V. Lombardi, et al, new poster: HERV in pDC as biomarkers & TLR defects

[Daffodil]

no one is this far ahead. we must support wpi/Lombardi/kdm

 

[serg1942]

Wow!, fantastic material guys!!! you are awesome! I cannot wait to look at this!

As for the siRNA sequences, I don't see the problem, it is a really smart way to inhibit the specific sequence you want to. So, if the proper controls are taken, I think it's a clever and a valid method...

By the way, in my first "layman explanation" of what I read from the article, I talked about XMRV, and I had to explain myself better in another place... I'm going to post below my explanation of why I think that the HERV proteins they are finding could actually be the same sequence they found in redlabs when the performed a PCR of my gut biopsy. I don't want to open a debate about this. I really don't mind, and it's completely irrelevant. But I rather explain just in case... Let's continue learning about this new trail that looks at least worth studying!

(I repeat, I don't want to give importance to this, but I saw necessary to properly explain this statement I did...).

let me explain myself in more detail: I did a stomach biopsy by Redlabs, and they found me "XMRV +", by PCR. This means that they took by biopsy my endothelial cells from my gut, then isolated the DNA, and with it, they performed a PCR. I don't see why they would use the22Rv1 cell line, because it wasn't a nested PCR, that is, there were not PBMCs to be cultivated previously to the PCR. But in anyway, they had to isolate the DNA from my tissue sample somehow, and then put it into the reactants for the PCR to take place.

What they looked for was for a known gag and env sequences, that they had identified as XMRV, and they did found these proteins. Where did these proteins came from? Did the sequence came from exogenous contamination? It could be. But, taking into account that for tissue PCR (not the PCR they performed in the Science article, nor in the NIH Lipkin paper) there's no need to use cell lines, I am not aware of other sources of contamination. Anyway, it could be contamination anyway.

What it is a fact is that they found gag and env sequences, that is, they found retroviral proteins from tissue from my gut. Ok? So what I am telling is that, it could be plausible that what they are finding now, that is, human endoretroviral proteins (HERVs) expressed by human endoretroviruses inside plasmacytoid dendritc cells of the gut of CFS patients and not in controls, could be the same sequence they previously found, and wrongly called it XMRV. Of course it was not XMRV, that is known to be a non infectious recombinant virus, but what I say is that it is possible that they found the very same proteins Dr. De Meirleir et al are finding right now.This is simple science, and it does make sense, at least to me... But of course I can be wrong and I probably am!

 

[Soundthealarm21]

Just when I think i'm smart and know a decent amount of stuff I read this conversation!

 

[anciendaze]

One slight correction, XMRV as found in laboratories, can infect individual human cells. Some of the ways it was spread in laboratories involved aerosols, others moving through liquid nitrogen. There is still significant concern that it could infect laboratory workers. So, in that sense it is wrong to say it is non-infectious. It could infect people, but so far does not appear to have done so.

It is unlikely to produce an infection with high-titers of virions in blood. However, if a virus is transmitted via dendritic cells, viral synapses, exosomes, etc. you may not find significant numbers of free virions. Infecting only a very special class of immune cells is another strategy which could result in chronic infection without producing high titers of antibodies or large numbers of nucleic acid sequences. I am not arguing that XMRV, as currently defined, does this, but I am saying we should be aware that slow infections operating this way are entirely possible.

Added: endothelial dysfunction is a factor not only in GI problems, but also is a risk factor for atherosclerosis. I'm looking for anything tying a wide range of problems together, and I'm particularly interested in those that cause cytotoxic T cells (CD8+ T cells) to invade nerves or endothelial tissue. A virus is a very likely suspect.

 

[NK17]

One slight correction, XMRV as found in laboratories, can infect individual human cells. Some of the ways it was spread in laboratories involved aerosols, others moving through liquid nitrogen. There is still significant concern that it could infect laboratory workers. So, in that sense it is wrong to say it is non-infectious. It could infect people, but so far does not appear to have done so.

It is unlikely to produce an infection with high-titers of virions in blood. However, if a virus is transmitted via dendritic cells, viral synapses, exosomes, etc. you may not find significant numbers of free virions. Infecting only a very special class of immune cells is another strategy which could result in chronic infection without producing high titers of antibodies or large numbers of nucleic acid sequences. I am not arguing that XMRV, as currently defined, does this, but I am saying we should be aware that slow infections operating this way are entirely possible.

