Plasma proteomic profiling suggests an association between antigen driven clonal B cell expansion and ME/CFS, 2020, Lipkin et al

We speculate that, at least in a subset of ME/CFS subjects, an increased level of IGHV3- 23 may be due to antigen driven clonal expansion, and that these patients might benefit from identification of the antigen driving B-cell receptor signaling or kinase inhibitors that interrupt signaling.
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Specifically, what types of kinase inhibitors do you think might benefit ME/CFS patients (to interrupt signaling)?
 
They don´t seem to have in mind the following findings on Protein kinase RNA-activated (PKR), at least these findings are not listed in their literatur:

Unravelling intracellular immune dysfunction in [CFS]: Interactions between protein kinase R activity, RNase L cleavage and elastase activity, and their clinical relevance
Meeus et al 2008

Confirmed by Sweetman et al 2018 [38]. which is referred to here:

Current Research Provides Insight into the Biological Basis and Diagnostic Potential for [ME/CFS]
Sweetman et al 2019

page 6
With the same study group, we have investigated the abnormal activation of protein kinase RNA-activated (PKR) as a potential biomarker for ME/CFS. This kinase has been described as a ‘universal immunological abnormality’ in ME/CFS [52]. ME/CFS often follows an acute viral infection, suggesting that the key role PKR plays in the innate immune response to infection may be significant in ME/CFS symptomology. The efficacy of PKR as a diagnostic biomarker for ME/CFS results from the fact that PKR is phosphorylated when activated. Healthy controls had undetectable phosphorylated PKR in protein extracts of PBMC cells using an in-house affinity purified antibody (two stage purification-positive and negative affinity steps). Phosphorylated PKR (pPKR) was in contrast detected in the protein cell extracts of ME/CFS patients.
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Maybe there has an imbalance arisen from to less b cells during infection?? (if I am allowed to speculate)

1. https://www.uniprot.org/uniprot/P19525

1.a Activity Regulation [my paragraphs and bold]
Initially produced in an inactive form and is activated by binding to viral dsRNA, which causes dimerization and autophosphorylation in the activation loop and stimulation of function. ISGylation can activate it in the absence of viral infection.

Can also be activated by heparin, proinflammatory stimuli, growth factors, cytokines, oxidative stress and the cellular protein PRKRA. Activity is markedly stimulated by manganese ions.

Activation is blocked by the viral components HIV-1 Tat protein and large amounts of HIV-1 trans-activation response (TAR) RNA element as well as by the cellular proteins TARBP2, DUS2L, NPM1, NCK1 and ADAR. Down-regulated by Toscana virus (TOS) and Rift valley fever virus (RVFV) NSS which promote its proteasomal degradation. Inhibited by vaccinia virus protein E3, probably via dsRNA sequestering
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5 publications​


1.b Function [my paragraphs]
IFN-induced dsRNA-dependent serine/threonine-protein kinase which plays a key role in the innate immune response to viral infection and is also involved in the regulation of signal transduction, apoptosis, cell proliferation and differentiation. Exerts its antiviral activity on a wide range of DNA and RNA viruses including hepatitis C virus (HCV), hepatitis B virus (HBV), measles virus (MV) and herpes simplex virus 1 (HHV-1).

Inhibits viral replication via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (EIF2S1), this phosphorylation impairs the recycling of EIF2S1 between successive rounds of initiation leading to inhibition of translation which eventually results in shutdown of cellular and viral protein synthesis. Also phosphorylates other substrates including p53/TP53, PPP2R5A, DHX9, ILF3, IRS1 and the HHV-1 viral protein US11. In addition to serine/threonine-protein kinase activity, also has tyrosine-protein kinase activity and phosphorylates CDK1 at 'Tyr-4' upon DNA damage, facilitating its ubiquitination and proteosomal degradation.

Either as an adapter protein and/or via its kinase activity, can regulate various signaling pathways (p38 MAP kinase, NF-kappa-B and insulin signaling pathways) and transcription factors (JUN, STAT1, STAT3, IRF1, ATF3) involved in the expression of genes encoding proinflammatory cytokines and IFNs. Activates the NF-kappa-B pathway via interaction with IKBKB and TRAF family of proteins and activates the p38 MAP kinase pathway via interaction with MAP2K6.

