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XMRV revisited (Split from Hornig/Lipkin lawsuit thread)

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Hip

Senior Member
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$50 approx per month depending on where you get it.

Alldaychemist I notice sell tenofovir cheaper than most, at $1.76 per 300 mg tablet (though ADC have a high $25 shipping charge). Most other pharmacies seem to sell it for $3 a tablet.

I have considered trying tenofovir myself, to see if its immunomodulatory effects might work for my active coxsackievirus B4 infection. In my case though, I find that many immunomodulators seem to significantly worsen depression symptoms (possibly through increasing interferon, which is known to cause depression). This makes longer term treatment untenable for me. Depression is listed as a common side effects of tenofovir: see Tenofovir Side Effects.
 

heapsreal

iherb 10% discount code OPA989,
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Tenofovirs immune modulation can work in away to improve immune function and kill pathogens, not necessarily just turning down inflammation.

Tenofovir also appeared to keep the balance of IL-12 and IL-10 stable. The drug enhanced the IL-12 levels, thus increasing their ability to respond to other infectious pathogens, and it kept IL-10 levels low, thus keeping the body from putting the brakes on the immune response.
https://www.poz.com/article/hiv-tenofovir-inflammation-20217-1101
 

Hip

Senior Member
Messages
17,824
Yes, it's tenofovir's immunomodulatory ability, that strongly reduces IL-10 (Th2 cytokine) but increases IL-12 (Th1 cytokine), in vitro at least, that might make it a good Th2 to Th1 shifter like oxymatrine. See:

Tenofovir selectively regulates production of inflammatory cytokines and shifts the IL-12/IL-10 balance in human primary cells

All Dr Chia's patients who responded to oxymatrine showed a measurable increase in their IL-12 / IL-10 ratio, and the patients that did not respond to oxymatrine saw no increases in this ratio. See this post.

So tenofovir might potentially be useful for the chronic enterovirus infections associated with ME/CFS.
 
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Hustler

I m a hustler
Messages
56
Location
Europe
one example of a fraudulent negative paper is the 2010 paper on XMRV by psychiatrist Simon Wessely.

Can we agree that that is fraud ?


It was one of the very very first negative papers .......
The tone at XMRV Workshop 2010 is horrific. Mikovits science falling on deaf ears.
It is all staged and a one big hoax.
McClure
Stoye
Coffin
All staGed.
One big joKe

You ask me for an example of fraud in the negative papers.
I deliver straight to your door.

PLoS One. 2010 Jan 6;5(1):e8519. doi: 10.1371/journal.pone.0008519.
Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome.
Erlwein O1, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare A.

Abstract
BACKGROUND:
In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.

METHODOLOGY:
Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

CONCLUSION:
XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.



Answer the question instead of deflecting attention away. Is the Wessely XMRV paper fraud ? He masterminded PACE. The 8 million dollar psych study that was being wrapped up and under peer review while Lipkin was definining HIS protocol for masterclass definitive neGaTiVe study oh yeahhh !


Who needs hollywood when you have COLUMBIA PICTURES and Generals like Wessely n Lipkin leading the troops !!!!! With a little forum help from the cdc. Negative words and negative tone can be very persuasive and deliberate and staged and infiltrate everywhere.

You call me unhinged? Maybe because I ve broken down so many doors and found out the truth. I ll gladly knock all your loose screws back into place. Measure for measure.

Or I tell you what, how about a game of pool? You want to take me on at pool? Come on I ll throw you the break and the five ball. What else do u want?
 
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Hip

Senior Member
Messages
17,824
one example of a fraudulent negative paper is the 2010 paper on XMRV by psychiatrist Simon Wessely.

Can we agree that that is fraud ?

Just to fill you in on the facts: Simon Wessely's name may have been on that paper, but the virology work was done by Prof Myra McClure, who in fact had never even met Wessely, and had no connection to Wessely's erroneous view that ME/CFS is "all in the mind".

McClure did everyone in the ME/CFS community a great favor by helping to bring the XMRV debacle to an end. And then the negative result was independently replicated by numerous completely different labs around the world, such that the XMRV story was completely blown out of the water.



You ask me for an example of fraud in the negative papers.
I deliver straight to your door.

You were asked for proof of your statements that the negative XMRV papers were fraudulent. Do you understand what the word "proof" means, or do you have some sort of comprehension difficulties?
 

Hip

Senior Member
Messages
17,824
You call me unhinged? Maybe because I ve broken down so many doors and found out the truth.

I think you are fighting the wrong battle here, Hustler. I am very pleased for you that you have found a treatment that has substantially improved your ME/CFS. And it would be interesting to see if other ME/CFS patients might also benefit from the drugs you used. But you are seeing conspiracies where there are none.
 

Hustler

I m a hustler
Messages
56
Location
Europe
Mikovits understood the limits of PCRs.

Hence she used complementary methods.

50% of the genome has sequences similar to known viruses.

We should never have been put in this situation by all the negative papers and they know that as smart people. Wessely and McClure knew that. They and nobody else used the complementary methods used by classic retrovirologists.

