Jarred Younger on Neuroinflammation in ME/CFS and Fibromyalgia

[Sushi]

But who knows what plasma levels are needed for minocycline to be an effective microglia activitation inhbitor and wether those levels can be reached with generally prescribed doses.

@Hip Any idea?

 

[Hip]

@Hip Any idea?

This study, which examined the ability of three tetracycline antibiotics (namely minocycline, doxycycline and tetracycline) to inhibit microglial activation during ischemia, used doses of 90 mg per kg of body weight in the animals, which are very high doses. That would correspond to a 7,200 mg dose in an 80 kg human. Normal doses of doxycycline and minocycline are around 200 mg daily. Though the authors did say that "we have also found that significantly lower doses of the compounds are neuroprotective in both global and focal brain ischemia."

Note that tetracycline antibiotics are not the only microglial activation inhibitors. I complied a list of dozens of microglial activation inhibitors in this thread:

Chronic Microglial Activation in ME/CFS, And Its Possible Treatment Using Microglial Inhibitors

 

[nandixon]

@Hip, you would probably want to incorporate a body surface area (BSA) conversion factor into the drug dosage calculation. See, e.g.:

Dose translation from animal to human studies revisited

So, as a guess for a gerbil to human BSA conversion, you might need to divide that 7200mg amount by about 9, giving a somewhat more reasonable 800mg.

 

[Jonathan Edwards]

Are there any studies showing defective B cells are the root cause of ME/CFS?

Seems to me that defective B cells and overactive microglia are very likely a downstream symptom of whatever the root cause is. If that's the case, one would think that the newly generated B cells will eventually become defective if the root cause isn't ever addressed and corrected.

So far there is no consistent evidence on B cell defects in ME. We are only going on the basis of the apparent benefit from rituximab.

In autoimmunity the production of bad B cells does not seem to be downstream of anything except itself. There are genetic risk factors but lots of people healthy their whole lives have these risk factors. The production of the bad B cells is a random event like in cancer. There is nothing upstream of the random generation of a particular cancer clone, except again background risk factors that anyone can have.

It is possible that in some cases there are addressable upstream factors for B cell misbehaviour but I am not sure what they could be.

 

[Jonathan Edwards]

@JonathanEdwards

Hypothetically, from a theoretical point of view, in the scenario presented above, would the introduction of glucocorticoids (at low replacement-level doses) have any impact?

I just ask because before commencing low-dose steroids, I was bed bound and my condition was declining week by week, but with the gluco- & mineralcorticoids I am not bed bound (unless intercurrently ill) and have better functioning.

Would the anti-inflammatory action of glucocorticoids impact on microglial activation or is that theoretically not possible?

glucocorticoids can produce some benefit in almost any situation so it is a bit hard to know what to make of a response. They have so many different actions.

 

[Jonathan Edwards]

I guess I'm wondering about the life cycle of how microglia activation would work normally and whether the timings of normal vs abnormal would tell us anything. I'm assuming that if a pathogen travels to the brain then microglia would normally activate clean up and then deactivate (or would something deactivate them).

If they fail to deactivate how long would a microglia cell live for before being replaced. But if one fails to deactivate would a drug help deactivate it say if a deactivation sensor was blocked.

If a drug treatment didn't tell work and scans before and after showed activation would that suggest the deactivation mechanism was failing? Or are we less sure how well the drugs work; or could the reactivation process work as fast as the drug based deactivation process. Could we tell the difference.

I agree. There are masses of unknowns - at least to me. I think all one could say would be that if a microglial activation inhibitor of some sort produced rapid symptom relief and scans improved but that things relapsed after stopping it might be logical to make use of the microglial drug while waiting for rituximab to have a longer term effect.

 

[JPV]

It is possible that in some cases there are addressable upstream factors for B cell misbehaviour but I am not sure what they could be.

Thanks for the response.

Is it possible that the B cells could become dysfunctional after constantly dealing with some type of chronic infection(s)?

 

[Jonathan Edwards]

Thanks for the response.

Is it possible that the B cells could become dysfunctional after constantly dealing with some type of chronic infection(s)?

