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Using RNASE-L as a marker ?

Discussion in 'Diagnostic Guidelines and Laboratory Testing' started by globalpilot, Oct 12, 2011.

  1. globalpilot

    globalpilot Senior Member

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    Hello all,

    I have tested positive for enterovirus in my stomach biopsy by Dr Chia.

    Unfortunately, with this virus there doesn't seem to be a marker to use to measure success in getting the viral load down. There are over 100 different types of enterovirus and ARUP only measures titres to 11 of these. I have elevated nagalase at 1.9 but Dr Chia says he thinks enteroviruses do not produce nagalase but rather the bacteria overgrowth in the small bowel as a result of the enterovirus does.

    So, I'm not able to use either antibodies or nagalase as a marker.

    I looked into RNASE-L recently. Wikepedia says "RNase L (Latent) is an interferon-induced ribonuclease which, upon activation, destroys all RNA within the cell (both cellular and viral)."

    Rich, in his recent talk in Sweden said "The persistent interferon-driven RNase-L activation is due to lack of a normal cell-mediated immune response, which normally takes over control of infections by viruses." and

    "The cleaving of RNase-L and formation of the LMW RNase-L is due to calpain activation in response to glutathione depletion."

    So, I'm wondering if I can use RNASE-L as a marker ? Thoughts anyone ? Would measuring RNASE-L be enough or would I need to do the LMW RNASE-L?

    Finally, where would I get this tested ?

    Regards,
    GP
  2. alex3619

    alex3619 Senior Member

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    Hi globalpilot, the wikipedia is wrong I think. RNase L does NOT destroy all RNA. It has specific RNA sequences it targets, sequences that are common in viruses. However, some human proteins will match those sequences - I am not sure which proteins those are, I have never read a study on this.

    There has been a lot of research into RNase L even a collection of research papers published as a book. Not everyone with ME or CFS test positive for RNase L or its 37 kDa variant. The 37 kDa (kilodalton) variant was once thought to be a biomarker. However, this was complicated by finding it in both multiple sclerosis and rheumatoid arthritis. What does appear to be a biomarker, but nobody seems to be researching it much, is the combination of elastase and the 37 kDa RNase L. Elastase cleaves RNase L into fragments resulting in the short form. Elevated elastase is not found in MS or RA apparently. This needs a lot more research to become a good biomarker I think. A patent was lodged in 2004 I think using a combination of short RNase L and elastase as a marker. So far nothing has come of it to my knowledge.

    One of the problems with RNase L is that it is variable - it goes up and down. If you have any kind of viral infection it will go up. The short form is even more variable, there was some suggestion that it is high in relapse, but I don't know if this research was ever confirmed or further investigated. It may be hard to use as a treatment marker.

    Most doctors will not know what to make of RNase L as a marker. For most patients, depending on their doctor, this would not be a good option without more research. Dr Chia being a leading researcher may well have the expertise to understand this. You should ask him.

    I too have suspected calpain (m-calpain?) as a cleaving agent based on my own research. It is another enzyme that can cleave RNase L. We know elastase is elevated commonly in us however - calpain may either represent a subset, is another pathway, or is not relevant. I would need to see good data to suspect calpain when elastase is more probable. It does warrant further study though - if calpain is involved it offers another target for therapy, and not just by treating methylation issues.

    Recent research implicates increased LPS translocation (Maes) as a primary cause of elevated elastase. This also needs more research to be sure.



    Bye
    Alex
  3. ukxmrv

    ukxmrv Senior Member

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    GP, MW Rnase - L was being touted as a marker around 2000 but I'm not sure how that panned out and particularly as a marker of response for treatment. Maybe one of the Ampligin people would know. I was tested for LMW Rnase L as there was a possibility of getting Ampligin here once.

    Could you test for the bacterial overgrowth instead? Would a breath or similar test pick that up?

    p.s. does Dr Chia have any faith in the VP1 blood test? We used to have that in the UK in the late 90's for entroviral infection.
  4. globalpilot

    globalpilot Senior Member

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    Hi, Thanks for all the info. I think I may it sound like I was looking for a marker for CFS. I'm not. I'm looking for a marker for viral activation and I was wonderng if RNASE-L could serve that purpose ? I don't have any other way that I can think of to track whether treatment is effective against this enterovirus that I have.


  5. globalpilot

    globalpilot Senior Member

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    I actually tested negative for bacterial overgrowth. I did the hydrogen test twice and the methane twice. I know something is in there b/c of the extreme bloating, burping and lousy feeling after certain fermentable foods. I really want a marker for the virus though. It may take a while for the overgrowth to go away even if the virus is treated due to the damage that may have occured.

    Does Ampilgen increase interferon ? Is the idea that some have too much LMW RNASE L and too little HMW RNASE L and the ampligen increases the latter ?

