17 Abstract: Invasion of eukaryotic cells by Borrelia burgdorferi requires β(1) integrins and Src kinase activity

Due to ASM's copyright terms on this journal, it's my understanding that I can't post the entire abstract for this publication here - even though abstracts are pretty much something I have long considered open source and commonly shared. Most scientists do - so to find out that some publishing companies and journals don't even want people to repost abstracts seems... well... strange to me.

How else does one promote their work to other people? I'd consider it free promotion for the full publication!

However, I wanted to share it with you however I could, because I think it's of value:

Invasion of eukaryotic cells by Borrelia burgdorferi requires β(1) integrins and Src kinase activity. Wu J, Weening EH, Faske JB, Höök M, Skare JT. Infect Immun. 2011 Mar;79(3):1338-48. Epub 2010 Dec 20.

Read The Abstract Here: http://www.ncbi.nlm.nih.gov/pubmed/21173306

This abstract relates directly to this post I made recently:
http://campother.blogspot.com/2011/07/fibroblasts-and-lyme-disease-sample.html

It's about how Lyme disease spirochetes may be able to hide from the immune system inside other cells, especially fibroblasts. The above study is more research related to this topic, so I recommend reading my recent post for background then looking at the above abstract on PubMed.

This work by Camp Other is licensed under a Creative Commons
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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

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0 Abstract: Interaction of Borrelia burgdorferi in coculture with human fibroblasts

I haven't seen this paper mentioned elsewhere - this is from a conference held last year. Need to find full text - not available on ASM or Scholar...

Interaction of Borrelia burgdorferi in coculture with human fibroblasts 
Daniel Wilfinger, Gerd Leitinger, Anna Maria Pabst, Helmut Schaider, Elisabeth Aberer

Kurzfassung/Background: B. burgdorferi (B.b.) can be recovered long after initial infection from antibiotic-treated patients; a protective effect of fibroblasts was assumed. Outcome of previous studies differ whether spirochetes are able to invade fibroblasts. Skin fibrosis has repeatedly been observed in European chronic Lyme borreliosis. So the aim of our study was to examine the interaction of B.b. with fibroblasts in coculture ultrastructually and to investigate key factors for fibrosis such as transforming growth factor ß (TGF-ß) and the production of type I collagen.

Materials and Methods: Human skin fibroblasts were propagated in Dulbeccos’s modified medium at 37°C and 5% CO2 up to 10 5 cells per culture. Bb sensu stricto and B. afzelii were cultured at 34°C in BSK-H medium up to 10 8 cells per culture. Borreliae and fibroblasts were then coincubated in RPMI medium at 37°C for 14 days. Cocultures and fibroblasts only were harvested between 2 and 14 days 3-4 times, fixed in glutaraldehyde and prepared for electon microscopy analysis. Supernatants were investigated for TGF-ß by ELISA. mRNA levels for type I collagen were measured by real time PCR.

Results: 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. Structural changes of extracellular borreliae due to the adverse culture condition were observed. Shedding of outer membrane blebs forming granules and tubules were seen as well as cystic degeneration of borreliae.

After 2 days of coculture with B.b.s.s. the total amount of TGF-ß in the supernatants was approximately the same as in fibroblast-cultures, after 14 days of coculture production of TGF-ß was decreasing in cocultures with B.b.s.s.. In supernatants of cocultures with B. afzelii TGF-ß was not detectable at any time.

The mRNA-levels for type I collagen were elevated after 2 days of coculture with B.b.s.s. as well as in cocultures with B. afzelii in comparison to fibroblast-cultures. After 7 days the mRNA-levels for type I collagen were decreasing in cocultures in comparison to fibroblast-cultures, a stronger decrease was observed in cocultures with B. afzelii.

Conclusions: 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.

We demonstrated differences in the two species B.b.s.s. and B. afzelii concerning production of type I collagen and TGF-ß. In contrast to cocultures with B.b.s.s. TGF-ß was not detectable in cocultures with B. afzelii at any time. In line with these findings in cocultures with B. afzelii a stronger decrease of collagen production was observed in comparison to cocultures with B.b.s.s. after 7 days of coculture. The elevation of mRNA-levels for type I collagen in cocultures after 2 days is confirmed by literature: Scleroderma-fibroblasts also show an increase in collagen production after 2 days of culture.

Schlagwörter/Keywords : Borrelia, B. burgdorferi sensu stricto, B. afzelii, electronmicroscopy, morphology, TGF-ß, mRNA collagen, morphea, fibrosis, ACA

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1 The Friday Four

In this week's Friday Four, we'll look at how some bacteria avoid antibiotics by shutting down and hiding until it's safe to come out again,  students who go bacteriophage hunting,  disrupting bacteria's communication or quorum sensing in future antibacterial treatments,  tests which use bacteria's scent to detect not only their presence but species, strain, and their antibiotic resistance profile.