There is no conspiracy.
Just plain conscious stupidity. A conspiracy is done more tactfully. Needs brains.

They caught only themselves out.

Sad but true to say that we are looking at a massive error by multiple teams.
Mikovits was always right.
If u look at the supplementary material appendixed to the Science 2009 paper you see very easily what the negative papers failed to do.
Catastrophy.
 

Hustler

I m a hustler
Messages
56
Location
Europe
No, that's not what the paper says. It says that the PCR method used can amplify certain parts of the animal/host DNA, and produce a false positive for XMRV.

If you think it's saying something else, please quote the relevant section of the paper.

That s why you need complementary methods.
The interest in sequencing only goes so far within known limits.
You need complementary methods.
Nobody did that
Total catastrophy not to listen what Mikovits said and what she wrote too.
 
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Hustler

I m a hustler
Messages
56
Location
Europe
You need complementary methods..
Not insufficient or rigged studies...


controls obviously have many problems in negative studies as they were contaminated with xmrv sequences..
patients are infected/carrying xmrv-related sequences – that is to say mlv related sequences that can infect humans which is actually what xmrv really stands for............

One example, qPCR for xmrv using a taqman probe will come up negative if there is even a single mismatch......
if the patient has one base different in that region, it won’t come up.....

the negative papers use cox2 and IAP as mouse-specific controls; they test these in the presence of lncap dna to show they are specific – that means they are specifically not detecting lncap dna, there may be some humans that they do detect (esp cox2).
IAP retrotransposons, are like little ancient retroviruses themselves – so if you contact mlv enough to catch it, you will also have contacted IAPs as well.

the technology they are using in some negative papers is also way outdated.

The real MLV infection in humans has been completely clouded by improperly run research, open to contamination for example because there have been two rounds of pcr not one round.

The rush to publication was always fraught with danger. Researchers did not live up to their responsibility.

Negative studies out there are just the finest purest manure. Crap crap and crap. Given the public health tragedy this is there can be no excuse.

And thus it will be shown before you show anything.

You want war.
You got it.

They thought they could invent crap and then play and pretend and even stay silent so everyone would forget.

X
MURINE LEUKEMIA RELATED RETROVIRUS
R
V
M
I
K
O
V
I
T
S

vs xmrvsilverman


HOW BLIND AND DEAF CAN YOU FEW PEOPLE REALLY BE ???
They played a very dangerous name again.

Mikovits said XMRVsilverman simply wasnt there. That s what her signature on the Lipkin study attests. Nothing more. Nothing less. For Lipkin to let the impression linger on that there is no Mikovits-retrovirus is PURE FRAUD.

At the elite level you often only have ONE PERSON who is right.

That person was Dr Judy Mikovits
 

Hustler

I m a hustler
Messages
56
Location
Europe
Mikovits understood the limits of PCRs.

Hence she used complementary methods.

50% of the genome has sequences similar to known viruses.

We should never have been put in this situation by all the negative papers and they know that as smart people. Wessely and McClure knew that. They and nobody else used the complementary methods used by classic retrovirologists.

There is no conspiracy.
Just plain conscious stupidity. A conspiracy is done more tactfully. Needs brains.

They caught only themselves out.

Sad but true to say that we are looking at a massive error by multiple teams.
Mikovits was always right.
If u look at the supplementary material appendixed to the Science 2009 paper you see very easily what the negative papers failed to do.
Catastrophy.

Anyone here have a direct line to the cdc ?
We re getting closer. Make it known.
 

Hustler

I m a hustler
Messages
56
Location
Europe
Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with
Chronic Fatigue Syndrome
Vincent C Lombardi, Francis W Ruscetti, Jaydip Das Gupta, Max A Pfost,
Kathryn S Hagen, Daniel L Peterson, Sandra K Ruscetti, Rachel K Bagni, Cari
Petrow-Sadowski, Bert Gold, Michael Dean, Robert H Silverman, Judy A
Mikovits*