    I wish I knew of your VP1 question before my consult. Unfortunately he didn't address that at all. I imagine if it shows up it's an issue but not showing up doesn't mean its not an issue.

    GP
  6. heapsreal

    heapsreal iherb 10% discount code OPA989,

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    Have u looked into immunoglobulin testing http://www.webmd.com/a-to-z-guides/immunoglobulins
    Also lymphocte subset test http://www.sydpath.stvincents.com.au/tests/ImmunoFrames/lymphocytosis.htm
    These tests arent specific to a particular infection but can tell you your immune system is trying to fight something, as u will read in the lymphocyte testing, it is commonly elevated in the herpes viruses like ebv,cmv etc
    redlabs also do some tests that may interest you http://www.redlabs.be/red-labs/our-science/chronic-fatigue-syndrome.php

    cheers!!!
  7. alex3619

    alex3619 Senior Member

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    Hi globalpilot, if RNase L is measured directly it might be a marker for viral infection. There are many variables in ME and CFS patients that could complicate this, as I discussed briefly.

    The simplest assay done though is RNase L activity - this by itself is NOT a marker for infection, as it seems to be high in many diseases. It might serve as a weak marker, but we need much more research to know if its reliable. This is especially true for MS, RA and ME/CFS which all have the LMW form of RNase L.

    I am still unclear what the relationship is between RNase L and the large tissue viral load in ME and CFS.

    The success rate of LMW RNase L as a biomarker was around 86% iirc, in its best study. It has problems.

    The best way to measure viral activity is tissue viral load - which means tissue biopsies for most of us. Even that is problematic as these viruses do not seem to cause constant high level viremia. We need to understand why that is.

    Cytokines as a substitute marker are even more problematic - they are even more variable, and subject to increase due to activity levels. They are not useful for this purpose.

    Chia would be the one to ask though - he is at the cutting edge of research and would know much more than almost anyone on this forum.

    Bye
    Alex
  8. Waverunner

    Waverunner Senior Member

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    Just want to throw in my 2 Cents. One of my former doctors told me he sent several blood samples to REDLABs in Belgium in order to test for RNase-L. He included a few controls of perfectly healthy practice employees. The results were somewhat interesting. Some PWCs and some of the healthy employees had increased values of RNase-L while at the same time other PWCs and employees had normal values. KDM doesn't run RNase-L as standard test anymore, he backed up a little bit from it. So it's difficult in my eyes to use RNase-L as a reliable marker.
  9. richvank

    richvank Senior Member

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    Hi, Alex.

    I had an email exchange in 2008 with Prof. Kenny de Meirleir and Marc Fremont, in his group, about which enzyme cleaves RNase-L. Here's what I wrote to them:

    "It is my understanding that inside living cells, the RNase-L is found in the
    cytoplasm and in the nucleus. Elastase, on the other hand, is found within
    macrophages and neutrophils, in granules and vesicles that are isolated from the
    cytoplasm and the nucleus by membranes that do not permit the passage of
    elastase. It seems to me that if elastase were not isolated in this way, it
    would cleave enzymes within these cells, and the cells would not survive. If
    elastase is indeed physically isolated from RNase-L by this compartmentalization,
    I do not understand how it could cleave RNase-L within these phaogocytic cells."

    This was the response I received from Marc Fremont:

    "Thank you for your comments. I think they relate to two questions that are actually still opened. The first one is the possible presence of elastase in monocytes; the second relates to the exact identification of the proteases involved in the cleavage of RNase L.

    "As you say elastase is produced mainly by neutrophils and macrophages. It is relatively unlikely (although not impossible, it happens in certain cell types) that elastase released in the plasma can cross the membrane and re-enter PBMCs, so if elastase is present in monocytes it was probably expressed there.
    In a normal situation elastase expression in monocytes is repressed by the transcriptional repressor Gfi1. However CFS is not a normal situation, and we can consider a number of pathological mechanisms that could lead to the abnormal expression of elastase. For instance, since Gfi1 is a known target of proteasome, one possibility would be that immune cell dysregulation (apoptosis) activates proteasome that will degrade Gfi1, leading to the synthesis of elastase. The involvement of Gfi1 is still speculative (we will investigate this point) but we have evidence for the activation of proteasome so this is already one plausible mechanism.