Added: endothelial dysfunction is a factor not only in GI problems, but also is a risk factor for atherosclerosis. I'm looking for anything tying a wide range of problems together, and I'm particularly interested in those that cause cytotoxic T cells (CD8+ T cells) to invade nerves or endothelial tissue. A virus is a very likely suspect.

As usual your reasoning is brilliant and exquisite @anciendaze!

 

[sidonie]

If the paper is so interesting, why is it presented as poster and not as an oral communication?

 

[WNM]

Slight warning here, those plasmacytoid dendritic cells were cultured with small interfering RNA sequences. These normally inhibit gene expression, though it is possible inhibiting expression of one gene can result in derepression of another, or that siRNA can sometimes promote expression. They are provoking this response from a particular class of immune cells. This will be easy for critics to dismiss as unrelated to infectious disease. The big question concerns why patients plasmocytoid dendritic cells respond differently from controls. I'm not sure the work described will be enough to convince anyone that this difference is real and not an artifact.

Ideally they would use siRNAs specific for inhibition of TLR7 and 9 mRNA. Genes code for proteins: gene transcription creates mRNA, then translation creates the protein. siRNA interferes with mRNA to inhibit translation. Use of specific siRNA for TLR7/9 mRNA in the study will lower levels of the TLR7/9 proteins, effectively inhibiting this signaling pathway right at the start. However it is notable that the function of TLR7 is actually to detect RNA and it can be activated by siRNAs; although I'm sure they used the appropriate materials.

The TLR7/9 signaling pathway uses common adapters (e.g. MyD88) and links to common second messengers, so the dysfunction in ME might actually be at the level of the TLR7/9 proteins themselves. Although it is possible that there may be some other unique downstream protein in this pathway which is actually affected.

The big warning is that scarcely anyone will pay much attention to WPI after the XMRV fiasco.

I don't think this is much of an issue. Science is about being curious, open-mined and agnostic. Everyone makes mistakes. New research will be appreciated on its individual merit; initially by the journal editor, then the peer review, and then the scientific community.

 

[Marco]

I have to declare that this isn't an area of primary interest to me (nor one in which I've any expertise) but I was searching for evidence of microglial activation via TLRs following common infections such as mono and came across a few papers which may be of interest to others in the context of this paper :

EBV activation of microglia via TLRs

http://www.theoneclickgroup.co.uk/d...t Epstein-Barr virus in active MS lesions.pdf

EBV HERV-W interaction in MS

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044991

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078474

EBV protein downregulates TLR9

http://www.ncbi.nlm.nih.gov/pubmed/21191071

Plus EBV infection in gastrointestinal disease

http://www.annalsgastro.gr/index.php/annalsgastro/article/viewFile/349/313

 

[anciendaze]

Hi Marco,

The two components of the immune system inside the CNS to watch are microglia and astrocytes. These have been on my radar for some time, though I'll admit the picture is still pretty fuzzy for me. In addition to actions by these, I'm looking for reports of CD8+ T cells invading the CNS. If you don't find the T cells themselves, they may leave damage in the form of nodules of Nageotte. (Unfortunately, the article on nodules of Nageotte has disappeared from Wikipedia. Jean Nageotte was an interesting neuroanatomist who suggested that glial cells participated in endocrine signalling in 1910. The length of time this was ignored in medicine is embarrassing. Perhaps he will be the next to disappear.)