Supporting Online Material

Supporting Online Materials and Methods
Patient samples. Banked samples were selected for this study from patients
fulfilling the 1994 CDC Fukuda Criteria for Chronic Fatigue Syndrome (S1) and
the 2003 Canadian Consensus Criteria for Chronic Fatigue Syndrome/myalgic
encephalomyelitis (CFS/ME) and presenting with severe disability. Samples
were selected from several regions of the United States where outbreaks of CFS
had been documented (S2). These are patients that have been seen in private
medical practices, and their diagnosis of CFS is based upon prolonged disabling
fatigue and the presence of cognitive deficits and reproducible immunological
abnormalities. These included but were not limited to perturbations of the 2-5A
synthetase/RNase L antiviral pathway, low natural killer cell cytotoxicity (as
measured by standard diagnostic assays), and elevated cytokines particularly
interleukin-6 and interleukin-8. In addition to these immunological abnormalities,
the patients characteristically demonstrated impaired exercise performance with
extremely low VO2 max measured on stress testing. The patients had been seen
over a prolonged period of time and multiple longitudinal observations of the
clinical and laboratory abnormalities had been documented.
DNA and RNA isolation. Whole blood was drawn from subjects by
venipuncture using standardized phlebotomy procedures into 8-mL green-
capped Vacutainers containing the anti-coagulant sodium heparin (Becton
Dickinson, Franklin Lakes, NJ). Plasma was collected by centrifugation, aspirated
and stored at -80 ºC for later use. The plasma was replaced with PBS and the
blood resuspended and further diluted with an equal volume of PBS. PBMC were
isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare,
Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and
washing it once in PBS. The PBMC (approximately 2 x 107
cells) were
centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS
and 10% DMSO at -80 ºC for further culture and analysis or resuspended in
TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 ºC for DNA and RNA
extraction and analysis. DNA was isolated from TRIzol preps according the to
manufacturer’s protocol and also isolated from frozen PBMC pellets using the
QIAamp DNA Mini purification kit (QIAGEN, Valencia, CA) according to the
manufacturer’s protocol, and the final DNA was resuspended in RNase/DNase-
free water and quantified using the Quant-iT Pico Green dsDNA Kit (Invitrogen,
Carlsbad, CA). RNA was isolated from TRIzol preps according to the
manufacturer’s protocol and quantified using the Quant-iT Ribo Green RNA kit
(Invitrogen, Carlsbad, CA). cDNA was made from RNA using the iScript Select
cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s
protocol.
PCR. To avoid potential problems with laboratory DNA contamination, nested
PCR was performed with separate reagents in a separate laboratory room
designated to be free of high copy amplicon or plasmid DNA. Negative controls
in the absence of added DNA were included in every experiment. Identification
XMRV gag and env genes was performed by PCR in separate reactions.
Reactions were performed as follows: 100 to 250 ng DNA, 2 µL of 25 mM MgCl2,
25 µL of HotStart-IT FideliTaq Master Mix (USB Corporation, Cleveland, OH),
0.75 µL of each of 20 µM forward and reverse oligonucleotide primers in reaction
volumes of 50 µL. For identification of gag, 419F (5’-
ATCAGTTAACCTACCCGAGTCGGAC-3’) and 1154R (5’-
GCCGCCTCTTCTTCATTGTTCTC-3’) were used as forward and reverse
primers. For env, 5922F (5’- GCTAATGCTACCTCCCTCCTGG-3’) and 6273R
(5’-GGAGCCCACTGAGGAATCAAAACAGG-3’) were used. For both gag and
env PCR, 94°C for 4 min initial denaturation was performed for every reaction
followed by 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 1 minute.
The cycle was repeated 45 times followed by final extension at 72°C for 2
minutes. Six microliters of each reaction product was loaded onto 2% agarose
gels in TBE buffer with 1 kb+ DNA ladder (Invitrogen, Carlsbad, CA) as markers.
PCR products were purified using Wizard SV Gel and PCR Clean-Up kit
(Promega, Madison, WI) and sequenced.
PCR amplification for sequencing full-length XMRV genomes was
performed on DNA amplified by nested or semi-nested PCR from overlapping
regions from PBMC DNA. For 5’ end amplification of R-U5 region, 4F (5’-
CCAGTCATCCGATAGACTGAGTCGC-3’) and 1154R was used for first round
and 4F and 770R (5’-TACCATCCTGAGGCCATCCTACATTG-3’) was used for
second round. For regions including gag-pro and partial pol, 350F(5’-
GAGTTCGTATTCCCGGCCGCAGC-3’) and 5135R (5’-
CCTGCGGCATTCCAAATCTCG-3’) was used for first round followed by second
round with 419F and 4789R (5’-GGGTGAGTCTGTGTAGGGAGTCTAA-3’). For
regions including partial pol and env region, 4166F (5’-
CAAGAAGGACAACGGAGAGCTGGAG-3’) and 7622R (5’-
GGCCTGCACTACCGAAAT TCTGTC-3’) were used for first round followed by
4672F (5’GAGCCACCTACAATCAGACAAAAGGAT-3’) and 7590R (5’-
CTGGACCAAGCGGTTGAGAATACAG-3’) for second round. For the 3’ end
including the U3-R region, 7472F (5’-TCAGGACAAGGGTGGTTTGAG-3’) and
8182R (5’-CAAACAGCAAAAGGCTTTATTGG-3’) were used for first round
followed by 7472F and 8147R (5’-CCGGGCGACTCAGTCTATC-3’) for second
round. The reaction mixtures and conditions were as described above except for
the following: For larger fragments, the final extension was done at 68°C for 10
min instead of 72°C for 2 min. All second round PCR products were column
purified as described above and overlapping sequences were determined with
internal primers.
Nested RT-PCR for gag sequences was done as described (5) with
modifications. GAG-O-R primer was used for 1st strand synthesis; cycle
conditions were 52o
C annealing, for 35 cycles. For second round PCR,
annealing was at 54o
C for 35 cycles.
PCR analysis performed on 20 of the identical patient PBMC DNA
specimens stored at the NCI (Frederick, MD) since 2007 confirmed nearly
identical gag sequences, thereby diminishing the possibility of laboratory
contamination as a source of XMRV.

