XMRV -Letter to Science.....

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IMEA: Letter to the editor of Science

regarding serious flaws in the

methodology of Paprotka et al.

by XMRV Global Advocacy

Tuesday, 28 June

Dear Dr Ash

We are a patient organisation and would like to make the following

observations regarding the study entitled *Recombinant Origin of

the Retrovirus XMRV*, in which Coffin and his team claim to

supply proof that XMRV arose during the passage of a xenograft

through strains of mice that could potentially have been used in

the formation of the 22Rv1 cell line and thus is a harmless

contaminant. Should you choose to reply to this letter we

undertake not to make the contents public without your express

written permission.

This review first highlights the quote below.

The claim is that XMRV was created as a result of recombination

of 2 proviral sequences dubbed PreXMRV-1 and PreXMRV-2.

*The complete sequence of PreXMRV-1 was

determined from the early passage xenografts, the

NU/NU and Hsd strains, and the CWR-R1 cell line.*

PreXMRV-1 is therefore a metaphor. There is no evidence that it

exists as an independent entity. A synthetic clone was

constructed from sequences amplified from NU/NU, Hsd and the

CWR-R1 cell line. All three are known to contain one or more

XMRV specific sequences. The sequences amplified in the early

xenografts had a sequence homology to 3.5 kb of XMRV, in a

region not unique to XMRV. The fact that the other sequences,

used to construct the synthetic clone called PreXMRV-1, could

actually have been XMRV specific sequences, must be obvious. It

is difficult to understand how this escaped the attention of a

competent peer reviewer.

The determination of XMRV concentration in 22Rv1, CWR-R1,

NU/NU and Hsd nude mice.

*To quantify the amount of XMRV DNA in the CWR22

xenografts, we developed a real-time PCR

primer-probe set that specifically detected XMRV env

and excluded murine endogenous proviruses present in

BALB/c and NIH3T3 genomic DNA (Fig. 1C). We used

quantitative PCR of 22Rv1 DNA to estimate 20

proviruses/cell and used the 22Rv1 DNA to generate a

standard curve. The CWR22 xenografts had

significantly fewer copies of XMRV env*

These primers were not used to search for XMRV in the other

strains of wild and lab mice despite being able to detect such a

low copy number of XMRV, nor were they used to examine the

early xenografts for the presence of XMRV despite demonstrating

such a high level of clinical sensitivity.

In order to examine the early xenografts the authors created 8

different primer sets. The CWR22 xenograft referred to above was

not examined using the new primer sets below:

*We used the same XMRV-specific primer sets to

amplify and sequence DNA from early passage

xenografts (736, 777, 8L, 8R, 16R, and 18R; Fig. 2B);

the results showed that XMRV env, but not gag

sequences were present (sequencing coverage

summarized in fig. S3), indicating that the early

xenografts did not contain XMRV.*

This statement is an opinion and not objective fact. An

examination of the primers used to examine the early xenografts is

enlightening. The data can be referred to in Fig. S3 (A) of the

supporting material. 8fsa-U5rsa can amplify env sequences when

XMRV and only XMRV, is present. 18f-13r, 8fsa-Ursa and 8f-U3r,

can amplify gag-pol sequences and env sequences when XMRV

gag, pol and env sequences are known to be present at high

concentrations in the late stage xenografts. There is no evidence

however, that 8fsa-U5rsa and 8f-U3r produce amplicons in any

situation other than when XMRV specific env sequences are

present.

Hence, the presence of amplicons produced via the use of that

8fsa-U5rsa and 8f-U3r primers, means that the presence of XMRV

in the early xenografts certainly cannot be ruled out. Indeed the

amplicon produced using 18f-13r primers, which amplifies gag pol

regions in XMRV, produces further evidence for the existence of

XMRV in the early xenografts. This would not be expected to

amplify the gag pol region of a proviral sequence PreXMRV-1, with

only some 90% homology to the corresponding region in XMRV.