Not that I can think of. The only dysfunction that would seem t matter is making an antibody against self or at least an antibody that causes problems. That is determined entirely by random mutation within the B cell itself. It cannot be made to happen by an infection or other outside influence.

 

[Hutan]

I'm learning about IL10 at the moment, so suddenly IL10 seems part of the answer to most of the questions I'm seeing. Apologies for almost certainly covering old ground.

The Light et al study found IL10 increases in people with CFS after exercise.
Perhaps it is possible that IL-10 makes the B cell go rogue? (see the bolded sentences in the reference below)

And, perhaps it is possible that a pathogen makes the body increase levels of IL-10, makes homologs of IL-10, and/or the body voluntarily increases IL-10 in order to avoid inflammatory damage from a pathogen?

The Role of IL-10 in Autoimmune Pathology
Andrew W. Gibson, Jeffrey C. Edberg, Jianming Wu, and Robert P. Kimberly.
http://www.ncbi.nlm.nih.gov/books/NBK6234/

High serum levels of IL-10 have been documented in human autoimmune diseases.

Interleukin-10 is produced by CD4 and CD8T cells, activated B lymphocytes, monocytes, macrophages, and keratinocytes.1,2 As an anti inflammatory cytokine IL-10 down-regulates the expression of Th1 cytokines, MHC class II and costimulatory molecules on macrophages. However, IL-10 also stimulates FcγR expression on the same cells,1,2 and has been shown to prolong B cell survival, to induce B cell differentiation, and to enhance B cell proliferation and antibody production.1-5 The effects of IL-10 on B cells, particularly on the stimulation and survival of autoreactive B cells are believed to be of great importance in autoimmune diseases. 2,5 Additionally, IL-10 may play an important role in influencing the balance of Th1 versus Th2 cytokines, which can influence the progression of autoimmune diseases.6-8

Role of Interleukin 10 Transcriptional Regulation in Inflammation and Autoimmune Disease
Shankar Subramanian Iyer1 and Genhong Cheng2,*
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410706/

...certain pathogens can promote a favorable environment for infection and persistence by expressing IL-10 homologs that bind the IL-10 receptor and exert immunological effects similar to that of the endogenous ligand. This is best characterized in the Epstein-Barr virus encoded IL-10 mimic, BCRF1

...several pathogens have evolved mechanisms that selectively up-regulate IL-10 during the course of an infection, presumably to create a more favorable microenvironment. For example, Toxoplasma gondii is capable of shutting down TLR4-mediated LPS signaling in a manner that specifically blocks TNFα expression but allows for production of IL-10

IL-10 functions as a potent B cell stimulator that enhances activation, proliferation, and differentiation of B cells. This relates to SLE <lupus>, which is characterized by high autoantibody production and decreased cellular immune responses. In SLE, high levels of autoantibodies generate immune complexes that exacerbate tissue damage. Compared with healthy individuals, levels of IL-10 in SLE patients are significantly higher and there is a correlation between IL-10 levels and clinical manifestation.77 Depletion of IL-10 by anti-IL-10 antibody in vitro treatment of SLE patient–derived PBMC significantly decreased autoantibody production.​

And if IL-10 is indeed part of the problem, what makes that disregulated? I have included the following paragraph from this same reference not because I understand it, but it seems to indicate that the regulation of IL-10 production is pretty complicated. Plenty of scope for problems upstream of the B cells I think.

IL-10 inducing signaling cascades have been studied less thoroughly in 3 cells than in macrophages and DCs. Stimulation of IL-10 expression can occur through one of three mechanisms: (1) instruction by APCs, (2) induction by IL-12 family cytokines, or (3) alternative means << !! >>. T cell receptor (TCR) and endogenous Il-12 have been shown to be essential for the differentiation of IL-10 producing 31 cells as well as for maximal expression of Il-10 following re-stimulation of these cells.106 Il-10 induction in 31 cells is STAT4 and ERK dependent. In 32 cells, Il-10 production appears to be regulated by 32 conditioning factors including IL-4, STAT6 and GATA3. <etc etc>​

 

[adreno]

Not that I can think of. The only dysfunction that would seem t matter is making an antibody against self or at least an antibody that causes problems. That is determined entirely by random mutation within the B cell itself. It cannot be made to happen by an infection or other outside influence.