Phylogenetic Analysis. Sequences were aligned using ClustalX (S3). Clustal
alignments were imported into MEGA4 to generate neighbor-joining trees using
the Kimura 2-parameter plus Γ distribution (K80+Γ) distance model (S4). Free
parameters were reduced to the K80 model, and α values were estimated from
the data set using a maximum likelihood approach in PAUP*4.0 (Sinauer
Associates, Inc. Publishers, Sunderland, MA, USA). The bootstrap consensus
tree inferred from 1000 replicates is taken to represent the evolutionary history of
the taxa analyzed. Accession numbers were acquired from GenBank.
(http://www.ncbi.nlm.nih.gov/Genbank): FLV (NC_001940), MoMLV
(NC_001501), XMRV VP35 (DQ241301), XMRV VP42 (DQ241302) XMRV VP62
(EF185282). Genomic Nonecotropic MLV Provirus Sequences were downloaded
from PLOS Genetics (S5).
Isolation, separation and culture of primary cells. Leukopaks of peripheral
blood from healthy donors were collected according to a NIH approved IRB #99-
CC-0168 protocol. Patients’ peripheral blood and plasma samples were from
frozen banked samples obtained under NIH exempt status. Mononuclear
leukocytes from both normal and patients’ cells were isolated by Ficoll-Hypaque
gradient centrifugation. The light density fraction (buffy coat) was collected and
washed twice with PBS. PBMC were activated by 1 µg/mL PHA (Abbott
Diagnostics, Abbott Park, IL) and after 72 hours the cells were cultured with 20
units/mL of IL-2 (Zeptometrix, Buffalo, NY) and subcultured every 3-5 days. For
isolation of CD4+T cells, CD8, CD11b, CD14, CD19, CD33 and CD56 positive
cells were removed using magnetic activated cell sorting (MACs) methods
according to the manufacturer’s instructions (Miltenyi Biotec, Inc., Auburn, CA).
After isolation, the CD3+, CD4+ T cells (>95% pure) were cultured in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM
sodium pyruvate and antibiotics. CD4+
T cells were activated by culturing with 20
units/mL of IL-2 and 1 µg/mL PHA.
In vitro expansion of primary B-cells. NIH 3T3 cells transduced with a
retroviral vector expressing CD40L (gift of Eugene Barsov, NCI-Frederick) were
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen,
Carlsbad, CA) supplemented with 10% calf serum (CS) (Lonza, Basel,
Switzerland) and 1% penicillin, streptomycin and L-glutamine (Invitrogen,
Carlsbad, CA) at 37°C with 5% CO2. To stimulate B cell expansion, ~3.5 x 106
NIH3T3-CD40L cells were trypsinized (0.25% trypsin with EDTA )(Invitrogen,
Carlsbad, CA), resuspended in 3 mL medium and irradiated with an absorbed
radiation dose (rad) of 9600 using a Cesium137 irradiator. Cells plus 7 mL medium
were added to a T25 cell culture flask (Corning, Corning, NY) and allowed to
adhere (2-3 h) to the flask surface (optimal density ~50%).
CD19+ B cells were isolated from PBMC using immunomagnetic bead
technology (Miltenyi Biotec, Auburn, CA). CD19+ cells were separated from 108
freshly isolated PBMC by positive selection according to the manufacturer’s
protocol. After magnetic separation, CD19+ B cells (>95% pure) were added to
an irradiated NIH3T3-CD40L monolayer and incubated at 37 °C with 5% CO2.
Cultures were monitored for B cell proliferation and split 1:5 every 72-96 hr onto
freshly irradiated NIH 3T3-CD40L monolayer. CD19+ primary B cells were
cultured and expanded in primary B cell expansion media: Iscove’s Modified
Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA) + 10% FCS (Atlanta
Biologicals, Lawrenceville, GA), 1% penicillin, streptomycin and L-glutamine
(Invitrogen, Carlsbad, CA), 40 ng/mL interleukin 4 (IL-4) (PeproTech, Inc., Rocky
Hill, NJ), 50 µg/mL holo-transferrin (Sigma, St. Louis, MO) and 5 µg/mL insulin
(Invitrogen, Carlsbad, CA).
Cell culture and reagents. Raji, SupT1 and LNCaP were obtained from
American Type Culture Collection (ATCC, Manassas, VA). The cells were
maintained in RPMI-1640 supplemented with L-glutamine (2 mM), penicillin (100
U/mL), streptomycin (100 ng/mL), and FCS (10%) and subcultured 1:5 every 4-5
days. HCD-57 cells are a mouse erythroleukemia cell line that expresses both
ecotropic and polytropic MLVs; HCD-57/SFFV are HCD-57 cells infected with
SFFV. BaF3-ER cells are a murine pro B cell line engineered to express the
erythropoietin receptor. BaF3ER-SFFV Env cells were derived and maintained
as described (S6).
Flow cytometry for viral proteins. Adherent cells were incubated in trypsin for
10 minutes at 37o
C. After additional washes, adherent and suspension cells
were incubated for 15 min at RT in 1 mL of paraformaldehyde (4% w/v in PBS),
washed in permeabilization wash buffer (0.5% saponin 0.1%, sodium azide, 2%
human AB sera in PBS) (PWB), and resuspended in 300 µL of permeabilization
buffer (PBS with 2.5% saponin) (PB). After incubating at 22o
C for 20 min, 5 mL of
human AB sera and either rat anti-MLV p30 mAb, rat anti-SFFV Env mAb, goat
anti-Rauscher MLV gp70 Env, p30 Gag, or p10 Gag, or the appropriate isotype
control (anti-rat IgG, rat myeloma supernatant, or preimmune goat serum) were
added, and the cells incubated at 4o
C for an additional 30 min. Cells were then
