Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndro

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RESEARCH ARTICLE
MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)
Robert D. Petty, Neil E. McCarthy, Rifca Le Dieu,Jonathan R. Kerr

Background
Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients.

Methods
miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets.

Results
Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology.

Conclusion
This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

Citation: Petty RD, McCarthy NE, Le Dieu R, Kerr JR (2016) MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME). PLoS ONE 11(3): e0150904. doi:10.1371/journal.pone.0150904

Editor: Partha Mukhopadhyay, National Institutes of Health, UNITED STATES



Received: August 20, 2015; Accepted: February 22, 2016; Published: March 11, 2016
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150904
 

IrisRV

Well-Known Member
This is great purely biomedical research out of the UK confirming abnormalities in NK cells. Even better, these abnormalities are consistent with defective NK cell function, which reliable researchers from other countries have been reporting for some time now. The case is getting very strong.

This group used a good patient cohort -- Fukuda + CCC and eliminating psych cases to avoid confounding the results. Eliminating those with psych issues is wise at this point in the research, although a group of CCC + psych conditions will eventually have to be studied to make sure the results are consistent over the full patient population.

It's good to see our old pal Jonathan Kerr again. :D I don't remember hearing about Robert D Petty before. It looks like he was the driving force behind this study. Does anyone know about him?

Kudos to the UK charities and the philanthropist who funded this research.
 

weyland

Well-Known Member
This is great purely biomedical research out of the UK confirming abnormalities in NK cells. Even better, these abnormalities are consistent with defective NK cell function, which reliable researchers from other countries have been reporting for some time now. The case is getting very strong.
The problem is it doesn't match the miRNA findings in NK cells coming out of Australia apparently. :(

Also seems a bit premature to mark these as ME biomarkers if the only control group was healthies. Would need controls from other diseases with low NK cell function such as PTSD or MS to make this more interesting.
 

Seanko

Well-Known Member
Copied & pasted from PlosOne: the Authors

Robert D. Petty, Jonathan R. Kerr
CFS Group, St George´s University of London, Cranmer Terrace, London, United Kingdom

Robert D. Petty, Rifca Le Dieu
Centre for Haemato-Oncology, Bart’s cancer institute, Queen Mary University of London, London, United Kingdom

Neil E. McCarthy
Centre for Immunobiology, The Blizzard institute, Queen Mary University of London, London, United Kingdom

Jonathan R. Kerr
Grupo de Salud Publica, Escuela de Medicine y Ciencias de la Salud, Universidad del Rosario, Quinta de Mutis, Bogotá 111221, Colombia

*Interestingly Dr Bansal of St Helier Hospital was involved with the patient cohort. He is the lead on the UK Rituxmab trail at University College London (UCL)

*Secondly you will see investigators from Queen Mary University (QMUL) & Bart's Hospital which are both intimately linked to PACE (albeit with different people & departments)
 

IrisRV

Well-Known Member
The problem is it doesn't match the miRNA findings in NK cells coming out of Australia apparently. :(
I don't know anything like enough about this field to answer the following. Maybe someone can help me. :D

Are they inconsistent or just different? Inconsistent would be unfortunate. That would need to be explained before either group could make a claim of having a reliable biomarker. Different, however, could just mean they're looking at somewhat different aspects and both could be right (or wrong :p).
 

weyland

Well-Known Member
While these abnormalities may not be unique to ME, I wonder if this technique could be utilized in place of the cytotoxicity assay for assessing NK cell function, which can only be done reliably on a handful of patients due to the stringent handling requirements. I know there are issues with RNA degrading in samples if they are left too long but I believe that can be worked around with preservative reagents.
 

weyland

Well-Known Member
I don't know anything like enough about this field to answer the following. Maybe someone can help me. :D

Are they inconsistent or just different? Inconsistent would be unfortunate. That would need to be explained before either group could make a claim of having a reliable biomarker. Different, however, could just mean they're looking at somewhat different aspects and both could be right (or wrong :p).
I haven't looked at either study personally, but someone on PR did and said there were no common miRNAs reported between the two studies.
 

IrisRV

Well-Known Member
I haven't looked at either study personally, but someone on PR did and said there were no common miRNAs reported between the two studies.
Well then, pending more information I'm going to hope that there's so many miRNAs that the two groups looked at different ones and each group found a subset of the larger group of abnormal miRNAs. :D
 

IrisRV

Well-Known Member
While these abnormalities may not be unique to ME, I wonder if this technique could be utilized in place of the cytotoxicity assay for assessing NK cell function, which can only be done reliably on a handful of patients due to the stringent handling requirements. I know there are issues with RNA degrading in samples if they are left too long but I believe that can be worked around with preservative reagents.
I'm not sure the handful of patients thing is quite right. Most INIM patients get NK cell function testing once or twice a year. I send samples overnight to my immunology lab 1300 mi (2100 km) away and it works fine.

Most people should be able to get cytotoxicity assays for NK cell function right now. According to this, even Quest claims to be able to do NK cell function tests using flow cytometry.
 

weyland

Well-Known Member
Well then, pending more information I'm going to hope that there's so many miRNAs that the two groups looked at different ones and each group found a subset of the larger group of abnormal miRNAs. :D
Yeah, unfortunately we only have a patent application to go off of for the Australian group so they were not specific about their methods. The assay used by the brits is mentioned in the Aussie patent application and the general procedure sounds about the same though.
 

weyland

Well-Known Member
I'm not sure the handful of patients thing is quite right. Most INIM patients get NK cell function testing once or twice a year. I send samples overnight to my immunology lab 1300 mi (2100 km) away and it works fine.
Works fine as in shows low or normal function?
 

weyland

Well-Known Member
Most people should be able to get cytotoxicity assays for NK cell function right now. According to this, even Quest claims to be able to do NK cell function tests using flow cytometry.
I've been to two of the top ME doctors in the US and neither ran NK function tests on me. If you're not a patient of INIM or OMI you may have difficulty arranging for this testing. None of my other normal doctors are willing to order any specialized testing for me. I have to imagine this isn't uncommon.
 

