its abstract:
"... The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus several eurkaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival."
Later on, within the study, the following statements are made:
"One of the regimens most commonly used clinically for treatment of Lyme disease is administration of ceftriaxone for 14 days. Our time course experiments showed that human skin fibroblasts can protect B. burgdorferi from ceftriaxone for 14 days. It will be of interest to examine the maximum duration of this protective effect."
and
"It has been previously demonstrated that B. burgdorferi penetrates endothelial cell monolayers and can be observed inside and between these cells; however, the viability of those potentially intra- and intercellular spirochetes was not assessed."
How many studies have been conducted since this study to see if spirochetes survive inside fibroblasts while exposed to more than 14 days of ceftriaxone? 28 days? 42 days?

Has anyone completed repeat studies which show Borreliae spirochetes surviving and replicating inside of other cell types?

How many intra- and intercellular spirochete studies have been completed, and what were the results?

A later, 1993 study by Klempner had this to say in its abstract:
"The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 μg/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable."[6]
What do all the studies on Borrelia's interaction with fibroblasts to date suggest about its intracellular behavior? Is it possible that this is a major cause of some patients' persistent symptoms - even after antibiotic treatment?

This is only a sampling of studies on Borrelia burgdorferi and fibroblasts - what is needed is a meta analysis of the data on this phenomenon and further studies to confirm Borrelia burgdorferi's intracellular nature.

With confirmation of Borrelia burgdorferi's intracellular nature and its proclivity for fibroblasts, new treatments could be developed that help patients more effectively fight off infection.

References:
[1] SJ Flavell, TZ Hou, S Lax, AD Filer, M Salmon, and CD Buckley. Fibroblasts as novel therapeutic targets in chronic inflammation. British Journal of Pharmacology. 153(S1): S241–S246.March 2008.
[2] Interaction of of Borrelia burgdorferi in coculture with human fibroblasts. International Conference of Lyme Borreliosis and Other Tick-borne Diseases. 2010.
[3] Chmielewski T, Tylewska-Wierzbanowska S. Interactions between Borrelia burgdorferi and Mouse Fibroblasts. Polish Journal Of Microbiology. Volume: 59 Issue: 3 Pages: 157-160. 2010.
[4] Franz JK, Fritze O, Rittig M, Keysser G, Priem S, Zacher J, Burmester GR, Krause A. Insights from a novel three-dimensional in vitro model of lyme arthritis - Standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts. Arthritis and Rheumatism. Volume:44 Issue:1 Pages: 151-162 Jan. 2001
[5] Kostis Georgilis, Monica Peacocke, and Mark S. Klempner. Fibroblasts Protect the Lyme Disease Spirochete, Borrelia burgdorferi, from Ceftriaxone In Vitro. Journal of Infectious Diseases. Vol. 166, pp. 440-444. 1992.
[6] Mark S. Klempner, Richard Noring and Rick A. Rogers. Invasion of Human Skin Fibroblasts by the Lyme Disease Spirochete, Borrelia burgdorferi. The Journal of Infectious Diseases. Vol. 167, No. 5 pp. 1074-1081.  May 1993. http://www.jstor.org/pss/30112679

5 comments:

  1. CO,

    Thanks for this wonderful post about a fascinating topic. I will need to return for a second (and possibly third) read.

    I just came across this and you came to mind immediately as I read that SLICE is -- "the first study in the U.S. to examine long-term health and outcomes of Lyme disease":

    http://ideha.dhmh.maryland.gov/CZVBD/pdf/ZDU_PTLS_Aucott.pdf

    Here are the links (an excerpts) that led to my search for more information about SLICE:

    http://www.wbaltv.com/health/22744251/detail.html

    Aucott said up to 20 percent of patients have lingering effects after treatment.
    "It's that group of people who get treated, but their health had changed and they don't fully recover. That's the group that has not been studied," he said.

    Aucott said he finds the controversy over Lyme disease distressing and hopes his fledgling Lyme Disease Clinical Research Center will find answers to help people who are suffering.

    "The bottom line is -- we don't know, so let's find out. Let's not argue about what we don't agree on. Let's figure it out," he said.


    http://www.lymemd.org/about.html

    "Dr. Aucott lectures widely and collaborates with internationally known Lyme disease researchers. He was a member of the 2010 Institute of Medicine of the National Academies of Science workshop on tick-borne infections and Lyme disease. He also directs the Lyme disease clinical research center and is Principal Investigator of the SLICE study at Johns Hopkins, the first study in the U.S. to examine long-term health and outcomes of Lyme disease".

    CO, I apologize that this isn't directly related to your post and if you've posted about SLICE already.

    RitaA

    Rita A

    ReplyDelete
  2. CO,

    I just remembered reading this (or something like it) and being intrigued that there was any association between mood disorders and blood vessels (i.e. endothelial function):

    http://www.ncbi.nlm.nih.gov/pubmed/16730331

    Biol Psychiatry. 2006 Oct 15;60(8):889-91. Epub 2006 May 30.

    Impairment of endothelial function in unipolar and bipolar depression.

    Rybakowski JK, Wykretowicz A, Heymann-Szlachcinska A, Wysocki H.
    Source
    Department of Adult Psychiatry, Poznan University of Medical Sciences, Poznan, Poland. rybakows@wlkp.top.pl
    Abstract
    BACKGROUND:
    Previous studies have suggested an association between abnormal endothelial function and depression. We therefore tested this hypothesis in patients with depression in the course of mood disorders and assessed the effect of antidepressant treatment.