washed in PWB, resuspended in 100 µL of PB with 3 µL (0.6 µg) of FITC-
conjugated goat anti-rat IgG or rabbit anti-goat antibody (BD PharMingen, San
Jose, CA) and incubated for 20 min at 4o
C. The efficiency of permeabilization
was determined using a FITC-conjugated anti-actin antibody. Cells were then
washed twice in PB, resuspended in 500 µL of sheath fluid (BD PharMingen, San
Jose, CA) to prevent clumping and analyzed by flow cytometry. For experiments
in which purified cell populations were examined, cells were stained with an anti-
CD3 or anti-CD19 antibody prior to permeabilization, and analyzed by gating on
the CD3+
or CD19+ subsets.
Western Blot (WB) analysis. Cells were pelleted, washed twice with PBS, and
lysed for 30 min on ice in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
0.25% deoxycholate,1% NP-40, and protease inhibitor cocktail (Sigma, St. Louis,
MO). Debris was removed by centrifugation for 15 min at 21,000x g at 4o
C.
Protein concentration was determined with the Bio-Rad Protein Assay reagent
and equal amounts of protein (70–200 ug) were separated by SDS-PAGE
electrophoresis on 4-20% Tris-Glycine gels (Invitrogen, Carlsbad, CA) and then
transferred to Immobilon-P membranes (Millipore, Billerica, MA). The
membranes were blocked with 5% non-fat dry milk/1x TBST (Tris-buffered saline
with 0.1% Triton X-100) for 1 h at room temperature, hybridized with the
appropriate antiserum diluted in 5% non-fat dry milk/1xTBST for 2 h at room
temperature or overnight at 4o
C, washed twice with 1xTBST, hybridized with the
appropriate horseradish-peroxidase conjugated antibody diluted 1:5000 for 1 h at
room temperature, and washed three times with 1xTBST. Hybridized bands
were visualized using HyGlo chemiluminescent HRP antibody detection reagent
(Denville Scientific, Metuchen, NJ) and exposed to film (Kodak, Rochester, NY).
Antibodies used were a rat monoclonal Ab to SFFV gp55 Env (7C10), diluted
1:100 and detected with peroxidase-labeled anti-rat secondary antibody
(Amersham, Waukesha, WI); goat anti-Rauscher MLV gp70 Env, p30 Gag and
p10 Gag (provided by NCI); and goat anti-NZB xenotropic MLV (provided by
NCI), all diluted 1:2500 and detected with peroxidase labeled anti-goat
secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Viral transmission. Frozen cell-free plasma and 0.22 µm filtered cell free
supernatants from PBMC and T cell cultures were diluted 1:1 with tissue culture
media and 600 µL aliquots were added to a six-well culture plate with the LNCaP
cell line (50% confluent) or a million primary activated CD4+ T cells isolated from
healthy donors. The plates were centrifuged for 5 min at 1500 RPM, rotated 180o
and centrifuged again for 5 min. The entire cycle was repeated once and cells
were then diluted in their growth media. For cell-cell transmission, 1 x 106 T cells
or PBMC without any IL-2 in the growth media were added to a six-well culture
plate with the LNCaP cell line (50% confluent) in 1 mL of media for 3 h. After 1
hr, T cells in suspension were removed and the LNCaP cells were grown for
several passages in the absence of IL-2 which caused any remaining T cells to
die. At the times after transmission indicated, protein analysis was done by
western blot and flow cytometry.
Genotyping. The rs486907 R462Q SNP was genotyped using Applied
Biosystems’ Taqman® 5' nucleotidase assays, Taqman® Universal PCR Master
Mix: No AmpErase UNG, and 5 ng of genomic DNA. The thermal cycling
conditions consisted of an initial hold at 95o C for 10 minutes followed by 50
cycles of a 15 second 95o C denaturation step and a one minute 60o C annealing
and extension step. A 7900HT instrument was used to detect fluorescent
probes, and Sequence Detection Software (SDS) v2.2 was used to discriminate
alleles and call genotypes (Applied Biosystems, Foster City, CA). The variant is
in Hardy-Weinberg equilibrium in both cases and controls. A Chi square test was
performed for both genotypes and alleles of RNASEL comparing XMRV negative
and XMRV positive controls. Both tests were not significant and the allele test is
displayed in Table S2. Homozygous R462Q variant of RNASEL is represented in approximately 13% of the human population (S6, S7).
Flow cytometry for detection of antiviral antibodies in CFS plasma. The
murine cell lines BaF3ER and BaF3ER-SFFV Env (S8) were grown in 2 units/ml
of Epo in RPMI 1640 plus 7% FCS. 500,000 cells per sample in log phase were
used as targets for direct staining. Cell lines were first washed in wash buffer (2%
FBS, 0.02% Na Azide, PBS) and resuspended in 200 µL of BSA staining buffer
(BD PharMingen, San Jose, CA). Patient plasma was thawed rapidly and used at
20 µL or 2 µL per tube (1:10 and 1:100 respectively) and incubated at 4o
C or on
ice for 30 minutes. Cells were then washed with 0.5mL of wash buffer. Tubes
were centrifuged at 800 rpm for 5 minutes, the supernatant was removed and
tubes blotted on a towel. Next, 100 µL of the following working solution was
added: 5 µL human A/B sera, 1 µL biotin labeled anti-human IgG (for human