CO message to readers: The Friday Four postings will be on hiatus for at least the month of May during Lyme Awareness Month.

1) 'Going off the grid' helps some bacteria hide from antibiotics

Link: http://www.sciencedaily.com/releases/2011/04/110425153611.htm

ScienceDaily (2011-04-25) -- Call them the Jason Bournes of the bacteria world. Going "off the grid," like rogue secret agents, some bacteria avoid antibiotic treatments by essentially shutting down and hiding until it's safe to come out again.

Comments:

I want to keep this one short and sweet: What if those few Borrelia burgdorferi left behind in collagen that some researchers say are not viable or non-dividing are just basically in stasis instead? What if they have shut down their metabolic processes and only look mostly dead? (This is starting to remind me of the scene in that movie, The Princess Bride, where Westley is... Oh, never mind, if you haven't seen it, I don't want to spoiler it for you. It's a fun movie. I will tell you the Bourne series is one of the best action series in my opinion - along those lines, I like Memento too...)

Source Reference:
Xiaoxue Wang, Younghoon Kim, Seok Hoon Hong, Qun Ma, Breann L Brown, Mingming Pu, Aaron M Tarone, Michael J Benedik, Wolfgang Peti, Rebecca Page, Thomas K Wood. Antitoxin MqsA helps mediate the bacterial general stress response. Nature Chemical Biology, 2011; DOI: 10.1038/nchembio.560

2) Phage hunting students find new bacteriophages in soils of St. Louis suburbs

Link: http://www.sciencedaily.com/releases/2011/04/110425135645.htm

ScienceDaily (2011-04-25) -- Twelve students who had participated in an unusual biology course as freshmen have found two bacteriophages, viruses that prey exclusively on bacteria, in the soil of two suburbs of St. Louis, Missouri. As the finders, they had the naming rights; the new phages are called Angelica and Uncle Howie.

Comments:

This is as awesome as being an amateur astronomer. If you're an amateur astronomer, if you find an object in the sky no one has discovered before, it can be named after you or you can decide what you want to name it. Here, students are discovering their own bacteriophages in the dirt and naming them anything they want.

I posted this mainly because I think it's cool, and I wish I had gotten the opportunity to do this in school, too. Well, who knows... maybe I'll go back to school someday, just to be able to take a course like this and name my own bacteriophage Camp Other. If I did, though, I'd try to find one that consumed Borrelia burgdorferi.

Source Reference:
Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, et al. Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution. PLoS ONE, 2011; 6 (1): e16329 DOI: 10.1371/journal.pone.0016329

3) Bacteria interrupted: Disabling coordinated behavior and virulence gene expression

Link: http://www.sciencedaily.com/releases/2011/04/110421122329.htm

ScienceDaily (2011-04-22) -- New research reveals a strategy for disrupting the ability of bacteria to communicate and coordinate the expression of virulence factors. The study may lead to the development of new antibacterial therapeutics.

Comments:

Bonnie Bassler is up to it again. I love her presentation on TED, and if you haven't seen it, you really should set aside 18 minutes of your time to watch her video on how to get bacteria to talk and how to get them to shut up.

And recently she was on a team that did more research on how to stop bacterial infections by shutting up them up.  Four points in turn outlined their strategy for how one could stop bacterial infection by stopping quorum sensing:

1. Quorum-sensing (QS) antagonists represent potential antibacterial therapeutics
2. They can bind LuxR-family transcription factors in competition with autoinducers
3. The antagonists stabilize a closed conformation incapable of binding operator DNA
4. This inhibition strategy may be generalizable to other multidomain receptors

Which means that there are antagonists which can bind to certain factors that normally autoinducers would bind to - the antagonists are competition for them,  much like Saccharomyces bouldarii can be competition for other yeasts and C. difficle. When the antagonists bind to the factors, they will not bind to operator DNA.

So to sum up: If you can stop autoinducers, you can stop the bacteria from communicating. You can shut it up. If you shut it up, you can tell it to stop having sex and the immune system police will evict it, much like a loud annoying neighbor.

You think I'm kidding, and making this story up? I'm not - I'm merely telling the story to illustrate a point: In order for gene transcription to be activated in the bacteria, the cell must encounter autoinducers secreted by other cells in its environment.

Here's a basic diagram of how Gram-negative bacteria engages in quorum sensing (noting that Borrelia burgdorferi is not exactly Gram-negative or Gram-positive here, it is somewhat closer to Gram-negative so I include that model here):

What you need to imagine here is that this oval represents a bacterium, and that initially a small number of bacteria are doing this all at the same time in their host, whether that be human or not.