Taken together, these results strongly suggest that that the early

xenografts did contain XMRV. This is especially so, when the fact

that the existence of PreXMRV-1, as a real in vivo entity, has not

been demonstrated. The conclusion, that the absence of gag

sequences invalidates this finding, is clearly erroneous. It fails to

take into the account that gag primers are some 10 fold less

sensitive when dealing with such copy numbers of XMRV. Indeed,

son et al. demonstrated that their nested PCR, using input

DNA of 600ng, could detect 1 XMRV provirus in 100,000 cells. Yet,

these authors were quite unable to detect gag sequences in

patients where env sequences were detected. Whereas Lombardi

et al. only detected gag sequences in 7% of patients tested,

using single round PCR, such as used in this study. They were

however able to detect gag sequences in 67% of people with

nested RT-PCR.

The concentration in the CWR22 xenograft was established as

being less than 1 copy per 100 cells by the authors themselves,

thus we would be dealing with concentrations even lower than

that because we are looking at a time before the xenografts were

fully formed. It is worthy of note that the primers and cycling

conditions used to determine the concentration of XMRV in the

CWR22 xenograft, were not at any time used to examine the early

passage xenografts, nor indeed in the attempt to detect XMRV in

other strains of wild and laboratory mice. The authors also make

no attempt to determine the concentration of the proviral DNA

sequence in the early xenografts.

Although MuLVs (henceforth called XMRV like viruses) can induce

tumours by inserting into the promoter regions of tumour related

genes, this is by no means the only mechanism by which XMRV

like viruses can induce the formation of tumours, as the example

below demonstrates. LP-BM5 is a mix of XMRV like viruses.

LP-BM5 murine leukemia retrovirus induces the excessive oxidative

stress and immune dysfunction leading to B cell leukemia and

murine AIDS with cytokine dysfunction. The cytokines upregulated

in this investigation were Il-4 Il-6 and TNf alpha. The cytokines

downregulated were INF gamma and IL-2(1).

Il-8 is a cytokine commonly elevated and deemed to be a

causative factor in many cancers (2). Some information regarding

the role of IL8 is given below:

" Our data show that IL-8 signaling increases AR

expression and promotes ligand-independent

activation of this receptor in two androgen-dependent

cell lines, describing two mechanisms by which this

chemokine may assist in promoting the transition of

CaP to the androgen-independent state. " (3)

Serum 1L-8 is elevated in men with prostate cancer (4).

" Aalinkeel et al. found that IL-8 was significantly

higher in the more metastatic PC-3 and DU-145

prostate cancer cell lines, when compared to the

poorly metastatic LnCAP cells. The results of our

study of IL-8 in men with prostate cancer support the

findings of Aalinkeel et al "

XMRV induces IL-8 expression in prostate cancer cell lines (5).

93% Xmrv infected people with ME have elevated il-8 levels (6).

Immune dysregulation and the production of oxidative stress in a

host in response of a XMRV like virus infection is well documented.

Thus, the claim that XMRV would have to infect many cells in

prostate tissue to induce prostate cancer, is an opinion only. The

cells shown to be directly infected may well have been

transformed by insertional mutagenesis, but the two mechanisms

are not mutually exclusive.

Summary

A PCR assay(1) with primers A and reagents B, using cycling

conditions C, was able to detect XMRV specific env sequences

when the copy number was as low as 1-3 provirus copies per 100

cells.

This PCR assay played no further part in the study.

Instead a new PCR assay(2) with different reagents and primer

combinations of D E F G H I J K were used to assay cell lines of

the CWR-R1 xenograft, the 22Rv1 cell line and the NU/NU and Hsd

nude mice. Primers A B and C were able to detect XMRV

sequences when only XMRV was present at a copy number of

greater than 2000 proviral copies per 100 cells. Primer J was used

to amplify an XMRV sequence from 100 ng of 22Rv1 cell DNA at an

unknown level of proviral concentration. The use of primer J

however, was not able to amplify any XMRV specific sequences in

the investigation cited above. Primer J was the primer chosen to

search the DNA of multiple mice species for the presence of

XMRV.

Hence, it is impossible to say that XMRV was not present in the

early xenografts or indeed the original prostate cancer tissue. It is

also not possible to determine whether gag and env sequences

were present in the multiple mouse species examined.

The explanation that XMRV entered the human population as a

result of recombination of two proviruses would at least require

that both proviruses did actually exist as in vivo entities. The

existence, of the proviral sequence dubbed as PreXMRV-1 as such

an entity, has not been established. The sequences are a

construct from sources known to contain XMRV.