plasma) or biotin-labeled anti-rat IgG (for SFFV Env mAb)(Ebioscience, San
Diego, CA), 1 µL of strep/avidin phycoerythrin (PE), 94 µL cold staining buffer.
Samples were then incubated at 4o
C for 20 minutes, washed with 0.5 mL of wash
buffer, and spun at 800 rpm for 5 minutes before being analyzed by flow
cytometry. For the competition experiments, 100 µL of cold staining buffer and 10
µL of human plasma were added to each tube prior to addition of either anti-
SFFV Env mAb (7C10) or Y3 myeloma supernatant (control). Samples were
incubated at 4o
C or on ice for 20 minutes, washed with 0.5 mL of wash buffer
and spun at 800 rpm for 5 minutes before being analyzed by flow cytometry.
Legends:
Figure S1. Gag sequences of XMRV in CFS patients. Partial sequences (nt 649-
1017) from CFS XMRV strains WPI-1130, WPI-1138 and WPI-1169 in
comparison to XMRV strains VP35, VP42 and VP62 derived from prostate
cancer patients. The yellow highlighting denotes the differences from the
reference strain (VP62).
Figure S2. Phylogenetic Analysis of XMRV in CFS patients. Neighbor-joining
analysis of CFS XMRV strains WPI-1104, WPI-1106 and WPI-1178 with
previously identified XMRVs and the nonecotropic MLVs xenotropic (Xmv),
polytropic (Pmv) and modified polytropic (Mmpv) (S5). Ecotropic MLVs FLV and
MoMLV were included to root the tree as outgroups in the phylogenetic
reconstruction. Bootstrap supports of >70% are shown next to branch nodes.
The scale measures evolutionary distance in substitutions per nucleotide.
Subgroup designations are marked with arrows. XMRVs (WPI and VPs) form a
distinct clade clustering together and separately from the xenotropic (Xmv)
proviruses (shown in red).
Figure S3. Detection of cloned XMRV-VP62 using a rat mAb to SFFV Env and a
goat antiserum to mouse NZB xenotropic MLV. A. Lysates were prepared from
XMRV-VP62-infected Raji (lane1), LNCaP (lane 2) or Sup-T1 (lane 3). Positive
controls used were HCD-57 cells, a mouse erythroleukemia cell line expressing
polytropic MLV gp70 Env (lane 4), and HCD-57 cells infected with SFFV, which
also express SFFV gp55 Env (lane 5). WB analysis was carried out using rat
anti-SFFV Env mAb 7C10. Molecular weight markers in kD are shown on the
left. B. Lysates were prepared from XMRV-VP62-infected Raji (lane 1), LNCaP
(lane 2) or Sup-T1 (lane 3). Lysates from SFFV-infected mouse HCD-57 cells
(lane 4) and from uninfected Raji, LNCaP and Sup-T1 are shown in lanes 5-7,
respectively. WB was carried out using goat antiserum to mouse NZB xenotropic
MLV. Molecular weight markers in kD are shown on the left.
Figure S4. Expression of XMRV proteins in PBMC from CFS patients. A.
Activated B cells from CFS patient WPI-1125, activated T cells from CFS patient
WPI-1105 or normal activatedT cells were incubated with goat antisera (black
area) against Rauscher MLV gp70 Env (top), p30 Gag (middle) and p10 Gag
(bottom) and analyzed by IFC. Preimmune goat serum (light area) was used as a
control. B. A B cell line from a CFS patient was incubated with rat anti-SFFV Env
mAb (right panel) or control myeloma supernatant (left panel) and then analyzed
by IFC. C. Lysates were prepared from B cells (lane 1) or T cells (lanes 2 and 3)
from CFS patients that had been grown for 42 days on CD40L or IL-2
respectively were analyzed by WB using rat anti-SFFV Env mAb (top panel) or
goat anti-NZB xenotropic MLV serum (bottom panel). Lane 4: normal T cells;
Lane 5: mouse HCD-57 cells; Lane 7: SFFV-infected HCD-57 cells. Molecular
weight markers in kD are shown on the left.
Figure S5: Infectious XMRV in CFS patients’ PBMC and plasma. A. The
indicated T-cell cultures from CFS patients were co-cultured with LNCaP as
described in the Methods. XMRV p30 Gag expression was detected in the
LNCaP cells using a rat anti-MLV p30 Gag mAb and IFC. Bottom panel: LNCaP
co-cultured with normal T cells. B. Plasma from the indicated CFS patients was
co-cultured with LNCaP. At the second passage, XMRV p30 Gag expression in
the LNCaP cells was detected by flow cytometry using a rat anti-MLV p30 Gag
monoclonal Ab. Co-culture with plasma from a normal healthy donor is shown in
the bottom panel.
Figure S6: Presence of antibodies in CFS plasma that recognize the cell
surface of SFFV Env expressing BAF3ER cells. A. Plasma from CFS patients
or normal healthy controls was diluted 1:10, reacted with BaF3-ER or BaF3ER-
SFFV Env cells and analyzed by IFC. Shown is the difference in mean
fluorescence intensity (MFI) between CFS and control plasma direct binding to
BaF3ER-SFFV Env cells versus BaF3ER (control) cells. B. Competition
experiment, carried out as described in the Methods, showing that plasma from a
CFS patient can block binding of a rat anti-SFFV Env mAb to BaF3ER-SFFV Env
cells. Left panel: CFS plasma diluted 1:10 (white area) eliminates most of the
anti-SFFV Env binding (striped area) and overlaps with the negative control
(black area). Right panel: CFS plasma diluted 1:100 (white area) eliminates less
of the anti-SFFV Env binding (striped area) and overlaps much more with the
positive than the negative control (black area).
 