IrisRV

Well-Known Member
Works fine as in shows low or normal function?
Yep. My most recent FedExed sample came back with low function. Others have been normal. I haven't mailed many yet because I try to get a draw when I'm down there seeing the doc, but the ones I have mailed are consistent with the ones drawn at the immunology lab.
 

weyland

Well-Known Member
Yep. My most recent FedExed sample came back with low function. Others have been normal. I haven't mailed many yet because I try to get a draw when I'm down there seeing the doc, but the ones I have mailed are consistent with the ones drawn at the immunology lab.
My point is that there is going to be a big chance for false positives on low function results if your blood sat in the hot Fedex truck too long or if the lab didn't quite get to it as fast as they should have. How cool would it be to have a test that wasn't limited by this possibility.
 

IrisRV

Well-Known Member
I've been to two of the top ME doctors in the US and neither ran NK function tests on me. If you're not a patient of INIM or OMI you may have difficulty arranging for this testing. None of my other normal doctors are willing to order any specialized testing for me. I have to imagine this isn't uncommon.
I imagine it isn't. :) I'm thinking, however, that if NK cell function is determined to be one of the biomarkers for ME it will become much easier to get the test ordered by doctors who are not ME specialists. I'm disappointed that top ME doctors are not ordering this test since low NK cell function is common (not universal) in ME patients and there are some treatments that might improve it. Perhaps they are just not comfortable dealing with immunology? Do you mind saying who the specialists are who didn't do NK function tests on you?

I doubt we could easily get miRNA testing now either even if it were readily available. I'm betting the authorities will go with the testing whose infrastructure is already in place than make the biomarker test be something that is not currently commercially available.
 

weyland

Well-Known Member
Do you mind saying who the specialists are who didn't do NK function tests on you?
Chia and Stanford.

I doubt we could easily get miRNA testing now either even if it were readily available. I'm betting the authorities will go with the testing whose infrastructure is already in place than make the biomarker test be something that is not currently commercially available.
Probably not but I'd be more willing to fight my doctors to get this type of test if it was more reliable, even if I had to ship my blood to Australia to get it.
 

IrisRV

Well-Known Member
My point is that there is going to be a big chance for false positives on low function results if your blood sat in the hot Fedex truck too long or if the lab didn't quite get to it as fast as they should have. How cool would it be to have a test that wasn't limited by this possibility.
Indeed! The good news is that INIM, at least, sends you a sample kit with a FedEx bag with all the right labels about proper handling of the biological sample. On my last test, FedEx did screw up the handling and the lab rejected the sample as a consequence. I had to do it all over again. :mad: Still, I was glad the lab was conscientious about checking for poor handling. They're not fools. They know how the sample needs to be handled and to get to it within the proper timeframe.

If Quest claims to be able to do the test, it should be even easier to get the samples there in a proper condition since they have experience shipping bio samples and there is likely a lab relatively close to where the sample is drawn.

Certainly, it would be great to have a test that didn't require special handling, but miRNA testing is probably no better -- at least within the next 10 years since they'd have to verify it as a biomarker, develop a commercial test, make it available in multiple labs, and deal with sample degradation like they do with NK cell function testing.

But who knows? It could get fast-tracked. I'll be happy with any reliable biomarker test they can get in place. :D
 

IrisRV

Well-Known Member
Chia and Stanford.
Chia runs a relatively small operation and is more tightly focused on entroviruses, so I suppose that's why he doesn't do NK cell function testing. I'm disappointed in Stanford, though. I don't see any significant limitations to them ordering NK cell function testing. It would be interesting to hear why they don't do it.

Probably not but I'd be more willing to fight my doctors to get this type of test if it was more reliable, even if I had to ship my blood to Australia to get it.
Absolutely! If it were more reliable, it would be worth fighting for. :)
 

weyland

Well-Known Member
Indeed! The good news is that INIM, at least, sends you a sample kit with a FedEx bag with all the right labels about proper handling of the biological sample. On my last test, FedEx did screw up the handling and the lab rejected the sample as a consequence. I had to do it all over again. :mad: Still, I was glad the lab was conscientious about checking for poor handling. They're not fools. They know how the sample needs to be handled and to get to it within the proper timeframe.
That's good, I'd trust INIM with that but personally I would not trust Quest to do the same.

It would be interesting to hear why they don't do it.
My guess is that it doesn't add much value to their overall treatment plan. Their main focus appears to be pathogen testing and treatment, so it wouldn't change their treatment plan if you had low or normal NK cell function. They would give antivirals/abx regardless and that is their main approach.
 

weyland

Well-Known Member
I really wish that all new ME research would take into account duration of illness in their cohort. For example it would be really interesting to know if these same miRNA abnormalities were present in both short and long duration patients.

The results here imply that there is persistent immune activation in at least NK cells, if not B cells, and monocytes too. It would be interesting to know if this was the case even in long duration patients, where the Columbia studies implied there was "immune exhaustion" due to low cytokine levels. I really don't buy the immune exhaustion idea, persistent immune activation even in long duration patients makes more sense given that overall the core symptoms remain the same as you pass the 3 year mark. It would also be interesting to know if short duration patients have the same exact NK cell gene expression as long term patients implying diminished cytotoxicity from the start which might put the finding more in the causal or contributory rather than consequence category.

Looks like the average illness duration in this cohort was 6.8 years.
 

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