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CROI: positive Spanish study - B cells XMRV reservoir

natasa778

Senior Member
Messages
1,774
http://www.retroconference.org/2 011/Abstracts/40987.htm


Session 43-Themed Discussion
TD: XMRV: New Findings and Controversies
Wednesday, 1-2 pm; Room 302-304
Paper # 217
Presence of XMRV Sequences in B Cells Are Restricted by APOBEC
Jorge Carrillo1, J Blanco1, E Garcia1, J Arreal2, B Clotet1, and C Cabrera1
1Fndn irsiCaixa, Barcelona, Spain and 2Hosp Univ Germans Trias i Pujol, Barcelona, Spain

Background: A link between xenotropic murine leukemia virus-related virus (XMRV) infection and different human diseases, such as chronic fatigue syndrome (CFS) and prostate cancer, has been recently established. Given that this retrovirus can infect different tissues and cell types both in vivo and in vitro, including cells from the immune system, the identification of the cellular compartment(s) where XMRV can establish a reservoir may be useful to understand the pathology associated with this virus.

Methods: In order to determine the presence of XMRV in B lymphocytes, we have screened EBV-transformed B-cell lines available in our laboratory. The origin of these cell lines include CFS patients (n = 11; fulfilling both Fukuda and Canadian criteria), HIV+ individuals (n = 4), prostate cancer patients (n = 1), and healthy donors (n = 5). DNA was extracted from cellular dry pellets and several XMRV genes were amplified by either real-time PCR (pol) or nested PCR (gag and env).

Results: We detected 7 positive samples from 14 individuals tested by RT-PCR (4 CFS, 2 donors, and 1 HIV+ individual). env amplification identified 4 positive samples out of 21 individuals tested (3 CFS-affected individuals and 1 healthy donor), whereas gag amplification showed only 3 positive samples (1 CFS-affected individual, 1 healthy donor, and one HIV+ patient). To confirm the presence of XMRV we performed sequence analyses of gag and env amplicons. The 3 gag sequences available showed a 100% homology with XMRV gag published sequences, including the XMRV characteristic 24nt deletion which is not found in any known exogenous murine leukemia virus. Furthermore, env sequences were also homologous to previously described XMRV sequences, showing none or low variability. Interestingly, most of the observed changes corresponded to multiple G to A mutations that were accumulated in 1 positive sample, resulting in a truncated env protein and suggesting that APOBEC-related restrictions operate in vivo during XMRV infection.

Conclusions: Despite the discrepancies observed in the different PCR approaches, our data provide evidence that EBV-transformed B-cell lines harbor XMRV-specific sequences, although the establishment of this infection could be modulated by the innate restriction factor APOBEC 3G . Our data suggest that, in vivo, B cells may represent a reservoir for XMRV contributing to its potential pathogenesis.
 

August59

Daughters High School Graduation
Messages
1,617
Location
Upstate SC, USA
They seem to have a technigue for finding XMRV. I don't have the knowledge to understand their reason for looking at EBV transformed B-cells, but I feel good that they are considering what co-infections could have on XMRV.

Their mention of the 24nt deletion rings a bell, but thats about it? Someone else??

Could we have a genetic malfunction that cripples APOBEC 3G? Could that be what our co-infections are doing, altering APOBEC 3G allowing XMRV to replicate?
 

Grape Funk

Senior Member
Messages
113
Location
USA
They seem to have a technigue for finding XMRV. I don't have the knowledge to understand their reason for looking at EBV transformed B-cells, but I feel good that they are considering what co-infections could have on XMRV.

Their mention of the 24nt deletion rings a bell, but thats about it? Someone else??

Could we have a genetic malfunction that cripples APOBEC 3G? Could that be what our co-infections are doing, altering APOBEC 3G allowing XMRV to replicate?

August, they might have taken a look there because a) a lot of people who come down with cfids will be the result of mono/EBV or b) B cells are known to be immature in PWC as stated in the last XMRV presentation from mikovits. Don't recall the 24nt but the APOBEC 3G is on cfidsresearch blog, he explains a possibility there and states "Once XMRV enters a cell, it could quite conceivably create an immune dysfunction that allows EBV to reactivate at a low level, fuelling a vicious cycle - through EBV producing a gene that downregulates APOBEC3, somewhat like the HIV vif protein does." APOBEC 3G is supposed to exhibit antiviral properties, you see. Just a thought
 

Grape Funk

Senior Member
Messages
113
Location
USA
I really do hope the continuance of these Spanish scientists prevail, especially with the relativity they are bringing with the herpes viruses. Even in the case of cell line contamination and allegedly endogenous RV XMRV recently stated from the same conference (diff study,author).