Here the LuxI protein makes the autoinducers (green pentagons) which then diffuse freely outside. Each bacterium doing the same, the concentration of external autoinducer is a measure of the size of the population (quorum).

When the autoinducer concentration is high (meaning the bacteria has reproduced to a certain population)  the autoinducer binds to a cognate receptor LuxR (cognate means having the same form and ad hoc characteristics to bind specifically to the molecule it receives).

This is quorum sensing.

The complex auto inducer-Lux R then binds at target gene promoters and activate their effect (transcription) which has behavioral consequences.

In other words, once the bacteria reaches a certain threshold, the level of autoinducers is very high, and the number of bacteria goes up. The high autoinducer level means more bacteria, and more bacteria means more autoinducers. It's a self-perpetuating feedback loop. If you can prevent the loop from even getting started, bacterial numbers will remain low.

So, you're probably wondering, does Borrelia burgdorferi engage in quorum sensing, and if so, can we get it to shut up also?

This has actually been somewhat under debate. Some research has stated that Borrelia burgdorferi has an autoinducing cognate receptor called LuxS, but it doesn't have the necessary autoinducer to bind to it, which in this case would be AI-2.

More recent research has shown that there might be a more complicated method for Borrelia burgdorferi involved for synthesizing its own autoinducers... Might.

To draw from this Polish research paper from 2009 (http://www.aaem.pl/pdf/16001.pdf):

"...the studies of von Lackum et al.[62] demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. According to these observations, B. burgdorferi was shown to synthesize the final product, 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). The addition of in vitro-synthesized DPD to the culture of B. burgdorferi manifested in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria for regeneration of methionine can utilize the other LuxS product, homocysteine, B. burgdorferi did not show such an ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD, and utilizes a form of that molecule as an AI-2 pheromone to control gene expression [4]."

Those cited papers are:

[62] Von Lackum K, Babb K, Riley SP, Wattier RL, Bykowski T, Stevenson B: Functionality of Borrelia burgdorferi LuxS: the Lyme disease spirochete produces and responds to the pheromone autoinducer-2 and lacks a complete activated-methyl cycle. Int J Med Microbiol 2006, 296, 92-102 -and-
[4] Babb K, von Lackum K, Wattier RL, Riley SP, Stevenson B: Synthesis of autoinducer 2 by the lyme disease spirochete, Borrelia burgdorferi. J Bacteriol 2005, 187, 3079-3087

I need to read more about it, at this point the above is currently hypothetical and an in vitro test, so the answer to your question is (unless you know something I don't): the jury is still out on this one.
.
Source Reference:
Guozhou Chen, Lee R. Swem, Danielle L. Swem, Devin L. Stauff, Colleen T. O'Loughlin, Philip D. Jeffrey, Bonnie L. Bassler, Frederick M. Hughson. A Strategy for Antagonizing Quorum Sensing. Molecular Cell, Volume 42, Issue 2, 199-209, 22 April 2011 DOI: 10.1016/j.molcel.2011.04.003

4) Get a whiff of this: Low-cost sensor can diagnose bacterial infections

Link: http://www.sciencedaily.com/releases/2011/04/110427171636.htm

Colorimetric sensor array
overlaid on petri dish

ScienceDaily (2011-04-28) -- Bacterial infections really stink. And that could be the key to a fast diagnosis. Researchers have demonstrated a quick, simple method to identify infectious bacteria by smell using a low-cost array of printed pigments as a chemical sensor. In only a few hours, the array not only confirms the presence of bacteria, but identifies a specific species and strain. It even can recognize antibiotic resistance -- a key factor in treatment decisions.

Comments: So the abstract for this paper is as follows:

"Rapid identification of both species and even specific strains of human pathogenic bacteria grown on standard agar has been achieved from the volatiles they produce using a disposable colorimetric sensor array in a Petri dish imaged with an inexpensive scanner. All 10 strains of bacteria tested, including Enterococcus faecalis and Staphylococcus aureus and their antibiotic-resistant forms, were identified with 98.8% accuracy within 10 h, a clinically important time frame. Furthermore, the colorimetric sensor arrays also proved useful as a simple research tool for the study of bacterial metabolism and as an easy method for the optimization of bacterial production of fine chemicals or other fermentation processes."

The full text requires paid access, however, just looking at what is known here between the article and abstract, I have to wonder how accurate a test this could be to detect Borrelia burgdorferi. I could see this rapid strain identification being useful for identifying bacteria for bacteriophage treatments and also for detecting the presence of bacteria on specific surfaces in hospitals or from open wounds. This wouldn't work well for something that is deeply embedded in collagen, but it might work from a synovial fluid sample better than current detection tests for Bb there.

