Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in ME/CFS - 2020 - Schreiner

[cassava7]

I believe this section is where a link between HHV6 and a subset of ME patients comes from - presence of sncRNA-U14

We have recently identified HHV-6–encoded sncRNA-U14 as a potent marker for viral reactivation (15).

Expecting that viral RNA numbers under reactivation/infection conditions can be higher than viral DNA numbers and that they should be easier to detect, we carried out FISH studies using concentrated PBMCs or blood clot sections from a fraction of these ME/CFS cases (n = 20) (Fig. 3). FISH analysis increased HHV-6–positive cases to 40% (n = 8 out of 20)

(Supplemental Table I). FISH image analysis confirmed our hypothesis that only a small fraction of the blood cells carried HHV-6 sncRNA-U14. None of the control cases, including the two iciHHV-6cases, showed positive staining for sncRNA-U14.

From methods section

Sure would be nice if someone could replicate the FISH analysis, say from the UK ME biobank samples. No idea what FISH entails, or if it requires fresh blood.

Thanks for pointing that out. I dismissed it as "it's only 8 out of 20 patients", but could that finding isolate at least a subset of patients? In his presentations at NIH'19, Prusty also mentioned similar small non-coding RNAs from other viruses. I wonder if checking for all of these sncRNA would yield a higher number of positive cases.

That diagram is a bit confusing. It is so easy to miss the "or" which shows they are separate experiments.

I found the title after a while

 

[Estherbot]

The omf should step in and finance Bhupesh, I have a feeling that will not happen.

*Depends on who makes the decisions at OMF eg is there there a bias towards Ron Davis's own lab at Stanford or is it done independently.
Robert Naviaux is one to the paper authors so it will be raised at the OMF at leasre

*Solve ME/CFS have funded Prusty's work and he has said he has applied to the NIH for a grant (whether that happens, we'll see)

*Prusty did raise 30 000 euros recently through crowd funding for this research but for that amount what you can with that is limited.

 

[Estherbot]

Resources for reading about HHV-6 & ME/CFS

HHV-6 Foundation

They have an overview of past HHV-6 research into ME/CFS
https://hhv-6foundation.org/associated-conditions/hhv-6-and-chronic-fatigue-syndrome

There links to papers from VanElzakaar, Komaroff, Montoya and others.

Looks a few years out of date but a useful starting point for your background reading

 

[Jonathan Edwards]

how common is mitochondrial fragmentation in ME (measure mitochondria in a bunch of samples) -- if there are identified biomarkers for mitochondrial fragmentation then validate these and test how common mitochondrial fragmentation is in ME

I was thinking this really needs to be established before anyone bothers to play with cell cultures and sera.

I am not aware of any clear indication that mitochondrial fragmentation is a feature of ME cells. If it isn't then this looks like a blind alley. Behan described mitochondrial changes in muscle decades ago but I think nobody could replicate this. Moreover, he talked of fusion and branching of Cristae. I have not read the whole paper again as the net version is paywalled but I don't remember him finding fragmentation.

I think we need mitochondrial fragmentation to be in a relevant tissue, too. Looking at peripheral blood white cells is I think too fraught with sampling problems and confounders and sorry mitochondria in white cells are not going to make you feel ill. The abnormality needs to be in something like muscle or liver. Getting biopsies is not that easy but there are probably various bits of ME people blocked up in paraffin in path labs stored away ready to be analysed for mitochondria. There might be some bits of Andy's gallbladder for instance.

 

[Hoopoe]

Looking at peripheral blood white cells is I think too fraught with sampling problems and confounders and sorry mitochondria in white cells are not going to make you feel ill.

So all these studies of white blood cell energy metabolism are like the man searching for his keys under the lamp post because it's easier to see there?

Speaking of the liver, this organ has not gotten much attention despite being a possible suspect.

 

[Sly Saint]

Behan described mitochondrial changes in muscle decades ago

I have not read the whole paper again as the net version is paywalled

do you mean this one?
Mitochondrial abnormalities in the postviral fatigue syndrome
W. M. H. Behan

sci-hub.tw/10.1007/bf00294431

 

[FMMM1]

I was thinking this really needs to be established before anyone bothers to play with cell cultures and sera.