HERV-W is one of the most confusing ERVs around. On the one hand, we have evidence it has been there for a very long time, possibly going back to the origin of placental mammals. (It would make sense that a virus promoting local immune tolerance to protect itself would be involved in creating a special immunological environment which allows an embryo to remain in the mother's body.) On the other hand, when we look at our closest relatives, great apes and chimpanzees, we find find considerable evidence that similar gamma retroviruses have been active recently. In gibbons there is even GALV, which causes chronic myeloid leukemia (CML). (In humans, the cause of CML remains unknown. Generally speaking, in humans beta retroviruses seem to have been more active recently.) HERV-W is not only activated in MS, but also in other autoimmune disorders and several mental illnesses. It has been called "the insanity virus". Trials of ARVs in treatment of MS have begun. The sequence of events which led to this is like those leading to trials of Rituximab in ME/CFS, clinicians noticed a patient treated for an unrelated condition (HIV+) showed dramatic improvement on ARVs. (Similar dramatic improvements took place in about 10% to 20% of ME/CFS patients who tried ARVs, but everyone knows these patients are crazy. Arguments about a particular retrovirus caused these scattered positive results to be ignored, even though several clinicians were convinced. Careful examination of the history of progress in medicine will show that just about every advance started with an idea that was incorrect in some important respect. In some cases researchers follow up anyway, in others they take the opportunity to trash the idea immediately. Bureaucratic science, where any mistake is fatal, is a recipe for stagnation.)

Everything I learn about EBV says this virus is very well adapted to humans, and performs sophisticated manipulation of human immune systems. The really appalling possibility here is that interactions between herpes-type viruses and gamma retroviruses can be much closer than previously considered possible. A retrovirus can be entirely inside a herpes virus. That is what I was arguing in my post about reticuloendotheliosis virus in fowl. That sequence of events, involving viruses from the echidna and diseases of birds (fowlpox and Marek's disease) is way too unlikely to be anticipated by researchers. As the Latin quote indicates, we are just a little late in making plans to deal with this possibility. Reticuloendotheliosis virus is now loose among wild fowl. The closest human equivalent of reticuloendotheliosis would likely be hairy-cell leukemia. (The convenient Wikipedia references to reticuloendotheliosis caused by retroviruses have also disappeared. Someone seems determined to provoke conspiracy theories about a cabal suppressing research on retroviruses. Published papers on these subjects are harder to disappear. If you need help, ask me.)

I do not claim to understand much about what EBV is capable of doing. It keeps surprising me. Perhaps those who claim to know what it is not capable of doing know better. If so, I may have some pointed questions in the future: "If you knew this, why didn't you do anything about it?".

Aside: I'm having a rocky start to this weekend, and will lie down again in a moment. Please take what I say with a large grain of salt.

 

[Aileen]

Ideally they would use siRNAs specific for inhibition of TLR7 and 9 mRNA. Genes code for proteins: gene transcription creates mRNA, then translation creates the protein. siRNA interferes with mRNA to inhibit translation. Use of specific siRNA for TLR7/9 mRNA in the study will lower levels of the TLR7/9 proteins, effectively inhibiting this signaling pathway right at the start. However it is notable that the function of TLR7 is actually to detect RNA and it can be activated by siRNAs; although I'm sure they used the appropriate materials.

What is siRNA please? And could you tell me in very simple language what its basic function is.
I know that mRNA = messenger RNA, mtRNA = mitochondrial RNA and rRNA = ribosomal RNA but this one is new to me.
Thanks

 

[anciendaze]

siRNA are short interfering RNA strands which typically block transcription of specific sequences into proteins. See this paper. They have been extremely useful in producing laboratory models in which particular genes are silenced. It is much easier for example to produce "knock-down mice" than to breed mice in which a gene is "knocked-out".

 

[Marco]

Hi Marco,

The two components of the immune system inside the CNS to watch are microglia and astrocytes. These have been on my radar for some time, though I'll admit the picture is still pretty fuzzy for me.

Aside: I'm having a rocky start to this weekend, and will lie down again in a moment. Please take what I say with a large grain of salt.

When you're feeling a little better I highly recommend the full version of this paper which does a very nice job of pulling together a wealth of research on neuroimmunity.


Pathological pain and the neuroimmune interface

Peter M. Grace, Mark R. Hutchinson, Steven F. Maier & Linda R. Watkins

http://www.nature.com/nri/journal/vaop/ncurrent/full/nri3621.html

'Pain' being just one of many danger signals that provoke a neuroimmune response so the scope of the paper is much more general.

 

[natasa778]

So they then looked at what else popped up when TLR7/9 signaling was suppressed and they found increased expression of several proteins, but most strongly the PI3K binding protein PLCL3 (phospholipase C-like 3). They then found this protein was also increased in ME pDCs, suggesting increased PLCL3 and HERV expression may be caused by a TLR7/9 deficit.