Source Reference:
James R. Carey, Kenneth S. Suslick, Keren I. Hulkower, James A. Imlay, Karin R. C. Imlay, Crystal K. Ingison, Jennifer B. Ponder, Avijit Sen, Aaron E. Wittrig. Rapid Identification of Bacteria with a Disposable Colorimetric Sensing Array.Journal of the American Chemical Society, 2011; : 110427110353066 DOI: 10.1021/ja201634d

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8 The Friday Four

In this edition of the Friday Four, we'll look at using bacteria itself to deliver antibiotics and treat cancer, a huge touchscreen microscope, the value of Vitamin A in B1 cell immunity, and the mystery of lateral gene transfer between Chagas disease pathogens and its host.

1) 'Bacterial dirigibles' emerge as next-generation disease fighters

Link: http://www.sciencedaily.com/releases/2011/03/110329134120.htm

Summary: ScienceDaily (2011-03-30) -- Scientists have developed bacteria that serve as mobile pharmaceutical factories, both producing disease-fighting substances and delivering the potentially life-saving cargo to diseased areas of the body. They reported on this new candidate for treating diseases ranging from food poisoning to cancer -- termed "bacterial dirigibles."

Comments:


I don't know how many people are aware how much genetic engineering already goes on. Once the industry took off... well, it took off like wildfire. It's pretty common to do exactly what is stated in this article: "...Traditional genetic engineering reprograms bacteria so that they produce antibiotics, insulin, and other medicines and materials. The bacteria grow in nutrient solutions in enormous stainless steel vats in factories. They release antibiotics or insulin into vats, and technicians harvest the medicine for processing and eventual use in people."


In this experiment, they programmed E. coli not to pump out antibiotics into a vat in some factory somewhere... No, they created a version of E. coli that could target a portion of the intestine and adhere to it, and begin sending out chemical signals that influenced the production of proteins in different cells around it.


So I'm trying to understand this. They took E. coli, the bacteria that often makes people sick, and made a version that delivers itself to a specific part of the body and is programmed to affect other cells near it. Crazy.


What if this sort of "bacterial dirigible" could seek out and find remote Borrelia burgdorferi in collagen-rich tissues, in the adventitia of the heart, and in the brain? Does this have potential for killing the remaining spirochetes that may survive the initial onslaught of antibiotics?


2) Researchers in Finland Build Giant Multitouch Microscope

This is just too cool.  I want one. I want to see my spirochetes on this sucker.  [Time 1:43]

I think Leeuwenhoek would have just about shit himself if he saw one of those...

3) In the absence of Vitamin A, the body loses immune cells that put the brakes on the earliest stages of infection

Link: http://www.physorg.com/news/2011-04-absence-vitamin-body-immune-cells.html

Summary: Scientists have recognized the immune-boosting capabilities of vitamin A for the better part of a century, even without fully understanding how it helps the body fight off bacteria and viruses. "Soon after its discovery, vitamin A was termed ‘the anti-infective vitamin’ and was widely used to enhance recovery; but with the introduction of antibiotics, the therapeutic use of vitamin A diminished," says Sidonia Fagarasan of the RIKEN Center for Allergy and Immunology in Yokohama, Japan.

Comments:


So these researchers fed these mice a Vitamin A-free diet, and when they did, the mice had lower levels of IgA and IgM. They were given pneumonia vaccines and produced zero response. And then, the researchers tried to transplant B1 cells from healthy mice to these deficient mice - only to find that the B1 cells deteriorated, didn't last that long, and died off over several days.


However, the good news is, they found out the stem cells in the deficient mice's bone marrow could give rise to B1 cells - but they wouldn't do it unless they had some Vitamin A.


The researchers found out that a transcription factor protein found in activated T cells (NFATc1), regulates expression of numerous important genes in B1 cells. The researchers observed reduced NFATc1 levels in the mice's deficient B1 cells, but found that expression could be largely restored if these mice were injected with ATRA, a product of cellular vitamin A metabolism. After this injection, B cells increased more than four fold in number in ten days.


Having a balanced diet is definitely important for the immune system, and being deficient in Vitamin A would be problematic. Something so simple.


Even though it sounds like a good idea to take lots of Vitamin A given the immune system benefit, it doesn't work that way: if you're deficient, you need more; if you're taking too much, you need less because it can damage your liver and by extension kidneys because of too much calcium there. (It's also bad to consume high quantities during pregnancy - it can lead to failure to thrive in newborns.)


So get a test to see if you're deficient in Vitamin A first - and if so, then it's pretty easy to find foods full of  Vitamin A.