I am not aware of any clear indication that mitochondrial fragmentation is a feature of ME cells. If it isn't then this looks like a blind alley. Behan described mitochondrial changes in muscle decades ago but I think nobody could replicate this. Moreover, he talked of fusion and branching of Cristae. I have not read the whole paper again as the net version is paywalled but I don't remember him finding fragmentation.

I think we need mitochondrial fragmentation to be in a relevant tissue, too. Looking at peripheral blood white cells is I think too fraught with sampling problems and confounders and sorry mitochondria in white cells are not going to make you feel ill. The abnormality needs to be in something like muscle or liver. Getting biopsies is not that easy but there are probably various bits of ME people blocked up in paraffin in path labs stored away ready to be analysed for mitochondria. There might be some bits of Andy's gallbladder for instance.

Thank you very much for your reply.

Further "mad idea" as you might describe it. Could investigation of mitochondrial fragmentation, if it it protective against coronavirus i.e. allowing you vital time to produce antibodies, be a potential research topic regarding coronavirus? I'm thinking of a way of linking ME research to the current coronavirus pandemic --- any ideas regarding that welcome!

@Michiel Tack

 

[FMMM1]

makes no sense at all ...

What I mean is that plasma electrophoresis (barely heard of it) might not take out this particular component e.g. if plasma electrophoresis "filters", i.e. on size basis, then the relevant factor in ME plasma might be too small - not removed.

Also if the factor is constantly produced (clearance via plasma electrophoresis may be ineffective) then the ME symptoms wouldn't necessarily disappear. If you removed it, and it didn't come back appreciably in say 1 week, then OK you should see a temporary improvement.

As I said --- barely heard of plasma electrophoresis.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854315/
removal of antibodies
Immunoadsorption

 

[Snow Leopard]

With the coronavirus, they can get studies done in a few weeks.

Yes, poor quality preprints, or quick and superficial genomic studies. The hard stuff is still unknown, like how is the virus killing people, what is the cause of the unusual symptoms?

 

[cassava7]

I think we need mitochondrial fragmentation to be in a relevant tissue, too. Looking at peripheral blood white cells is I think too fraught with sampling problems and confounders and sorry mitochondria in white cells are not going to make you feel ill. The abnormality needs to be in something like muscle or liver. Getting biopsies is not that easy but there are probably various bits of ME people blocked up in paraffin in path labs stored away ready to be analysed for mitochondria.

Yes! Could an abnormality in the liver or muscle happen in PBMCs too, but to a lesser extent, or would it be detectable exclusively in those tissues? If there effectively is a "factor in the blood" that floats around, I am thinking that it could affect different types of cells, but not in a clinical relevant way for some (most?) of them.

I would be willing to do a muscle biopsy for a study, or even a liver one but I'm afraid that's more involved / less common (is it?). Wish researchers had brain, liver and muscle tissue from people with ME readily available...

 

[Jonathan Edwards]

So all these studies of white blood cell energy metabolism are like the man searching for his keys under the lamp post because it's easier to see there?

One way of putting it.

 

[Jonathan Edwards]

Yes! Could an abnormality in the liver or muscle happen in PBMCs too, but to a lesser extent, or would it be detectable exclusively in those tissues?

I think it is all too difficult to predict.

Looking at white blood cells for signs of a general metabolic shift might be a bit like in a financial crisis due to wildfires looking at the bank balances of firemen. They might be the one lot of people doing better than usual.

Interferon and similar responses that shut down cells that might get infected can drive immune cells crazy.

And all white cells in the blood are more or less by definition not doing anything anyway because they are only in the blood on their way to somewhere the might do something. So to mix metaphors you might be looking at firemen while going down their greasy poles, not actually doing any firefighting.

 

[sebaaa]

Yep. The answer to that doesn't really matter at this stage. The question is, can you identify the factor(s) in ME/CFS serum that cause it, could those factors lead to the next clue, and could those factor(s) be unique to ME/CFS. This work leads to more questions and that is what is truly exciting. Will it lead to the next clue?

Bhupesh Prusty has said on twitter to have a reasonable numerically powered study he needs Eur 400K for 3 years to fund the work and Phd's.