    "Another thing is that elastase is not the only enzyme that can cleave RNase L. We can very efficiently prevent the cleavage of recombinant RNase L by PBMC extracts, by adding several elastase inhibitors or purified alpha-1-antitrypsin (AAT), the natural inhibitor of elastase in plasma. However these inhibitors (even AAT) have a relatively broad specificity so this does not rule out the possibility that other enzymes contribute to the cleavage. The initial study reporting the involvement of elastase was the 2002 publication (Demettre et al, JBC 277(38), p35746-51). In this publication, the possible implication of other proteases such as calpain or cathepsin G was already mentioned. Other enzymes (metalloproteinases, proteasome-related proteases...) could also be involved. Since most of them are biochemically related to elastase they're not easy to distinguish; in fact the RNase L fragments we do observe are most likely produced by several proteases, not only one. There is currently a research project ongoing that should, I hope, clarify this point.

    "Finally, regarding the assay itself elastase inhibitor, as well as other protease inhibitors, are added during the extraction to make sure that the cleavage we measure is not generated during the assay process, but was already present in the living cells.

    "All these are complex but very interesting questions. You are right in saying that elastase should not cleave RNase L, since it is contained in vesicles. The question is, why does this happen in CFS? Activation of proteasome is one possibility but there are others. Implication of other proteases is also possible. Actually it may be that different dysregulations occur in different patients, depending on the initial trigger of the disease (viral infection, chronic inflammation...) and that the proteases that cause the cleavage will not be the same in different patient sub-groups. Identification of these mechanisms is the main focus of our research at this time.

    "As you see I cannot give a definitive answer to your comment, because these are still open questions that we currently investigate. I'm confident we will one day get a clear picture of what happens in the disease."


    I haven't seen any new results concerning this issue since then. I continue to doubt that elastase is responsible for the cleavage of RNase-L, for the reason I gave. Compartmentalization will prevent it, in my opinion.

    Best regards,

    Rich
  10. alex3619

    alex3619 Senior Member

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    Hi Rich, Thank you for that information. The claim that elastase is elevated is independent of the RNase L research though. Also, I think it is likely it does target other proteins - especially elastin.

    Hyperelastasemia is recognized medical issue, typically occurring in alcoholic hepatitis. "Slight clouding of consciousness, fever, and jaundice were evident on his admission to our hospital." See for example:

    http://www.mendeley.com/research/tw...d-intravenous-infusion-urinastarine-miraclid/

    So loss of elastin could lead to brain fog. It does not escape my attention that we also have alcohol intolerance. You are right though, this is an open question, and I would really like to know the answer.

    There may also be a link here to EhlersDanlos syndrome (EDS), as I wonder if the collagen damage in this disorder might not affect synthesis of elastin and related pathways, but this is very speculative. I wonder if EDS does not predispose to ME but exacerbates it severely so that even mild subclinical cases in the average person present as severe cases in EDS?

    Elastin is also essential for keeping blood vessels flexible, this fits with ME very well and may assist causing OI.

    However against this we have Williams syndrome in which reduced elastin makes the voice harsh, and loss of elastin should be visible in skin, and we don't see either. However, this may be related to the location of the elastase production - it wont travel far.

    The question of extracellular/intracellular elastase is a problematic one and I do hope they can clarify this. However since elastase can induce similar symptoms to ME, I still think its a good candidate. What is intriguing though is that m-calpain may indeed target the same or similar proteins, so might also induce similar symptoms.

    There were three enzymes originally found to cleave RNase L. I always forget the name of the third one for some reason.

    Bye
    Alex
  11. Daffodil

    Daffodil Senior Member

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    they dont do LMW RNase L test anymore in nevada, saying that elastase testing will tell you the same thing.

    montoya found no correlation between Rnase L activity levels and illness severity.

    interestingly, my Rnase L was in the thousands but came down to almost normal on valtrex, even though i was still sevrely ill the whole time.

    however, my elastase never normalized until i got on antiretrovirals.
  12. alex3619

    alex3619 Senior Member

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    Hi Daffodil, interesting example, thanks.

    Where did you get the information about RNase L from Montoya if you recall the source?

    With allowance for the limitation of one isolated example, here is a possible explanation. Elastase cleaves RNase L. However for it to do so there must be RNase L. RNase L is induced by viral RNA I think, or anything that can trigger the same pathway (an interferon pathway). The 37 kDa variant requires both RNase L and also a specific protease such as elastase.

    When your RNase L was measured, did they measure the chemical or its RNA cleaving function? Usually they do an RNase L activity assay - find out how fast it cleaves RNA.

    That elastase came down on antiretrovirals is very interesting. A presumed stealth retrovirus will not produce enough RNA to trigger much interferon, but co-existing or opportunistic viruses might. The question is why elastase comes down on antiretrovirals. Is it a side effect, or is it because a presumed stealth retrovirus such as an HGRV is directly or indirectly inducing elastase production? My best guess at the moment, presuming an HGRV or similar retrovirus, is that gut immune cells are compromised leading to LPS translocation to the blood, which induces the elastase. This is only speculation of course, but its an interesting question. I look forward to seeing the science evolve on this issue.

    Bye
    Alex

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