One thing that comes to mind after reading this is that recently I've read a paper, 'The Important And Diverse Roles of Antibodies in Host Response to Borrelia' by Laroca and Benach. In it, it mentions that B1 cell or x-linked immunodeficiency leads to more severe spirochetemia with B. hermsii... B1 b cells are needed for IgM antibody response.

Source publication:

Maruya, M., Suzuki, et al. Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells. Proceedings of the National Academy of Sciences USA 108, 722–727 (2011). http://www.pnas.org/content/108/2/722.short

4) Two new studies seek to validate the results of a retracted 2004 paper on parasite-to-host gene transfer, but skepticism lingers

Link: http://www.the-scientist.com/news/display/58093/

Do not let this bug kiss you - it can
carry Chagas disease parasites...

Summary: The microparasite that causes Chagas disease really can integrate bits of its genetic material into its host's genome, where it can then be inherited by the host's offspring, according to two studies published in PLoS ONE and PLoS Neglected Tropical Diseases (PLoS NTD).

Comments:


So this is kind of insane. Interesting and insane. The claim is being made for what might be the first documented instance of lateral gene transfer from the parasite that causes Chagas disease to not only its host but also a following vertical transfer to the host's offspring.


WTF. This is almost as far out as Lynn Margulis' claims about Borrelia burgdorferi.


These two recent studies are supposed to confirm the research found in a 2004 paper published in Cell which was later retracted. That paper showed - or supposedly showed - that University of Brasilia researchers found that T. cruzi could transfer genetic material to its rabbit, chicken, and human hosts. This sort of gene transfer - specifically of mitochondrial kinetoplast DNA (kDNA) - may contribute to the disease by disrupting host gene function and causing an autoimmune response.


Those looking at the newer research are eyeing it cautiously because of the earlier publication's retraction, which was done because Cell's staff made the determination that certain important information was missing from the paper. Speculation was that it was because identification and analysis of the specific sites of DNA integration were omitted.


I think this study and the two subsequent studies recently done will need to be repeated by another party not related to them, since this would be pretty big news if it's true. Also, someone needs to make sure their PCR methods don't create weird chimeras in passing.

Source publications:

M.M. Hecht et al., "Inheritance of DNA transferred from American trypanosomes to human hosts," PLoS ONE, 5: e918, 2010. 

A.R.L. Teixeira et al., "Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism," PLoS Negl Trop Dism, 5: e1000, 2011. 

---

And here's a bonus link set for my readers who are interested in aberrant and unusual contrails in the sky:

http://www.nature.com/nclimate/journal/v1/n1/full/nclimate1078.html

Here is the study to which the above article refers:

http://www.nature.com/nclimate/journal/v1/n1/full/nclimate1068.html

It was posted at the source on March 29, so I reassure you that it was not an April Fool news item.

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17 Video: Shortcuts To Learning Immunology

I realize I posted a 14 part mini-series on Immunology not long ago and know that could be a bit much to bite off at once in order to learn more about the immune system.

So I've been spending time looking for shortcuts - ways for readers to quickly get up to speed on some of the terms and processes used when discussing the immune system.

I figure videos usually are the best way to begin learning for many people - so I have been watching them on Youtube to decide what to post here.

Let me just say that as entertaining as some of them are, I wish more of them were factually correct or just had more educational content in them.

I love the idea of using battle scenes from The Lord of the Rings movies as an analogy for the immune system - some creative guy did this, and it was hilarious. But most of it had no mention of the immune system or how it worked, beyond "the good guys are these immune cells, and the orcs are the invading pathogens". And then there was the guy who went so far as to make a 1950's style documentary on angry macrophages, with retro props and a fake ad for Solomon cigarettes... this documentary compared pathogens to invading Communists.

Unique. Original. Points for style. And yet, next to no substance.

What could have been both an entertaining and educational clip ended up an abysmal failure. C'mon guys, you can do better.


Anyway, I found what I hope are some of the better videos on Youtube that you can watch to learn more about the immune system, and the material on the first one will help you advance to the next video. Also, each video reinforces what you've seen in an earlier one - it helps familiarize you with the terms and concepts used.

Let me know if you have any trouble following these. For those of you who are already more advanced students of immunology and have gotten past all of this, I will be posting more intermediate and advanced videos on the immune system later. (Those who have watched these basic and intermediate videos first will be able to move on to the advanced section, which will mention toll-like receptors and interleukins.)

If you are a more advanced student and already understand those, then I would like to encourage you to comment more here and perhaps start your own blog to let people know more about the science of the immune system and infectious diseases like Lyme disease.