According to my brief e-mail conversation with Prusty, he said that he's already found one these "factors" and that there are many others varying from patient to patient. If we take the assumption that mitochondrial fragmentation is central to causing the symptoms in ME/CFS, then finding the "factors" that cause it could be massive. Now, I have absolutely no clue when he's going to publish this research. It could take many months or even a year. That's also assuming that he finds the 400k from somewhere. That's why I think it's imperative to try to find another extremely fast and cheap way to find what the "something in the blood" is and release the information to everyone.

The omf should step in and finance Bhupesh, I have a feeling that will not happen.

I think they're already collaborating. Though, I'm not sure if they're directly funding him.

 

[Estherbot]

Cort Johnson has written up a summary of the paper.

https://www.healthrising.org/blog/2020/04/26/explaining-chronic-fatigue-syndrome-naviaux-prusty/

He has provided some key points

• When the low viral loads of HHV-6 have suggested it may not be involved in ME/CFS, some researchers have suggested that a smoldering infection may be present.
• Bhupesh Prusty has provided evidence that a low, smoldering infection may indeed be present in a subset of ME/CFS patients.
• Prusty believes that very early in HHV-6’s reactivation phase, HHV-6 attempts to cripple a cell’s mitochondrial output by producing small non-coding RNAs that cause mitochondrial fragmentation and metabolic decline.
• In response to the mitochondrial damage the infected cell senses, it amps up its antiviral defenses, putting it, Bob Naviaux believes, into a hypometabolic state – in which most of the energy of the cell goes to antiviral defense – leaving little energy left over for anything else.
• The infected cells also appear to secrete something which puts other cells around them in a similar “cell danger response”. This is an important step as few cells appear to be directly infected.
• The kicker came when Prusty and Naviaux showed that same process occurred when serum from ME/CFS patients’ cells was added to healthy, uninfected cells; mitochondria began to fragment and the formerly healthy cells developed a strong antiviral response.
• Prusty’s and Naviaux’s study could help explain 2016 findings suggesting that putting ME/CFS patients’ cells together with healthy cells causes the energy production or other factors in the healthy cells to drop.
• They also believe the cell danger stance ME/CFS patients cells are in could explain why some ME/CFS patients rarely get colds.
• Attempts to determine the nature of the mysterious substance that may be putting other cells on high alert and sapping their energy production at the same time are ongoing.
• Naviaux and Prusty are also examining ways to block the signal that they believe sends uninfected cells into “cell danger mode”.
• Given the novelty of their research, it was no surprise to see that it was supported by private foundations including the Solve ME/CFS Initiative and the HHV-6 Foundation. Private foundations play a pivotal role in supporting researchers with creative approaches to ME/CFS.

 

[Estherbot]

Cort has also supplied the press release for the study.

I'm guessing that he has liased with Bhupesh Prusty & Robert Naviaux over the study.

 

[unicorn7]

I see this paper more as a hypothesis-kind of paper, but I'm happy that someone has followed up on the serum swap idea. Because to me that seems like a relevant finding.
What I find interesting about the serum swap, is that this means there is nothing wrong with the cell or the mitochondria THEMSELVES, it's only when the signal from the serum joins in that "something" detrimental happens. I think the symptoms suit a fluctuating "something" in the serum, otherwise I could never have "almost normal days"? Plus it would explain why people haven't found much in the past? Because you need that serum component?

I was thinking this really needs to be established before anyone bothers to play with cell cultures and sera.

I am not aware of any clear indication that mitochondrial fragmentation is a feature of ME cells. If it isn't then this looks like a blind alley. Behan described mitochondrial changes in muscle decades ago but I think nobody could replicate this. Moreover, he talked of fusion and branching of Cristae. I have not read the whole paper again as the net version is paywalled but I don't remember him finding fragmentation.

I think we need mitochondrial fragmentation to be in a relevant tissue, too. Looking at peripheral blood white cells is I think too fraught with sampling problems and confounders and sorry mitochondria in white cells are not going to make you feel ill. The abnormality needs to be in something like muscle or liver. Getting biopsies is not that easy but there are probably various bits of ME people blocked up in paraffin in path labs stored away ready to be analysed for mitochondria. There might be some bits of Andy's gallbladder for instance.