Can act as both a positive and negative regulator of the insulin signaling pathway (ISP). Negatively regulates ISP by inducing the inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) at 'Ser-312' and positively regulates ISP via phosphorylation of PPP2R5A which activates FOXO1, which in turn up-regulates the expression of insulin receptor substrate 2 (IRS2). Can regulate NLRP3 inflammasome assembly and the activation of NLRP3, NLRP1, AIM2 and NLRC4 inflammasomes. Can trigger apoptosis via FADD-mediated activation of CASP8. Plays a role in the regulation of the cytoskeleton by binding to gelsolin (GSN), sequestering the protein in an inactive conformation away from actin
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24 publications​
(I underlined cytoskeleton because Lipkin et al mentioned actin. NLRP3 might be interesting in a whole context, though difficult to judge at, of course.)

2. a) Manganese induces apoptosis of human B cells: caspase-dependent cell death blocked by bcl-2
N Schrantz et al 1999 May;6(5):445-53. doi: 10.1038/sj.cdd.4400508. https://www.nature.com/articles/4400508

abstract, my pragraphs
Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells.

We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis.

Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis.

These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.
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The following finding involves dopamine, which Lipkin et al, somehow, do refer to, too (page 2). (->?)

2. b) Manganese-Induced Toxicity in Normal and Human B Lymphocyte Cell Lines Containing a Homozygous Mutation in Parkin
Jerome A. Roth et al 2012, doi: 10.1016/j.tiv.2012.07.005 PMID: 22841634

Mutations in the parkin gene are linked to development of juvenile onset of Parkinson’s disease and recent studies have reported that parkin can protect against increased oxidative stress and mitochondrial dysfunction caused by a variety of oxidative and toxic insults. Overexpression of parkin has also been reported to selectively protect dopaminergic neurons from Mn toxicity. Accordingly, in this paper we compare the effect that mutations in parkin have on Mn toxicity and associated apoptotic signals in normal and human B lymphocyte cell lines containing a homozygous mutation in the gene.

Results of these studies reveal that Mn toxicity was similar in both control and mutant parkin lymphocyte cells indicating that cell death caused by Mn was not altered in cells devoid of parkin activity. In contrast, Mn did inhibit mitochondrial function to a greater extent in cells devoid of active parkin as indicated by a decrease in ATP production although mitochondrial membrane potential was essentially unaffected. Consistent with inactive parkin influencing the Mn response is the observation of increased activity in the down-stream apoptotic signal, caspase 3. In summary, results reported in this paper demonstrate that mutations in parkin can lead to functional changes in potential signaling processes known to provoke Mn toxicity. The selectivity and magnitude of this response, however, does not necessarily lead to cell death in lymphocytes which are devoid of dopamine.
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strategist said:
Sounds like they interpret this as an autoimmune problem.

But how plausible is B cell autoimmunity after the negative Rituximab trial?
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One thing @Jonathan Edwards has been highlighting is that rituximab does not kill all (antibody producing) B-cells i.e. it does not kill long lived (antibody producing) B-cells.

Another thing that Jonathan has highlighted is that symptoms similar to ME would be produced if B-cells, responsible for command & control, were dysfunctional; however, that supposes that this type of (command & control) B-cells exist.
 
Jaybee00 said:
If you have any questions, I will forward them and they will try to have their researchers answer them on their web site.
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I have to be honest, the technicality of this paper is way beyond me, but i have a few questions:
1) does their work warrant further study in this specific topic, what would it be and where would that lead?
2) if the clonal expansion leads to suggestion of autoimmunity, would this be grounds to target it via biologic drugs other that Rituximab, or is it still too early?
 
FMMM1 said:
Another thing that Jonathan has highlighted is that symptoms similar to ME would be produced if B-cells, responsible for command & control, were dysfunctional; however, that supposes that this type of (command & control) B-cells exist.
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I don't think I said that! I have no idea what a command and control B cell would mean.
 