Knowing more about the immune system opens the door to understanding research out there done by the IDSA guidelines panel, scientific researchers in microbiology and molecular biology outside of the IDSA panel, the statements LLMDs have made about Lyme disease and its treatment, and claims other Lyme patients have made online.

Okay, without further delay, here are four videos which may prove useful for beginners - each under 10 minutes:

Immunology Overview [Time: 4:42] - overview of basic parts and terms of the immune system

The Immune System [Time: 9:36] - Basic explanation of the immune system and how it works.

Immune System, Part 1 [Time: 7:59] - Barriers and Non-Specific Defenses
Note that this video is accompanied by this easy to view PDF:
http://www.kirkwood.edu/pdf/uploaded/695/immune_and_lymphatic2.pdf

The Immune Response (Garland Science) [Time: 1:43]

That should be good to get you started.

Note that each one has slightly different information about the immune system, but the core material is the same. Being exposed to this information in different ways over time makes it easier to learn.

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2 The Friday Four

1) Stress affects the balance of bacteria in the gut and immune response

Low stress & bacterial biodiversity:
The key to better health?

ScienceDaily (2011-03-22) -- Stress can change the balance of bacteria that naturally live in the gut, according to new research.

This study funded by the NIH shows that stress dysregulates the immune system, changing the natural flora of one's intestines and leaving people more susceptible to infections such as C. difficile. The more biodiverse intestinal flora is, the healthier one's immune system generally is.

Intestinal bacteria have been linked to diseases like inflammatory bowel disease and asthma, and a future goal of the study is see if changes in gut bacteria is related to such diseases worsening when people are under more pressure.

Comments:

Lyme disease patients are already pretty savvy about taking probiotics inbetween taking antibiotics - but is there something that can be done to diversify the number and kind of bacteria in our guts that would reflect the right balance of helpful organisms? Which combination of organisms is most beneficial to have, and how close are common probiotic blends to this beneficial mix?

Original Source Reference:
Bailey. Exposure to a social stressor alters the structure of the intestinal microbiota: Implications for stressor-induced immunomodulation? Brain, Behavior, and Immunity, 2011; 25 (3): 397 DOI: 10.1016/j.bbi.2010.10.023

2) 'Knowing it in your gut' is real: Cross-talk between human gut bacteria and brain

ScienceDaily (2011-03-23) -- A lot of chatter goes on inside each one of us and not all of it happens between our ears. Researchers have discovered that the "cross-talk" between bacteria in our gut and our brain plays an important role in the development of psychiatric illness, intestinal diseases and probably other health problems as well including obesity.

This study showed that genes linked to learning and memory are altered in germ-free mice and, in particular, in the hippocampus - one of the key brain regions for learning and memory.

"The take-home message is that gut bacteria influences anxiety-like behavior through alterations in the way the brain is wired," said Jane Foster, associate professor in the Department of Psychiatry and Behavioural Neurosciences of the Michael G. DeGroote School of Medicine.

Foster's team has a hypothesis that the state of your immune system and your gut bacteria influence your personality - and in this case, influences anxiety.

Comments:

This reminds me of a video I posted in a Friday Four a while ago that showed personality changes in mice based on whether they had cultivated bacteria or not.

One fascinating thing to consider here is if researchers find out that certain bacterial flora combinations create different psychological states and can be directly implicated in mental illness that new treatments involving probiotics may improve conditions that to date have been treated with psychiatric medications. Perhaps these new treatments will avoid some of the more troubling side effects of anti-depressants and anti-psychotic drugs.

Original Source Reference:
K. M. Neufeld, N. Kang, J. Bienenstock, J. A. Foster.Reduced anxiety-like behavior and central neurochemical change in germ-free mice.Neurogastroenterology & Motility, 2011; 23 (3): 255 DOI:10.1111/j.1365-2982.2010.01620.x

3) Biofilm  reorganization: Back to the theoretical drawing board

Staphlococcus aureus biofilm

"In a surprising new study, researchers using image-analysis methods similar to those employed in facial-recognition software have made a startling discovery that rules out the two main theories scientists had created to explain how bacteria self-organize into multicellular aggregate mounds. The study by researchers from Rice University and the University of Georgia has implications for biofilm research and appears online this week in the Proceedings of the National Academy of Sciences."

What scientists did was make a microscopic movie of Myxococcus xanthus, common soil bacteria, while it was forming aggregates or spore forms with up to 100,000 cells. In this way, the bacteria could survive more easily - just as many other bacteria survive in biofilms to evade antibiotics.

They discovered that the size of the aggregates led to a higher survival rate, and not other factors they predicted such as individual chemical signaling between cells.

So in this case: size matters.

Comments:

More studies on how biofilms form and what can be done to break them up are needed to prevent resistant infections. Studying bacteria and how it organizes itself can tell us more about what makes biofilms work and how to target them for treatment in the future.