Hustler

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Thereafter follow the phylogenetic trees clearly showing XMRV was never just one virus.

Those who believed Coffin + Lipkin were totally fooled
 

Hustler

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Dr Judy Mikovits responds to the ‘Science’ editorial expression of concern, 30 May 2011

May 30, 2011

Dr. Bruce Alberts
Editor-in-Chief
Ms. Monica Bradford
Executive Editor Science
1200 New York Avenue, NW
Washington, DC 20005


Dear Dr. Alberts and Ms. Bradford:

As the corresponding author of the Lombardi et al. study I want to express my deepest concern about the proposed issuance of your editorial expression of concern regarding our XMRV findings and its association with chronic fatigue syndrome. This is especially so in light of the gross disregard for the integrity of the scientific process by the apparent willful breach of your embargo by one of the authors or their collaborators. This has resulted in the apparent public knowledge of the contents of your request that we retract our seminal paper. I would respectfully ask that you focus on the following key facts and reconsider your position. We share your deep concern over the number of negative non-replication studies in this new area of research. However, the publication of your editorial expression of concern over the validity of Lombardi et al. findings are premature and would have a disastrous impact on the future of this field of science. Please do not proceed down a path that could be detrimental to the scientific exploration of human retroviruses in infectious disease, cancer, and, therefore, the future health of millions around the world.

First, the title and substance of the Lombardi et al. study Detection of an Infectious retrovirus, XMRV, in blood cells of patients with Chronic Fatigue Syndrome is accurate, and not one reported study has been able to show why it is not. Using four different methods including PCR (of cultured and co-cultured cells), detection of human gammaretroviral (HGRV) viral proteins (culture and co-culture detected by Western Blot and flow cytometry), anti-gammaretrovirus Env antibodies in human serum (competed by 7C10 rat monoclonal antibody), and virus isolation from primary cell and co-cultures, we reported evidence of human gammaretrovirus infection in at least 67 out of 101 CFS patients. In addition, we reported that 3.7% of the control population had evidence of infection.

Second, this significant study was conducted over eight months and conducted in five different laboratories. It resulted in the first isolation of a human gammaretrovirus from the blood of humans and concluded that this virus may be a contributing factor in the pathogenesis of CFS. Electron micrographs of gammaretroviruses isolated from patients cells were shown in the Lombardi et al. study and support these conclusions. These electron micrographs do not show VP62 plasmid contamination. In addition, we have maintained the viral isolates of five patients from which the electron micrographs were derived. Moreover, data presented in Lombardi et al. suggested additional strains of gammaretroviruses and viral Gag proteins could be directly immunoprecipitated from the blood of patients supporting a finding that additional strains of HGRV were isolated. In fact, subsequent work by Jones et al. presented at Cold Spring Harbors supports the presence of more than one strain of HGRV. The original manuscript submitted to Science discussed DG75, a human B cell line, from which a MLV-related virus was fully sequenced. This raises the possibility of many viruses originating from recombination events as human tissue has been passed through mice for more than five decades. PCR would not have detected a DG75 isolate but the rat monoclonal Env antibody used in these studies can detect DG75 isolates. This raises the question of how many gammaretroviruses are circulating in the human population with the potential of contributing to human disease.