"The 3 gag sequences available showed a 100% homology with XMRV gag published sequences, including the XMRV characteristic 24nt deletion which is not found in any known exogenous murine leukemia virus. Furthermore, env sequences were also homologous to previously described XMRV sequences, showing none or low variability. Interestingly, most of the observed changes corresponded to multiple G to A mutations that were accumulated in 1 positive sample, resulting in a truncated env protein and suggesting that APOBEC-related restrictions operate in vivo during XMRV infection."

Science bloggers bitch about the homologous state of XMRV though, as they say it is highly unlikey for a retrovirus to have the same sequence in unrelated cases. Anyone want to throw their 2 cents in feel free.
 

free at last

Senior Member
Messages
697
Hi Grape, isnt this the group who recently failed to find the virus, If this is indeed the group who have had trouble finding the virus then this presentation is more important than first realized, AS OTHERS ARE FAILING TOO, see the importance of that, they seem confident they are finding a virus. It almost feels like its a man made virus that has great difficulty mutating, as is noted in nature viruses often mutate, this appears somewhat hindered, both in mutation, and replication, often lying, dormant. though activation does occur as we know, when it gets the right catalyst, if this is contamination, its a live viral contamiante that has infected humans, OR THE MONKEY STUDIES JUST WOULD NOT BE, any nay sayers what to try and explain that lol. hence the contamination connections maybe. Just throwing ideas here, i will probably get shot down through lack of knowledge. Ahh well i do try, Think this is my new pet theory, yes it is contamination, or started out as such but somehow its now a live rather hindered REAL VIRUS ABLE TO SET UP INFECTION IN BOTH MONKEYS AND HUMANS
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
Science bloggers bitch about the homologous state of XMRV though, as they say it is highly unlikey for a retrovirus to have the same sequence in unrelated cases. Anyone want to throw their 2 cents in feel free.

XMRV is nothing like one single strain, there are many. Some testing protocols just don't go looking for the various strains. At some point I think we will be talking about HGRVs not XMRV. If you only look for an identical bit in your testing, that is all you will find. However, its good just to read someone else found it.

Bye
Alex
 

kurt

Senior Member
Messages
1,186
Location
USA
This study surprised me until I re-read. They did not collect fresh samples, they tested cell lines. Who knows how old those lines are, how many opportunities there were for contamination, cell lines have to be maintained long-term. But still, this is an interesting report. Will be interesting to see where they go with this.
 

Enid

Senior Member
Messages
3,309
Location
UK
Many thanks natasa778. Just feel we are really going places now as more and more is revealed/understood.
 

taniaaust1

Senior Member
Messages
13,054
Location
Sth Australia
This didnt make me happy as they didnt find it in large numbers in the CFS people and they found it in high amounts then we expected in the other groups. This could be used by that pesky psych group against the idea that its XMRV affecting us.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
This is the same study that was presented in September 2010 at the 1st International XMRV Workshop. So it's not really something new. I would be interested what they have done in the meantime and what their plans for further studies are.

A Spanish ME/CFS group recently wrote a call for more donations to this institute, so they can continue their research and i helped circulate this and tried to advertise for it.
I was of the opinion that this is a very good place to do that kind of research and that it's the best XMRV/CFS research we currently have in Europe.

But to be honest, they have presented what they think to be a biomarker for ME/CFS in December, if i remember correctly, and i thought they will officially present it at this CROI, but so far i have not heard about it. So i want to hear more from them. I still hope i was right and they are our best bet here and we should support them, but i want to know what they have done since Sept and what their biomarker is. We need to know more, so we can decide wheter to support them or not.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
This didnt make me happy as they didnt find it in large numbers in the CFS people and they found it in high amounts then we expected in the other groups. This could be used by that pesky psych group against the idea that its XMRV affecting us.
I agree Tania, i was thinking the same thing. Unfortunately i don't have a direct contact to IrsiCaixa (this institute), i "only" have a contact to a person who knows researchers there. So it's a bit hard for me to get a statement about these results from IrsiCaixa. I also would like to know what they think about these percentages.
 