Speculation... is some infection evading the immune system by suppressing the TLR7/9-PI3K pathway on DCs? A similar scenario occurs with Yersinia enterocolitica infection. Evidence of enterovirus (82%) and Parvovirus B19 (40%) have been identified in the gut of many with ME. Of note despite the gastritis, H. pylori infection is apparently not present in ME/CFS (see and see).

Edited.

Wouldn't EBV be capable of exactly that? Eco posted this on another thread wrt B cells

Furthermore, our study provides insight into the way in which EBV can subvert the human immune response, as we show that BPLF1 can remove ubiquitin tags from proteins in the TLR signaling cascade. This inhibits TLR signaling and decreases the expression of immune response genes. ....Since EBV can activate TLR2, TLR3, TLR7, and TLR9, it would be of benefit if EBV were to evade innate immune activation. Here, we examined whether EBV DUB activity was present during productive infection in B cells and whether it could inhibit innate immune signaling...
.
... ...BPLF1 is expressed during the late phase of productive EBV infection and is incorporated into viral particles...
...During EBV infection, EBV components are sensed by host TLRs (a). ...The EBV-encoded DUB BPLF1 counteracts TLR-mediated NF-κB activation ...BPLF1 is expressed as a full-length protein during the late phase of productive EBV infection, is incorporated into the tegument of viral particles (f), and can subsequently be released into newly infected cells ...BPLF1 could exert its immunomodulatory functions towards TLR signaling not only in EBV-producing B cells

 

[natasa778]

or any retrovirus with a gp120-like tool in its kit

HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells

Taken together, these data demonstrate that the direct interaction of HIV-1 gp120 with pDCs interferes with TLR9 activation resulting in a decreased ability of pDCs to secrete antiviral and inflammatory factors that play a central role in initiating host immune responses against invading pathogens.

 

[serg1942]

@anciendaze, Thanks for explaining other sources of XMRV contamination. Actually I don't really care much about it. For my assumption to be true, obviously the XMRV should share a very similar sequence of that of the HERV proteins found (it wouldn't be surprising as they are very conserved though... Who knows, I am not going to go into this now as the subject of the thread is really interesting! ) (I agree BTW about slow infections as a possibility).

@WNM, Actually what I think they are trying to demonstrate, slowly, as the science requires, is that inhibition of TLRs by pathogens or toxins could actually be the source of the HERVs expressions, and so on... And, actually, by inhibiting the TLR7 and 9 with competitive inhibitors is a way to prove this as possible. Also, it ensures that the siRNAs are working as they were supposed to!

Thankyou very much @Marco for these compilation of papers! Well, I knew about EBV, but what actually draws my attention is that in this paper:

http://www.google.es/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0CEYQFjAB&url=http://www.dovepress.com/getfile.php?fileID=5213&ei=jCEuU8flBMTS0QXfo4DIDQ&usg=AFQjCNGypKdCKknTroECZZO34Y-yYuhszg&sig2=YAv1jGEWtRvX4SLhgxHl7w&bvm=bv.62922401,d.d2k

The TLR 9 is shown to be activated by bacteria, so I am of course thinking of Borrelia, as roughly an 80% of PWCFS here in Europe are testing positive for Borrelia...
Actually, Borrelia is recognized by both TLR7 and 9:

http://www.ncbi.nlm.nih.gov/pubmed/19794067

It seems that also, tick saliva inhibits TLR2 in order for the Borrelia to infect:

http://www.ncbi.nlm.nih.gov/pubmed/22709526

But, this wouldn't match with Lombardi's poster so I thought in the protein MYD88, that is common to TLR2, 7 and 9, in their signaling cascade, and actually it seems to be the key protein allowing Borrelia B. to infect us, by altering specific immune response:

http://www.ncbi.nlm.nih.gov/pubmed/24452685

http://iai.asm.org/content/76/6/2341

I do look forward to see this paper published, with the numbers and statistics, and I of course hope that it draws the proper attention, as it needs to be replicated. They are not just finding potential biomarkers for ME, but also provide a plausible explanation for its pathogenesis, and what it's more important, targets for specific treatments...
We'll see, I hope sooner than later,

Sergio

 

[serg1942]