It was recently discovered that the microbiome has influence on antibody repertoire. I wonder how this might influence autoantibody production?

Early B Cells Found in Gut, Schooled by Microbes

Study in mice shows distinctive antibody repertoire in gut-bred B cells

CAROL CRUZAN MORTON

Although T cells have long dominated the immunology of multiple sclerosis (MS), B cells have recently emerged as major players in disease pathology and therapeutic targets. A new study extends the potential influence of the microbiome on immune function with the discovery of a small but distinctive population of B cells in young mice that acquires its antibody diversity in the intestines under the influence of the bacteria there.

Early in development, millions of new B cells randomly assemble a seemingly endless variation of antibodies by shuffling DNA pieces from three gene segments. The random V(D)J process results in some molecules that react to the body's own antigens. One way to purge self-reactive B cells is called receptor editing, which salvages the B cell by signaling it to continue its V(D)J shuffle until it can produce an acceptable antibody, Alt told MSDF. In mice, as in people, this was thought to happen exclusively in the bone marrow.

But in people and in mice, the “B” in B cells might as well stand for bone marrow, where most of them are still likely to develop. "The idea our study raises is that the receptor-editing process can occur in the gut, where antigens from the gut microbes are available to interact and have a window of opportunity to shape the receptor-editing process," Wesemann said.

In fact, the role of editing in gut B cells is unknown, Alt said. Editing in bone marrow B cells is considered a process to eliminate autoreactive receptors. "While editing could serve a similar role in gut B cells," he said, "by analogy to what happens in other species, we also speculate that editing in the gut may diversify potential antibody repertoires of B cells that develop there."

"This is an intriguing thing to think about," Goverman said. "It's complete speculation, but could this be a way of explaining how susceptibility to certain autoimmune diseases like MS can be shaped early in life even though the disease is manifested later in life?" She added that it's bound to be even more complicated than it now appears.

http://www.msdiscovery.org/news/new_findings/7379-early-b-cells-found-gut-schooled-microbes

 

[Sasha]

https://www.facebook.com/permalink....total_comments=6&comment_tracking={"tn":"R2"}

I find it interesting (though I know it's already well known) that mitochondria are only inherited from the mother. Anecdotally, we have noted ME tends to follow the mother's line, where there are several family members with the disease. Of course there could be other explanations for this like close proximity or exposure to the same agents.

@Jonathan Edwards, are we talking about an acquired mitochondrial problem in which heritability would play no part? If not, does it make sense to do a PR survey on which family members have ME, like the one you did on thyroid issues?

 

[Bob]

@Jonathan Edwards, are we talking about an acquired mitochondrial problem in which heritability would play no part? If not, does it make sense to do a PR survey on which family members have ME, like the one you did on thyroid issues?

Such a survey would be skewed towards mothers because more women than men have ME. But it might be interesting anyway.

 

[Sasha]

Such a survey would be skewed towards mothers because more women than men have ME. But it might be interesting anyway.

That's a good point...

 

[Marky90]

So far there is no consistent evidence on B cell defects in ME. We are only going on the basis of the apparent benefit from rituximab.

In autoimmunity the production of bad B cells does not seem to be downstream of anything except itself. There are genetic risk factors but lots of people healthy their whole lives have these risk factors. The production of the bad B cells is a random event like in cancer. There is nothing upstream of the random generation of a particular cancer clone, except again background risk factors that anyone can have.

It is possible that in some cases there are addressable upstream factors for B cell misbehaviour but I am not sure what they could be.

Could autoimmunity be due malfunction from other immune cells, which does maintenance-work on the B-cells? (and is this only T-cells?) A norwegian immunological researcher explained to me that autoimmunity may be caused by malfunction of such regulatory processes on "bad B-cells".. I thought that sounded plausible.. That would implicate that the b-cells aren't necessarily doing anything wrong, however the control that is supposed to find place during e.g the different maturation stages of the b-cells is potentially inadequate.