August59

Daughters High School Graduation
Messages
1,617
Location
Upstate SC, USA
This is the same study that was presented in September 2010 at the 1st International XMRV Workshop. So it's not really something new. I would be interested what they have done in the meantime and what their plans for further studies are.

A Spanish ME/CFS group recently wrote a call for more donations to this institute, so they can continue their research and i helped circulate this and tried to advertise for it.
I was of the opinion that this is a very good place to do that kind of research and that it's the best XMRV/CFS research we currently have in Europe.

But to be honest, they have presented what they think to be a biomarker for ME/CFS in December, if i remember correctly, and i thought they will officially present it at this CROI, but so far i have not heard about it. So i want to hear more from them. I still hope i was right and they are our best bet here and we should support them, but i want to know what they have done since Sept and what their biomarker is. We need to know more, so we can decide wheter to support them or not.

The biomarker was suppose to be announced or presented at this conference!
 

free at last

Senior Member
Messages
697
True Kurt but some of the contamination studys might also be using non fresh samples ( i might be wrong ) apolgies if i am. but i havent heard that complaint from you, about those studys if true ?
 

Grape Funk

Senior Member
Messages
113
Location
USA
Free at last, i have to agree along the lines when you said it is somehow contamination yet a real virus is able to infect monkey tissue and human tonsil when infected with xmrv. It is very contradictory. Now the other report came out that just said it is the cell line contaminate. So maybe people were already supposedly affected by the "endogenous RV" found in the prostate. But why are cdc studies not showing any xmrv then? How about substantial percentage differences?

So alex you are saying they are finding different strains, because i believe that is main concern for scientists. The blog i was looking at was straight up trashing xmrv, giving it no shot at all, though a sizable blog with resonable comments. But not skeptics just doubters.
 

SOC

Senior Member
Messages
7,849
Can someone explain "EBV-transformed B cells" to me? Are the B cells of everyone who has ever had EBV transformed somehow? Or is this some special situation?
 

Grape Funk

Senior Member
Messages
113
Location
USA
Can someone explain "EBV-transformed B cells" to me? Are the B cells of everyone who has ever had EBV transformed somehow? Or is this some special situation?

B cells are the cells EBV normally lies dormant in, throughout the human body. But in our case they could be activated, by another antigen, or suppression of the immune system. EBV is also associated with cancers and other diseases, but as with most unknown diseases of etiology, it is under speculation.
 

lansbergen

Senior Member
Messages
2,512
Can someone explain "EBV-transformed B cells" to me? Are the B cells of everyone who has ever had EBV transformed somehow? Or is this some special situation?

http://en.wikipedia.org/wiki/Epstein-Barr_virus

Transformation

When EBV infects B-lymphocytes in vitro, lymphoblastoid cell lines eventually emerge that are capable of indefinite growth. The growth transformation of these cell lines is the consequence of viral protein expression.
 

SOC

Senior Member
Messages
7,849
B cells are the cells EBV normally lies dormant in, throughout the human body. But in our case they could be activated, by another antigen, or suppression of the immune system. EBV is also associated with cancers and other diseases, but as with most unknown diseases of etiology, it is under speculation.

In this study, they used "EBV-transformed B cells". I assume that healthy people have EBV-transformed B cells (if they've been exposed to EBV) since they had healthy controls in this study.

Are they saying they didn't test all the B cells, just those infected with EBV? Or are B cells transformed some other way by EBV?

I don't really understand why they tested only EBV-transformed B cells. Did they have some reason to think those specific B cells were more (or less) likely to show XMRV infection.
 

lansbergen

Senior Member
Messages
2,512
In this study, they used "EBV-transformed B cells". I assume that healthy people have EBV-transformed B cells (if they've been exposed to EBV) since they had healthy controls in this study.

Are they saying they didn't test all the B cells, just those infected with EBV? Or are B cells transformed some other way by EBV?

I don't really understand why they tested only EBV-transformed B cells. Did they have some reason to think those specific B cells were more (or less) likely to show XMRV infection.

EBV is used to make cell lines. They tested the cell lines.
 

SOC

Senior Member
Messages
7,849
EBV is used to make cell lines. They tested the cell lines.

So this is standard procedure for making cell lines, in general? Because the EBV viral protein expression makes the cells "immortal" after a fashion and this is somehow beneficial for maintaining cell lines? :confused: