Exosomes

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Ron

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Exosomes Frequently Asked Questions (FAQs) - EXCERPT

General info from the same site
Exosomes are small vesicles (30–150 nm) containing sophisticated RNA and protein cargos. Exosomes are now viewed as specifically secreted vesicles that enable intercellular communication and have become the focus of exponentially growing interest, both to study their functions and to understand ways to use them in the development of minimally invasive diagnostics

Exosomes have been shown to transport a range of molecules from one cell to another. Their cargo includes proteins, lipids, mRNA (fragments and full length), rRNA, miRNA, and various ncRNA.

Visual identification of exosomal vesicles can be challenging, and free exosomes alone are too small to be detected in flow. There are a few methods, though, which can be used for analysis.

How are exosomes defined?
The current definition of exosomes is complex, with no absolute consensus in the field. Typically, exosomes are defined as vesicles floating in sucrose solutions at a density ~1.13 to 1.19 g/ml during ultracentrifugation-based isolation, with an expected size of 30–150 nm (based on electron microscopy analysis). Exosomes can also be defined and identified by their surface protein markers, which include: tetraspanins (CD63, CD81, CD9) and others like ALIX. Currently, there are no appropriate tools nor sufficient knowledge in the field to set a clear and simple definition of exosomes that would differentiate them from other micro-/nanovesicles.

What are exosomes composed of?
Exosomes are tiny vesicles (30–150 nm) containing protein and/or RNA cargo, within a lipid bi-layer membrane. Exosomes can differ extensively in both their cargo and surface proteins, and different cell types can secrete different —sometimes multiple—types of exosomes.

How can exosomes having different origins be differentiated if the sample is serum?
In terms of exosome characterization and comparison, this can be performed as we did it in our publication (Oksvold et al., Clinical Therapeutics 2014 June 36, 847-862.e1, PMID:24952935). We characterized 4 different B-cell lymphoma derived exosomes by starting to compare the expression of typical exosomal markers, such as tetraspanins and B-cell specific markers. Characterization was then performed after pre-enrichment of the exosomes and again after isolating exosomes with Dynabeads magnetic beads that target CD81+ and CD63+ exosomes. Interestingly, the expression of both exosomal markers and B-cell markers differed between all 4 B-cell lymphoma derived exosomes. The complex nature of serum as a starting sample will add to the complexity. Here a pre-clearing step (e.g., using size-exclusion chromatograph prior to Dynabeads isolation) is recommended.

How are exosomes visualized and/or counted?
Exosomes are too small (30–150 nm) to be seen using a regular microscope, as this is limited to objects that are at least several micrometers in size. Structures as small as bacteria, and even very large viruses such as vaccinia virus (approx. 0.5µm in size), can be observed by light microscopy. Yet the resolution of the light microscope is too low for observing exosomes.

The typical methods of analysis for exosome size distribution include the NanoSight instrument and electron microscopy. Although very different in methodology, both technologies allow you to study nanoparticles down to 10 nanometers in size.
To determine the concentration of exosomes in the sample, you can use the NanoSight instrument or the Izon instrument. The NanoSight instrument enables counting and sizing of nanoparticles (10–1000 nm) using light scattering and browning motion, while the Izon instrument accomplishes the same thing using nanopore analysis.

Is it known if factors such as CD9 or CD81 are specific for exosomal origin?
Currently there is no consensus about a general exosome marker. The current recommendation from the research community is to combine detection of many membrane-bound or membrane-associated proteins in order to verify the presence of membranes. Our own experience is that targets CD63 and CD81 are found in many different exosome preparations, as is CD9. However, we detected at least 2 different cell lines releasing exosomes which were CD9 negative (Jurkat cells and several B-cell lymphoma cells, Oksvold et al 2014). Equally important is to document the absence of contaminating vesicles by staining for markers known to be expressed in compartments such as ER, Golgi or nucleus. It is also important to characterize the host cell in terms of the same markers.

Which markers can be used for exosome characterization?
As far as I know (even after consulting with many key opinion leaders in the field of extracellular vesicles), there is currently no consensus about a general exosome marker that will be found on all exosomes. The current recommendation from the research community is to combine detection of many membrane-bound or membrane-associated proteins in order to verify the presence of membranes. In our own experience, targets such as the CD63 and CD81 are found in many different exosome preparations, as is CD9. However, we could detect at least 2 different cell lines releasing exosomes which were CD9 negative (Jurkat cells and several B-cell lymphoma cells). In house, we usually label for CD9, CD81 and CD63 to verify the presence of membranes in our preparation using the following detection antibodies:

Exosome—anti-Human CD9 (for Western) (Cat. No. 10626D)
Exosome—anti-Human CD81 (for Western) (Cat. No. 10628D)
Exosome—anti-Human CD63 (for Western) (Cat. No. 10630D)

These detection antibodies have been validated for use with both pre-enriched exosomes and those isolated with Dynabeads magnetic beads. Tsg101 and annexins have also been mentioned as useful targets.
It is also important to characterize the host cell in terms of different markers to determine the level of contaminating vesicles from these organelles:

ER: HSP90B1, calnexin
Golgi: GM130
Mitochondria: cytochrome C
Nucleus: Histones

How much RNA can be recovered from the exosomes?
This can vary depending on the sample type, volume of sample, isolation method, and exosome content/concentration. Listed below are some examples:

When exosomes are isolated from 30 mL of HeLa cell culture media using the Total Exosome Isolation (from cell culture media) reagent, it is possible to recover ~8 ng exosomal RNA.
For exosomes recovered from 4 mL serum using the Total Exosome Isolation (from serum) reagent, ~2 ng exosomal RNA can be obtained.
In both cases, these amounts of RNA are sufficient for RNA library prep for Ion Torrent PGM or Proton sequencing. For real-time PCR analysis, substantially smaller amounts of RNA are needed and much lower sample volumes can be utilized. For example, RNA recovered from 3 μL serum or 30 μL cell media is enough for one RT-qPCR reaction.

What is the mechanism of exosome formation?
Exosomes are classically described as vesicles originating from the endocytic pathway through fusion of multivesicular bodies with the plasma membrane. They are a part of a larger family of vesicles secreted by cells—including microvesicles, ectosomes and shed particles–which originate by direct budding from the plasma membrane. It is extremely challenging to separate these entities using currently available techniques and instruments, due to overlap in their size, density and overall composition similarities.

How do I know that I’ve isolated exosomes, and not other types of vesicles?
In addition to their 30–150 nm size, to be categorized as exosomes, the vesicles should be positive for certain surface protein markers such as tetraspanins. The most widely accepted marker is CD63, but CD81, CD9 are utilized as well. Western blotting for these targets on the sample of interest is a relatively simple way to confirm that the vesicles are indeed exosomes. However, the current definition of exosomes is not set in stone, as there is no absolute consensus in the field. It will probably take another several years to come up with the exact specification and nomenclature for all nano-/microvesicles (including exosomes)

According to the literature it seems that 18S mRNA is absent from exosomes. Can you comment on that
We have analyzed exosomal RNA content by sequencing and qPCR, and these vesicles definitely include some 18s RNA. In our opinion, the early papers were misleading. One team claimed that exosomal cargo is miRNA- and mRNA-negative, and everyone else accepted this as a dogma. In reality, exosomes contain ribosomal RNA, tRNA, many non-coding RNAs, and a large fraction of miRNA is actually associated with proteins (e.g., Ago 2) not exosomes.

What controls should be included to ensure maximal signal/noise ratio for miRNA isolation only from exosomes?
I did not see any papers yet on the topic of separately recovering different entities from the same body fluid source (protein-miRNA, exosomes, microvesicles, etc). However, know that a number of teams are looking into fractions vs. analysis of whole serum or plasma RNA.
 
extracellular vesicles (EV), exosomes and parasites

is it the ace in their sleeve ?

The ability of parasitic helminths, such as H. polygyrus, to release EVs identifies a newly recognized vehicle to mediate cross-species communication as part of the host-parasite relationship.

While uptake by macrophages might initially be interpreted as a host mechanism for the removal of parasite EVs, we show that the nematode-derived EVs exert functional properties on the recipient macrophages.

Indeed, EVs generate potent suppression of type 1 and type 2 effector molecules when administered during or after the onset of macrophage activation. Among the molecules suppressed, arginase-1 is an essential component of protective immunity to intestinal helminths.

We do not yet know which parasite molecule(s) within the EV cargo is responsible for these effects, as there is a suite of protein, lipid, and RNA candidates that merit further study.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457486/
 
Here we report for the first time that ciprofloxacin induced the release of both mitochondrial and chromosomal DNA associated with the surface of Exosomes (EXOs).
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Published: 15 August 2017
Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes.

Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin.

Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Extracellular vesicles (EVs) can alter signaling of recipient cells by either cell surface receptor-ligand interactions or upon uptake by cells.

EVs have been shown to deliver specific mRNAs and various small RNAs as well as DNA to healthy cells. They modify the genetic composition of recipient cells and alter their functions

The presence of a clinically relevant dose of ciprofloxacin has been reported to cause oxidative damage, mitochondrial dysfunction and mtDNA depletion in mammalian cells.

We also demonstrate that this exofacial DNA facilitates Exosomes (EXOs) binding to the extracellular matrix protein fibronectin.

https://www.nature.com/articles/s41598-017-08392-1
Formation of Exosomes can be induced by certain substances like the antibiotic Ciprofloxacin

"Die Bildung von Exosomen kann weiterhin durch bestimmte Stoffe wie dem Antibiotikum Ciprofloxacin ausgelöst werden"
https://de.wikipedia.org/wiki/Exosom_(Vesikel).
 
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Sunshine3 said:
Is it possible that the identification of the 'something in the blood' could be an important Breakthrough. If it were identified could we then be on track for effective treatments?
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Once we isolate what exactly is the culprit, oh yeah.