4) Breakthrough in delivering drugs to the brain

Alzheimer's plaque

A team of researchers Oxford removed exosomes from mouse dentritic (immune system) cells. Then they attached specific proteins from the rabies virus (not the virus itself) to these exosomes -  proteins which bind to acetylcholine receptors in brain cells.

Then they filled these exosomes with the genetic code, siRNA, and injected them back into the mice.

In doing so, the siRNA got delivered to the mice's brain cells and turned off a gene (BACE1) which is involved in Alzheimer's disease. There was a 60% reduction in the gene's activity.

Comments:

If there is more than one cause for Alzheimer's disease - if it can be treated by using the body's own natural defenses and systems - this could be ground breaking.

Treatment systems similar to these exosome injections could also potentially be used to deliver medicine past the blood brain barrier for other conditions including cancer and infectious diseases.

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3 The Friday Four

In this edition of the Friday Four: how llamas are helping the fight against C. Diff infection, a new strategy to reduce antibiotic-resistant infections, how antibiotics may make fighting the flu harder,  a chart on US Gov't R & D funding for 2011 - plus two bonus links.

(And apologies for the island time posting - still Friday here, but not much longer.)


1) Researchers step closer to treatment of virulent hospital infection: Unique antibody from llama provides weapon against Clostridium difficile

Llamas

Researchers from the University of Calgary, Canada discovered that a simple antibody found in llamas may be the answer for future drug development against C. difficile. C. difficile is becoming increasingly resistant to existing antibiotic treatment such as metronidazole and vancomycin.

Llamas have antibodies which are very similar to human antibodies, and also another class of antibodies which are about 1/10th the size of human antibodies and are easier to engineer into drugs.

These antibodies - known as single-domain antibodies - bind to the C. difficile toxins with high affinity and interfere with the toxins' ability to damage cells.

Dr. Jamshid Tanha, the corresponding author of the study from the National Research Council in Ottawa says that understanding how camelid antibodies work will ultimately allow researchers to develop a new treatment for this important disease and potentially others.

"We are currently working with Dr. Ng's group to determine why these antibodies are successful," says Tanha.

Comment: Research in this field is especially important to patients who use high doses or long courses of antibiotics and run the risk of infection with C. difficile. Next to antibiotic resistance, C. difficile infection is one of the biggest problems with long-term antibiotic use, whether administration is oral or intravenous.

Original Source Publication:
The article, Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell-receptor binding domain, is published in the Journal of Biological Chemistry http://www.jbc.org/ and written by Greg Hussack (NRC and University of Ottawa), Mehdi Arbabi-Ghahroudi (NRC and Carleton University), Henk van Faassen (NRC), Glen Songer (University of Arizona), Kenneth K.-S Ng (Alberta Ingenuity Centre for Carbohydrate Science, and University of Calgary), Roger MacKenzie (NRC and University of Guelph), Jamshid Tanhan (NRC, University of Ottawa and University of Guelph).

2) Economics and Evolution Help Scientists Identify New Strategy to Control Antibiotic Resistance

Pseudomonas aeruginosa

In the March 2011 issue of Genetics, the scientists show that bacterial gene mutations that lead to drug resistance come at a biological cost not borne by nonresistant strains. They speculate that by altering the bacterial environment in such a way to make these costs too great to bear, drug-resistant strains would eventually be unable to compete.

A team of scientists from the University of Oxford, U.K. have taken lessons from Adam Smith and Charles Darwin to devise a new strategy that could one day slow, possibly even prevent, the spread of drug-resistant bacteria.

"Our study shows that concepts and tools from evolutionary biology and genetics can give us a boost in this area by identifying novel ways to control the spread of resistance," said Alex Hall, PhD, researcher from the Department of Zoology at the University of Oxford.

The research team measured the growth rates of resistant and susceptible Pseudomonas aeruginosa bacteria in a wide range of laboratory conditions. They found that the cost of antibiotic resistance has a cost to bacteria, and can be eliminated by adding chemical inhibitors of the enzyme responsible for resistance to the drug. Manipulating the cost of resistance may make it possible to prevent resistant bacteria from persisting after the conclusion of antibiotic treatment.

Comment: As the IDSA is moving to tighten the use of antibiotics in the US (and possibly worldwide) in order to prevent growing antibiotic resistance, it's important to research how to inhibit resistance. Research such as this could allow hospitals to continue to prescribe antibiotics with less concern about resistance to potentially deadly infections such as MRSA. It will also help in the fight against various tickborne infections.