We are thus left with at least two competing explanations.

On the one hand we have the explanation that a PCR assay, of

unknown sensitivity below the level of 2000 XMRV proviral copies

per 100 cells, was simply unable to locate very low copy numbers

of XMRV. This is supported by the fact that the use of primers J

were unable to amplify XMRV sequences when proviral copies

were as high as 3000 proviral copies per cell. Alternatively, we

have the authors preferred explanation, that XMRV was formed by

a recombination event which is so rare that it could only have

happened once and the odds of it happening at any other time

(by the authors own admission) are over a billion to one against. I

leave the reader to judge what is the most parsimonious,

conciliate hypothesis.

References

1) Lee, J.M.; Dehydroepiandrosterone Sulfate Inhibited Immune

Dysfunction Induced by LP-BM5 Leukemia Retrovirus Infection

through Regulating Th1/Th2 Type Cytokine mRNA Expression and

Oxidative Stress in Murine AIDS Model; Journal of The Korean

Society of Food Science and Nutrition (Dec 2006)

2) Yuan A, Chen JJ, Yao PL, Yang PC. The role of interleukin-8 in

cancer cells and microenvironment interaction. Front Biosci. 2005

Jan 1;10:853-65. Print 2005 Jan 1.

3) Seaton, a Scullin, Pamela J. Maxwell,

, Johanna Pettigrew, Gallagher, Joe M. O'Sullivan,

G. ston and J. J. Waugh: Interleukin-8 signaling

promotes androgen-independent proliferation of prostate cancer

cells via induction of androgen receptor expression and activation;

Carcinogenesis (2008) 29 (6): 1148-1156.

4) Lehrer S, Diamond EJ, Mamkine B, Stone NN, Stock RG.; Serum

interleukin-8 is elevated in men with prostate cancer and bone

metastases.; Technol Cancer Res Treat. 2004 Oct;3(5):411.

5) H. Silverman, Carvell Nguyen, J. Weight &

A. Klein; The human retrovirus XMRV in prostate cancer and

chronic fatigue syndrome; Nature Reviews Urology 7, 392-402

(July 2010)

6) V.C. Lombardi, K. S. Hagen, K. W. Hunter, J. W. Diamond, J.

-Gagen, W. Yang And J. A. Mikovits; Xenotropic Murine

Leukemia Virus-related Virus-associated Chronic Fatigue Syndrome

Reveals a Distinct Inflammatory Signature; in vivo 25: 307-314

(2011)

These following extracts from the paper clearly show that the

argument rests on highly subjective interpretations. One could

easily reverse the conclusions of the authors and the whole study

would fall.

" ( PCR and sequencing of PreXMRV?1. The complete

PreXMRV?1 genome was cloned and sequenced from

the indicated sources using primers that specifically

amplify XMRV or PreXMRV?1 but exclude known

endogenous MLV sequences (Fig. S2). We amplified

PreXMRV?1 from the CWR?R1 cell line, but not the

22Rv1 cell line, indicating the absence of PreXMRV?1

from these cells. Partial PreXMRV?1 (env divergent

region) was also amplified from xenografts 2524 and

2274, showing that both XMRV and PreXMRV?1 are

present in these samples. "

" We used the same XMRV-specific primer sets to

amplify and sequence DNA from early passage

xenografts (736, 777, 8L, 8R, 16R, and 18R; Fig. 2B);

the results showed that XMRV env, but not gag

sequences were present (sequencing coverage

summarized in fig. S3), indicating that the early

xenografts did not contain XMRV. "

Due to the serious implications that this paper will have on further

research into this retrovirus, it is therefore necessary to amend

these flaws, so as to allow your readership the opportunity to

judge the data in their usual manner.

Yours sincerely

Louise Gunn

CEO

IMEA

[Guest guest]

After reading that, I just sat in awe of the content and the writer.

In a message dated 6/29/2011 7:01:20 P.M. Pacific Daylight Time,

j.van.roijen@... writes:

_http://on.fb.me/lGallx_ (http://on.fb.me/lGallx)

IMEA: Letter to the editor of Science

regarding serious flaws in the

methodology of Paprotka et al.

by XMRV Global Advocacy

Tuesday, 28 June