Why are mitochondria in white blood cells not going to make you feel ill? I don't get the reasoning for that?
Feeling ill is the byproduct of you own immune system producing cytokines, so I don't see why liver or muscles should be involved for this. Plus we don't know if this mitochondrial fragmentation doesn't happen in other cells.

 

[mariovitali]

@Estherbot

From the link you provided on Phoenix Rising, we read :

In people who don’t have ME/CFS, this phenomenon is normal and only lasts a few days at the beginning of a new infection or after exposure to certain environmental chemicals, or after physical injury. However, in ME/CFS, they believe HHV-6 infected cells continue to secrete a substance which inhibits cellular energy production, leading to fatigue, and all the other symptoms of the disease.

My question is : Does this mean that ME patients cannot get inhibited cellular production because of chemicals or physical injury (so from non-viral events)? In other words, the text in bold is this only applicable to non-ME patients?

Also, we will have to keep in mind that we have patients that got ME from a non-viral cause.

 

[wigglethemouse]

Just to be clear with all the talk about PBMC's. Prusty used U2-OS and A549 model cells that you can buy and grow for the experiments as they have specific properties that he used to control the experiment.

From Health Rising article

In order to ensure that replication was not a factor, the authors chose a cell-line (U2-OS) that has the CD46 receptor that enables HHV-6 to enter the cell and integrate its DNA, but which does not allow the virus to carry out its life-cycle. This enabled the researchers to focus on the very early stages of reactivation.

The A549 lung carcinoma cells were virus naive and could be grown in the lab for two days in the presence of Healthy control or ME serum.

Regarding PBMC's, both the Newcastle and Cornell teams reported that cells needed to be activated to see differences between cells from healthy and ME patients, and then the difference is not that great.

In his talk and paper Paul Fisher explained that lymphocytes generate a fair percentage of quiescent energy in the cystol so in general you will not see the difference in ATP generation from the mitochondria, and as they are small cells the measurements are at the lower end of the instrument detection range giving you larger measurement errors. For this reason he converted lymphocytes to much larger lymphoblasts by exposing them to EBV.

As @Hutan posted Lymphocyte basal OCR is difficult to measure as the level is approaching the measurement limit of the instrument used. Lymphoblasts OCR is 100x or more, so good repeatable measurements are possible.

 

[Andy]

“These findings are important because they show for the first time that there is an antiviral activity in the serum of patients with ME/CFS that is tightly associated with an activity that fragments the mitochondrial network and decreases cellular energy (ATP) production,” said Robert Naviaux, MD, PhD, professor of medicine, pediatrics and pathology at UC San Diego School of Medicine.

https://health.ucsd.edu/news/releas...s-viral-immunities-come-at-lifelong-cost.aspx

 

[Jonathan Edwards]

Why are mitochondria in white blood cells not going to make you feel ill? I don't get the reasoning for that?
Feeling ill is the byproduct of you own immune system producing cytokines, so I don't see why liver or muscles should be involved for this. Plus we don't know if this mitochondrial fragmentation doesn't happen in other cells.

I agree that feeling ill tends to be due to cytokine release. But if that is the case then the guiding idea that PWME feel ill because their mitochondria cannot produce enough energy is wrong for starters. We should be looking for cytokines. But if we say maybe you can feel ill, or at least weak and fatigued, because mitochondria are unable to produce energy, which seems a very popular idea with these researchers, then you are only going to feel weak or fatigued because of poor energy production in cells that help you move around, like muscle, or provide background support for that energy production, like liver. Both muscle and liver cell failure make people feel weak and fatigued.

But white cell malfunction has no reason to make you feel weak. People who suddenly lose all their white cells from e.g. drug reactions do not feel weak or fatigued in general. They are usually unaware of the problem until they get an infection. Poorly performing mitochondria in white cells might make you develop an opportunistic infection but that seems to bear no relation to the situation in ME.

I absolutely agree that we do not know if mitochondrial fragmentation occurs in other cells. That was my point. We have no evidence that it occurs in cells that might be responsible for a feeling of lack of energy.