Original Source Publication:
A. R. Hall, J. C. Iles, R. C. MacLean. The Fitness Cost of Rifampicin Resistance in Pseudomonas aeruginosa Depends on Demand for RNA Polymerase. Genetics, 2011; 187 (3): 817 DOI: 10.1534/genetics.110.124628

3) Antibiotics may make fighting the flu harder

H1N1 flu virus

Scientists knew that friendly bacteria in the intestines could help stop disease-causing bacteria from setting up shop in the gut. And this is one of the reasons Lyme disease patients take lots of probiotics between antibiotic doses - to prevent disease-causing bacteria such as C. difficile from setting up shop and producing toxins.

Some previous experiments hinted that gut microbes could influence how well the immune system works, but researchers thought the effect was mainly confined to the digestive system. Now there's evidence that friendly, or “commensal,” bacteria help defend against viruses affecting other parts of the body by keeping the immune system on alert for viral invaders, a research team discovered.

“What’s fascinating about this [new study] is that there’s a distant regulation of resistance to viruses by gut microbiota,” says Alexander Chervonsky, an immunologist at the University of Chicago.

Researchers found that the presence of "friendly" bacteria helped fight off viral infections that could affect the lungs - something that came as a total surprise.

Antibiotic treatment impaired the mice’s ability to make an important flu-fighting molecule called interleukin-1 beta or IL-1 beta, which is necessary to combat influenza and other viruses. Gut bacteria are constantly priming the immune system to make IL-1 beta, keeping the immune system vigilant against flu and other viruses. The researchers aren’t sure yet which bacteria in the gut are responsible for the virus-defense mechanism, but they are looking at Lactobacillus as a potential candidate for study.

Comment: Here is more evidence that keeping one's gut and intestinal flora in balance is important to one's overall immune system. Patients who are taking antibiotics are encouraged to keep up their probiotics to maintain a healthier balance, and more research is needed to find out which commensal bacteria are the most beneficial for combatting both bad bacterial and viral infections.


Original Source Publication:
T. Ichinohe, et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proceedings of the National Academy of Sciences. Published online ahead of print, March 2011.

4) Not so much a link, but something to think about: Where US Gov't R & D Funding Is Spent...


Source:LiveScience

Comment: So $32.09 billion goes to the NIH, and everything else gets a smaller slice of the research pie.  Why is that? And are contracts in other areas that are awarded potentially falling under that mysterious "All Other" slice of the pie?

Bonus links for today: The Daily Kos Series on Lyme Disease Awareness 2010 raised the issue of Chronic Lyme disease and March 10, 2011's Chronic Tonic Column focused on one person's Lyme disease experience.

It's interesting to see people writing about Lyme disease at The Daily Kos - not your typical Lyme disease discussion venue. I don't know when or if to expect any Lyme Disease Awareness posts for this May, but I plan to check out the site then and see if there are new ones.

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0 Free Video Lectures & Podcasts: UC Berkeley Molecular and Cell Biology

For those of you who use iTunes to download podcasts and have the ability to learn by listening - or hope to absorb information through repetition and osmosis - there are FREE audio podcasts on Molecular and Cell Biology from UC Berkeley you can download from iTunes.

There are also some video podcasts of the classes available on iTunes and both audio and video podcasts can be found on UC Berkeley's webcast site.

If you're new to studying biology, I highly recommend starting with a basic biology course first, - such as this Biology 1A lecture at Berkeley and/or these Biology video podcasts from MIT - then this class:

Molecular and Cell Biology 110, 001|Fall 2009|UC Berkeley
by Qiang ZHOU, qing zhong, Thomas C. ALBER
Download up to 41 classes (start with session 1 at bottom of podcast list!)
http://itunes.apple.com/itunes-u/molecular-cell-biology-110/id354820350


Followed by its more advanced class:

Molecular and Cell Biology 130, 001|Spring 2009|UC Berkeley
by Randy W SCHEKMAN, Kunxin LUO, David G. DRUBIN
Download up to 42 classes (start with session 1 at bottom of podcast list!)
http://itunes.apple.com/us/itunes-u/molecular-cell-biology-130/id354820424

If you do not have iTunes, you can also go directly to UC Berkeley's webcast site and WATCH and listen to these classroom lectures for free, on a variety of topics.


I realize this may be challenging for many Lyme patients dealing with cognitive issues and "brain fog", but I put the information out there because it can be useful to learn to decipher the studies and research you may come across from the IDSA, ILADS, and other groups. Knowledge is power, and the great things about these videocasts and podcasts are:

• You can play and replay each podcast as often as you like.
• You can learn at your own pace.
• You can share these links with others and talk about what they learned at their pace.
• You can watch some and listen to others - work with your best learning style.
• They are absolutely FREE - do you have any idea how much each unit at UC Berkeley costs?

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