Answers to questions.


  1. Can you please explain the biological significance of the quadratic transformation of the data?
The quadratic transformation of data allows us to understand and interpret the connections between protein analytes and ME/CFS, since these connections are not linear. The data suggest that both high and low levels of a certain protein are associated with an increased risk of ME/CFS in our sample. This is what we call a U shaped association. On the one hand, the people withME/CFS in our sample have higher levels of IGHV3-23 than controls, and on the other hand, subjects with ME/CFS in our sample have low levels of IGHV3-23. However, very few subjects with ME/CFS in our sample had very low levels of IGHV3-23, so we want to be cautious about drawing conclusions.There may be a biological reason for this U-shaped connection, which we don’t yet understand, but may be worth pursuing. Overall, these results suggest that there is a potential role for IGHV3-23, and more work should examine it.
  1. To demonstrate B cell clonal expansion, do you need to sequence the antigen binding section of the antibody (CDR or complementarity determining region)?
Yes, in order to demonstrate B cell clonal expansion, we would need to sequence the variable regions of B lymphocytes using one or more techniques, such as next generation sequencing or single cell B-cell receptor sequencing (sc-BCRseq).

3) "We speculate that, at least in a subset of ME/CFS subjects, an increased level of IGHV3- 23 may be due to antigen driven clonal expansion, and that these patients might benefit from identification of the antigen driving B-cell receptor signaling or kinase inhibitors that interrupt signaling." Specifically, what types of kinase inhibitors do you think might benefit ME/CFS patients (to interrupt signaling)?

B-cell malagnancies have been shown to be sensitive to kinase inhibitors that disrupt BCR signaling. The use of the kinase inhibitor Ibrutinb has had promising results in early trials among patients suffering from marginal zone lymphoma and chronic lymphocytic leukemia. If, indeed, a subset of people with ME/CFS suffered from a similar B cell dysfunction, targeting BCR signaling could have apositive potential. However, our results are preliminary,and further work will need to be done in different and larger samples before therapies can be considered.
 
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Jaybee00 said:
Answers to questions.
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Thanks for asking these questions, very useful.

During the Massachusetts ME/CFS conference Lipkin said their findings were confirmed by a Japanese group. I assume he was referring to this paper by Sato et al. Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome, 2021, Sato et al | Science for ME (s4me.info)

The Japanese group found higher levels of IGHV3-30 using next -generation sequencing, especially in those with an infectious onset. They also report that "B cell clones using IGHV3-30 and IGHV3-30-3 genes were more frequent in patients with an obvious infection-related episode at onset".

 
In one of the recent NIH ME/CFS CRC update webinars Ian Lipkin gave a general update for their NIH ME/CFS center and in the Q&A at 27-28mins he described in mid-1990's finding high antibody levels to a wide range of "things" in a swedish cohort while working on a project to look for Borna virus.


Here is the video transcript:
"My first engagement in this field was in the mid-1990s when we were asked to look at whether or not a virus called borna might be implicated and in those days we went back we used pcr we used uh antibody tests and we found no evidence for borna virus infection but i was impressed at that point as were my colleagues by the observation that people with mecfs in the swedish cohort had very high levels of antibodies to a wide range of things some of them that you don't even encounter in nature so at the tail end of our paper we said we find no evidence that this particular virus is important in mecfs but we do find evidence of abnormalities in the immune system suggesting that this is a biologically based disorder"

I think this is the 1999 Lipkin CFS paper regarding the work he mentioned in his talk.
Paper : Absence of evidence of Borna disease virus infection in Swedish patients with Chronic Fatigue Syndrome
https://www.jneurovirol.com/pdf/5(5)/495-499.pdf

I thought this text in the discussion section of the 1999 paper key:
"Although serum immunoreactivity to BDV proteins observed in Swedish CFS patients by ELISA may reflect infection with related microbial agents that induce cross-reactivity with conformational determinants on BDV proteins (Kliche et al, 1996) and b-galactosidase, the serologic findings are also con-sistent with nonspecific polyclonal B-cell activa-tion."
 
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