This is the thing that is blocking pharmaceutical research. They need a target to get started. Plus the technology is rapidly maturing to automatically parallel test thousands of compounds and measure the effect. The nanoneedle seems perfect for that automation since it's a simple electrical signal and is cheap.

The only thing I'm not sure is how fresh the samples have to be to produce the signal. Do we know this?
 
an exosome could be responsible for high/special signal in cfs patients (Ron Davis/Stanford)

Forbin said:
Going by the chart below, there were at least 20 patients and twenty controls. It looks like all of the CFS patients produced a signal and none of the controls. According to Dr. Davis, the odds that this does not represent a real difference between the two groups is a billion to one. [I'm not sure if he was referring to this specific chart, or if there might have been more patients/controls added later.]
Click to expand...

nn-jpg.6787
 
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My maths brain is offline but surely he could have drawn the green line anywhere he chose, that caused all the blue endpoints to be seperate from all the red endpoints? I'm not sure about any effect of 'odds' that might have in this case.

The odds of any endpoint being either red or blue are surely 50:50, they did 40 tests, all matched with no overlaps (subject to the above point) so surely that means the odds were 1:2^40? That's quite a big number, and probably wrong as it doesn't, in this case, matter what order they hit the 'correct' endpoints.

I used to know this stuff, I didn't even have to try and work it out, it was 'know' :banghead::banghead::banghead::banghead:
 
What are the sizes of exosomes? Relatively to what Ron Davis described the "something in the blood" as large?
 
junkcrap50 said:
What are the sizes of exosomes? Relatively to what Ron Davis described the "something in the blood" as large?
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I don't think Ron Davis has given a size estimate other than to say "it's big." My impression when he talks about breaking it apart and seeing what it's made of is that he's talking about a "particle" made up of many molecules as opposed to just one large molecule.

I found some quotes on the size of exosomes.
Since the size of exosomes is limited by that of the parent MVB [multivesicular body], exosomes are generally thought to be smaller than most other EVs [extracellular vesicles], from about 30 to several hundred nm [nanometers] in diameter: around the same size as many lipoproteins but much smaller than cells.
https://en.wikipedia.org/wiki/Exosome_(vesicle)
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The majority of EVs occur within the sub-micron range (30–1000 nm), where exosomes are believed to be most abundantly present in the lowest size range (30–150 nm).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533132/
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The range of 30-150 nm would make them 273-55 times smaller in diameter than a red blood cell (8200 nm). Their relative volumes would be much, much smaller still compared to the blood cell.

Exosomes.jpg
 
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in mecfs: more and smaller EVs

We found that the amount of EV-enriched fraction was significantly higher in CFS/ME subjects than in HCs (p = 0.007) and that EVs were significantly smaller in CFS/ME patients (p = 0.014).

A growing body of evidence suggests that extracellular vesicles (EVs) play a role in cell-to-cell communication, and are involved in both physiological and pathological processes.

J Extracell Vesicles. 2018; 7(1): 1453730.
Published online 2018 Mar 22. doi: 10.1080/20013078.2018.1453730
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912186/
 
It's possible that exosome cargoes will be found to be biomarkers for ME/CFS, but we don't know the answer about that yet. I've attached a paper that showed differences between RNA content of vesicles in healthy people vs. Alzheimer's patients, to show the potential usefulness.
 

Attachments

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Cipro info is interesting. Cipro is reported to cause cfs or cfs like syndromes. On the other hand someone on here reports that they improve on one of the flox antibiotics (levaquin). So maybe the levaquin results in a shift of the production of exomes to ones that are bad albeit less bad than “regular” CFS exomes. Also, perhaps the quinolone antibiotics could be used to develop an animal model of mecfs.
 
I was put on Cipro for what a doctor thought I had was bacterial prostatitis for a month (2x per day). When that didn't work, I was trialed on doxy, amoxy, and Bactrim. Turns out it was a nerve instead causing the pain. I have been in the worst crash ever and I am almost a year into it to no avail. I already had CFS but it was manageable, now I am bed bound/housebound with incredibly terrible abdominal pain, nausea, IBS, and fatigue.
 
from above
Here we report for the first time that ciprofloxacin induced the release of both mitochondrial and chromosomal DNA associated with the surface of Exosomes (EXOs).

Formation of Exosomes can be induced by certain substances like the antibiotic Ciprofloxacin.
Click to expand...

fluoroquinolones are super powerful, world wide super often prescribed since very helpful.
(something, changed later in the recipe, makes their efficiancy outstanding, forgot what it was)
some ppl get super harmed on fluoroquinolones.

now, the fluoroquinolones "do something" about exosomes.

perhaps, its something helpful ?
not necessarily bad ?
 
Exosomes may protect us and could deliver drugs

Exosomes 'swarm' to protect against bacteria inhaled through the nose
Date: November 12, 2018
Source: Massachusetts Eye and Ear Infirmary

A research team describes a newly discovered mechanism. The findings shed new light on our immune systems -- and also pave the way for drug delivery techniques to be developed that harness this natural transportation process from one group of cells to another.

https://www.sciencedaily.com/releases/2018/11/181112082414.htm
 
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