I apologize in advance, if i have misunderstood something basic here.

 

[Jonathan Edwards]

Could autoimmunity be due malfunction from other immune cells, which does maintenance-work on the B-cells? (and is this only T-cells?) A norwegian immunological researcher explained to me that autoimmunity may be caused by malfunction of such regulatory processes on "bad B-cells".. I thought that sounded plausible.. That would implicate that the b-cells aren't necessarily doing anything wrong, however the control that is supposed to find place during e.g the different maturation stages of the b-cells is potentially inadequate.

I apologize in advance, if i have misunderstood something basic here.

You would be in good company! In a sense the issue is basic but the context is so complicated that it is quite difficult to see the wood for the trees. Everyone in immunology is obsessed with the idea that B cells are innocent and just make mistakes because of some other cells like T cells or Tregs. But the mistakes we find in autoimmunity arise from random mutation in the B cells. Other cells might help such bad B cells survive but then you have to give a reason for those cells misbehaving that would specifically help bad B cells. The old molecular mimicry idea simply makes no sense if you look at it carefully. So although it is easy to find hundreds of reviews saying there are upstream mechanisms involving IL-10 or T cell subsets or whatever, nobody actually has a thoery based on that which would generate the specific sorts of bad B cell we find. On the other hand it is quite easy to see how these particular bad B cells might encourage their own survival by producing antibody that fed signals back onto their sisters.

 

[Marky90]

You would be in good company! In a sense the issue is basic but the context is so complicated that it is quite difficult to see the wood for the trees. Everyone in immunology is obsessed with the idea that B cells are innocent and just make mistakes because of some other cells like T cells or Tregs. But the mistakes we find in autoimmunity arise from random mutation in the B cells. Other cells might help such bad B cells survive but then you have to give a reason for those cells misbehaving that would specifically help bad B cells. The old molecular mimicry idea simply makes no sense if you look at it carefully. So although it is easy to find hundreds of reviews saying there are upstream mechanisms involving IL-10 or T cell subsets or whatever, nobody actually has a thoery based on that which would generate the specific sorts of bad B cell we find. On the other hand it is quite easy to see how these particular bad B cells might encourage their own survival by producing antibody that fed signals back onto their sisters.

Hah! I see. Such upstream-processes must be really hard to research as well i assume?

Would you be able to illuminate your interesting idea in your last sentence some more? What kind of signals e.g?

 

[Jonathan Edwards]

Hah! I see. Such upstream-processes must be really hard to research as well i assume?

Would you be able to illuminate your interesting idea in your last sentence some more? What kind of signals e.g?

The self-perpetuating signals are various and complicated and not very easy to explain in an internet post. The general idea and several examples were given in a paper I wrote with G Cambridge and V Abrahams in 1999. You shuld find it at
http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2567.1999.00772.x/full

 

[Marky90]

I imagined! Thanks!

 

[FancyMyBlood]

This study, which examined the ability of three tetracycline antibiotics (namely minocycline, doxycycline and tetracycline) to inhibit microglial activation during ischemia, used doses of 90 mg per kg of body weight in the animals, which are very high doses. That would correspond to a 7,200 mg dose in an 80 kg human. Normal doses of doxycycline and minocycline are around 200 mg daily. Though the authors did say that "we have also found that significantly lower doses of the compounds are neuroprotective in both global and focal brain ischemia."

Note that tetracycline antibiotics are not the only microglial activation inhibitors. I complied a list of dozens of microglial activation inhibitors in this thread:

Chronic Microglial Activation in ME/CFS, And Its Possible Treatment Using Microglial Inhibitors

It's much less actually. When translating an animal dose to a human equivalent dose (HED), you need to take into account the animal's body surface area. The entire HED formula is explained here:http://www.fda.gov/downloads/Drugs/Guidances/UCM078932.pdT

The study used gerbils and I'm not sure of their BSA, but if it used mice the HED would translate to 90 x 0.081= 7.2 mg/kg. Obviously this formula is just a starting point and the accuracy depends on a lot of factors. Still, it's at least double the generally prescribed doses of minocycline.