    METHODS:
    Psychometric evaluation and hemodynamic and endothelial function studies using pulse wave analysis were performed on a group of 31 patients with unipolar or bipolar depression. The control group consisted of 18 healthy subjects, age- and gender-matched.

    RESULTS:
    Arterial endothelial function was impaired in patients compared with that in control subjects, both during a depressive episode and when in remission after pharmacological treatment. The diagnosis, intensity of depression, and type of antidepressant drugs did not influence the results.

    CONCLUSIONS:
    The impairment of endothelial function might constitute a trait marker of the biological make-up of patients with mood disorders and might contribute to the increased frequency of cardiovascular conditions observed in these patients.

    PMID: 16730331 [PubMed - indexed for MEDLINE]

    Here's a more recent one that is specifically concerned with Vascular Endothelial Growth Factor (VEGF) -- something that has been brought up in the past with reference to Lyme disease and quite a few other illnesses, including MS:

    http://www.sciencedirect.com/science/article/pii/S0304394010004866

    Vascular endothelial growth factor (VEGF) polymorphism is associated with treatment resistant depression


    CO, I must admit that I don't know what to make of all this.

    Rita A

    ReplyDelete
  3. Amazing that the IDSA can keep saying we are right because we said so.

    It might take 50 years for their propoganda to be worn down.

    John S

    ReplyDelete
  4. Quick responses to you both here...

    One thing I want anyone reading this to be aware of is that the above studies are all in vitro not in vivo - and what is really needed is a longer term in vivo study of spirochetes to see what they do and where they go. I would think a long term mRFP/GFP study could demonstrate what happens over time in vivo... someone just has to do so.

    Occasionally intracellular spirochetes have been detected in cardiac microfibers by electron microscopy. Not sure which researcher to look at for that - try Armstrong, Barthold, Pachner, and Zimmer.

    Rita A:

    Thanks for the link to the study. I think I'll need to reread it as I have more questions about it. I'm with Dr. Aucott on wanting to stop arguing and rolling up our sleeves and figuring out what's going on.

    Re depression and endothelial dysfunction:

    This has been an issue in research and another topic of controversy - something that's not well-understood. It is known that the neurotransmitter, serotonin, is largely created in our guts and from cells known as enterochromaffin cells. Here, they're not looking at dysfunctional enterochromaffin cells but endothelial cells - which have been studied in relation to cardiovascular disorders for some time. Their connection to depression is a more recent development, along with studying homocysteine and cytokine roles in depression.

    I'm not sure what's going on there, Rita A, but one can hypothesize about what could be happening. Right now this intersects with what I've been thinking about what is known about Bb's behavior in attaching to endothelial cells, and about Bb's metabolic needs.

    John S,

    If you are referring to this post, remember that in vivo proof of intracellular behavior makes more the point for longer/different treatment and in vitro, not so much.

    The IDSA guidelines panel's statements are problematic for me on a number of counts, including the fact that when interfacing with the public and Lyme disease patients, they emphasize the autoimmune hypothesis without explaining their basis for it and without offering any patient group willing to try it clinical trials and treatment studies.

    The guidelines don't acknowledge how limited they truly are in observing how strain variability can affect outcome, such as patient X can get over Lyme infection quickly yet patient Y does poorly due to one being infected with a nonpathogenic strain while the other is.

    To quote from p. 501 of Borrelia (Radolf):
    "Unfortunately, methodologies to distinguish patients who have cleared "benign" asympomatic infection from those who may be persistently infected and at risk for subsequent late complications do not exist. The management of asymptomatic persons found to be seroreactive for Lyme disease, therefore, is an important unresolved issue for practitioners. Current treatment guidelines do not include recommendations for this common situation."

    This is only the tip of the iceberg. The rest we can't see yet.

    ReplyDelete
  5. To add to the above research:

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067508/

    Invasion of Eukaryotic Cells by Borrelia burgdorferi Requires β1 Integrins and Src Kinase Activity. Jing Wu,1 Eric H. Weening,1† Jennifer B. Faske,1§ Magnus Höök,2 and Jon T. Skare1*. Infect Immun. 2011 March; 79(3): 1338–1348. Published online 2010 December 20. doi: 10.1128/IAI.01188-10

    Abstract

    Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most widespread tick-borne infection in the northern hemisphere that results in a multistage disorder with concomitant pathology, including arthritis. During late-stage experimental infection in mice, B. burgdorferi evades the adaptive immune response despite the presence of borrelia-specific bactericidal antibodies. In this study we asked whether B. burgdorferi could invade fibroblasts or endothelial cells as a mechanism to model the avoidance from humorally based clearance. A variation of the gentamicin protection assay, coupled with the detection of borrelial transcripts following gentamicin treatment, indicated that a portion of B. burgdorferi cells were protected in the short term from antibiotic killing due to their ability to invade cultured mammalian cells. Long-term coculture of B. burgdorferi with primary human fibroblasts provided additional support for intracellular protection. Furthermore, decreased invasion of B. burgdorferi in murine fibroblasts that do not synthesize the β1 integrin subunit was observed, indicating that β1-containing integrins are required for optimal borrelial invasion. However, β1-dependent invasion did not require either the α5β1 integrin or the borrelial fibronectin-binding protein BBK32. The internalization of B. burgdorferi was inhibited by cytochalasin D and PP2, suggesting that B. burgdorferi invasion required the reorganization of actin filaments and Src family kinases (SFK), respectively. Taken together, these results suggest that B. burgdorferi can invade and retain viability in nonphagocytic cells in a process that may, in part, help to explain the phenotype observed in untreated experimental infection.

    (Fulltext at the above link.)

    ReplyDelete

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