Just for what it is worth, here is the gist of Lombardi's poster, that actually have been perfectly explained by @anciendaze , but in bullet points format:


- Patients with ME produce less type I IFN than controls.
- pDCs are the primary source of all forms of type I IFN.
- pDCs play a crucial role in promoting immune tolerance as well as orchestrating events during viral infection, autoimmunity, and inflammation
- pCDs from gut ME patients express HERV proteins, but not gastritis, crohn or heathy controls. This suggests that the expression of HERVs may be unique from ME and not a result of inflammation.
- It's been reported that HERV expression is activated in a TLR -3 -7 -9 knocked out mouse.
- cPDs cultured with siRNA to TLR 7 and 9 results in expression of HERV proteins
- Could a toxin or a pathogen cause the same?: The same was done but using TLR7 and TLR9 competitive inhibitors. So Yes.
- Is HERV expression a result of TLR inhibition?: Transcriptional analysis by RNAseq on cultured human pCDs in the presence and absence of siRNA to TLR7 and TLR9:

RESULTS: when siRNA to TLR7 and TLR9 present: PI3K binding protein PLCL2 was identified by IHC in ME gut bipsies. So pDCs from ME expressed PLCL2 proteins, in contrast to control biopsies which did not express this protein by their pDCs

This data supports a disregulation of the TLR 7/9 in at least a subset of ME patients.

 

[anciendaze]

Serg1942, I think a mouse model would more likely have the gene "knocked down" through RNA interference to prevent expression of proteins, rather than "knocked out" by destroying the gene. I think it would be fairly difficult to breed knock-out mice with defective TLR genes in every cell. These are fairly important genes.

The results from the poster strike me as mostly in vitro, based on cultured cells without a complete animal model. This is one of the advantages of using siRNA.

As always, we will be certain about more things when the paper becomes available.

Comment: I've been noticing quite a number of diseases where genetic causes have long been sought turning up evidence of specific SNPs in genes related to immune function. This strongly suggests to me that these diseases are precipitated by an immune challenge like an infection which exposes a preexisting defect.

With all the evidence that has emerged of active HERVs and retroelements, I am no longer impressed with studies which assume all inheritance is Mendelian. If we pay attention to what the data are actually telling us I think we will disregard most newspaper accounts about "the gene" which causes a disease. Most of human genetics and most diseases do not follow the player-piano model of genetics used by journalists. You can learn a great deal from genetic studies, but not if you persist in forcing them into the Procrustean bed of your preconceptions. When the dust settles from the revolution in genetics I predict that a great many "facts" about genetic diseases will be found to be wrong. A great deal goes on between the inheritance of a particular allele and the expression of the corresponding protein. It is quite possible some of us are "knock-down" humans with RNA interference blocking the expression of genes we could use. It is also possible methylation has epigenetically repressed genes we actually possess. It is even possible some genes thought to be ours actually belong to our microbial tenants.

There is careful genetic work now being done which meets high standards, but not all the rubbish from earlier times has been cleaned up. If this process does not produce dramatic reversals of previous thinking I will be the most surprised person around.

 

[Katherine]

Speculation... is some infection evading the immune system by suppressing the TLR7/9-PI3K pathway on DCs? A similar scenario occurs with Yersinia enterocolitica infection. Evidence of enterovirus (82%) and Parvovirus B19 (40%) have been identified in the gut of many with ME. Of note despite the gastritis, H. pylori infection is apparently not present in ME/CFS (see and see).

I noticed from a few recent posts that KDM seems to have started testing patients for Yersinia, so you could well be hitting the nail on the head there.

I heard that Yersinia enterocolitica was thought to be implicated in the Lyndonville outbreak, but apparently Dr. David Bell said that it was not published.

 

[anciendaze]

Has anyone else here been disturbed by the fairly close relation between yersinia enterocolitica and yersinia pestis? The latter had a substantial effect on human history.

 

[SOC]

Has anyone else here been disturbed by the fairly close relation between yersinia enterocolitica and yersinia pestis? The latter had a substantial effect on human history.

Crossed my mind since I just had to read up on the bubonic plague in order to teach the science in support of my students' history lesson. Nasty stuff. Kids loved it.

Quite different diseases, though, aren't they?