Edit: Just noticed @nandixon already explained it earlier.

 

[Hip]

@Hip, you would probably want to incorporate a body surface area (BSA) conversion factor into the drug dosage calculation. See, e.g.:

Dose translation from animal to human studies revisited

So, as a guess for a gerbil to human BSA conversion, you might need to divide that 7200mg amount by about 9, giving a somewhat more reasonable 800mg.

Thanks for pointing that out. I never realized this. Very interesting.

I have often seen body surface area being used in dose calculations, especially for chemotherapy dosing, but was always puzzled why they used this rather than body volume (which in animals is more or less proportional to body mass, since most animals have similar density) .

But from reading the papers you guys provided, it seems you must also take other metabolic "processing speed" factors like an animal's metabolic rate, oxygen utilization, energy expenditure and renal function into consideration in order to determine the drug correct dose (and the paper said these factors correlate quite well to body surface area, which is why you use surface area as a rule of thumb measurement).

Taking an animal's metabolic "processing speed" into account makes sense when you think about it: an animal like a rat has a high overall metabolic processing speed compared to a human, and so this extra speed will be using up and excreting a drug at a higher rate than a human. Thus small animals like rats will require either higher per kg doses of the drug, or more frequent dosing, to keep up with their metabolic speed.

In that FDA document on page 7 there is a very useful conversion table between various animal mg/kg doses, and human mg/kgdoses.



Conversion Between Micromolar (μM) Concentrations In Vitro, and a Human Oral Dose

The other type of dose conversion always I wanted more info on, but could never find any, is the conversion between micromolar (μM) concentrations — which are usually used to express the concentration of a drug in solution applied in cell line experiments in vitro — and the equivalent human oral dose of that drug that would achieve the same level of drug concentration in human tissues.

Sometimes you read one of these in vitro studies, and you want to try the same drug or supplement yourself, but need to convert from micromole concentrations to an oral dosage in grams.

I came up with my own formula for this, which is based on the assumption that a normal weight person will have around 40 liters of "accessible" water in their body, and that the drug will distribute in this 40 liters. On that assumption, the conversion formula from micromolar (μM) concentrations to an oral dose in milligrams grams would be:

Formula for when the concentration C of the solution in vitro is expressed in μM (micromoles per liter = μmol/L):

Dosage in milligrams = 400 x C x W / ( B x (100 - P))

Where:
C = concentration of the solution in μM, used in the in vitro study
B = percentage bioavailability
P = percentage plasma protein binding
W = the molecular weight of the drug or compound in grams per mole

Or:
Formula for when the concentration C of the solution in vitro is expressed in μg/ml (micrograms per ml):

Dosage in milligrams = 400,000 x C / ( B x (100 - P))

Where:
C = concentration of the solution in μg/ml, used in the in vitro study
B = percentage bioavailability
P = percentage plasma protein binding


The plasma protein binding percentage P specifies what percentage of the supplement or drug binds to the proteins in the blood. You can find the plasma protein binding percentage of many drugs by searching Google. This percentage can be anything from 0% to 100%. Only the unbound (free) drug is active in the body (usually); the drug bound to plasma proteins becomes inactive; thus the above formula takes this into account, factoring P into the equation.


Just how valid these formulas are, I am not sure. I expect they will at least provide a rough guide.

In this discussion they say that there is no easy way to reliably convert from in vitro micromolar concentrations to an oral dose in grams, because too many factors are at play which affect the final concentration that an oral dose achieves. However, I think the formula I devised probably works for getting a rough ballpark figure for the oral dose.



Note that:

A molar (M) solution is 1 mole of the chemical dissolved in 1 liter = 1 mol/L
A millimolar (mM) solution is 1 thousandth of a mole dissolved in 1 liter = 1 mmol/L
A micromolar (μM) solution is 1 millionth of a mole dissolved in 1 liter = 1 μmol/L