Third, this study showed that human gammaretrovirus was transmissible, and a more recent study has confirmed these data in an animal model and has shown that there could be different routes of entry and difference in blood reservoirs between acute and chronic infection.

Fourth, this study conducted the following tests to insure that the reported data were not as a result of contamination, including the detection of a human antibody response to the virus, the screening of all reagents and cell lines for any evidence of gammaretrovirus contamination, human or otherwise. The antibodies used to detect viral proteins in Lombardi et al. were rat monoclonal and goat polyclonal antisera, all of which were negative for murine contamination; all reagents used in PCR and tissue culture were lot tested for contamination. In addition, it was clearly stated in the Supplemental Methods which taq enzyme manufactures were shown to be contamination free. None of the negative papers, which demonstrated contamination, used the enzymes or antibody reagents used and recommended by Lombardi et al.

All samples and controls were processed in the exact same way and placed in a clean lab free from any other cell line. Only five human cell lines were grown in the WPI laboratories during the time these studies were conducted: Raji, SupT1, HFF, LNCaP and HSB2 and all were shown at the initiation of and throughout these studies to be free of XMRV/VP62 and all were used as negative control tested weekly by every method (including pelleting of supernatant over glycerol for virus isolation).

No murine cell line was grown in the WPI labs prior to the submission of the Lombardi et al. manuscript. The murine BAF cell line was cultured and used after the July 22, 2009 NCI closed meeting on XMRV during which all of the data of Lombardi et al. was shown not only to one of the reviewers of the original manuscript but also to John Coffin who wrote the accompanying commentary. We were requested at that time to run the mouse mitochondrial assay to show absence of mouse contamination. We conducted this assay on samples from all 101 patients in Lombardi et al. and published these data in the subsequent Virulence addenda, a copy of which is attached hereto. While we have been advised by you that Paprotka et al. suggest a recombinant origin of an XMRV, it says nothing about the human gammaretroviruses detected and isolated from patient samples in Lombardi et al. They cannot have any data to support the conclusion “that laboratory contamination with XMRV produced by a cell line (22Rv1) derived from these early xenograft experiments is the most likely explanation for detection of the virus in patient samples.” In fact, the authors of this paper know full well that this explanation cannot explain XMRV integration in human tissue, in situ hybridization, or antibodies reported in prostate cancer or CFS patients. Furthermore, all strains of wild rodents have not been examined and other examples of ancestral XMRV can be found. Neither 22Rv1 nor any of the cell lines reported to be contaminated with XMRV or cell lines growing the VP62 infectious molecularly cloned virus was in the laboratories where the patient cells were isolated. This can also not in any way explain the Env antibodies demonstrated in patient plasma in Lombardi et al. The reactivity demonstrated to Spleen Focus Forming Virus (SFFV) Env was competed by the rat monoclonal antibody which detects all known xenotropic, polytropic and ecotropic MLVs. This again suggests that we have, in fact, detected more than one strain of human gamma retroviruses in these patient samples. Clearly data presented in Lombardi et al. where samples were PCR negative but Western blot positive, using the 7C10 antibody, further support the notion of a family of gammaretroviruses. These data must be appreciated as a complete body of evidence and not in the context of individual pieces, such as PCR amplification using primers designed to an arbitrary reference strain.

All of these data led Harvey Alter, in the NIH State of the Knowledge Workshop (April 2011), to draw the conclusion that there existed no evidence of contamination in ‘either the Mikovits or Lo labs’.

The authors are aware of ten negative CFS papers listed in PubMed on the subject of XMRV. Most of the negative studies failed to find any evidence of XMRV in any sample type. This would suggest that the methods and materials used in the non-replication studies are insufficient to use when attempting to detect human gammaretrovirus in the blood of human samples. The methods, processes, and materials of Lombardi et al. need to be followed precisely. The Alter and Lo study is the only study which has attempted a partial replication of the methods and materials of the Lombardi study, which confirmed evidence of MLV related viruses. Studies using multiple different methods are not replication studies, and studies optimized to detect murine gammaretroviruses and not human gammaretroviruses must be seriously questioned. See, Virulence attachment.

Scientific research of human gammaretroviruses is in its infancy. Studies of XMRV and macaques are beginning to reveal information concerning the life cycle of the virus, multiple tissue reservoirs, and a description of factors that induce viral activation. These studies are critical to understanding gammaretroviruses in human disease. Other human studies such as the one by Fischer et al., reported the detection of XMRV in the respiratory tract of immunocompromised individuals pointing to the potential for gammaretroviruses to be more easily transmitted than all other known retroviruses. WPI researchers are contributing to the development of more accurate clinical testing methods with others in the blood working group. Without the participation of Drs. Alter, Lo and the WPI, who have proven gamma retroviral detection methods, it may be impossible to discover whether or not gammaretroviruses are a threat to human transfusion and transplantation medicine.

In summary, human retroviruses are not known to infect individuals according to their sex or age therefore there can be no excuse as to why it would be acceptable to study the viruses in cancer but not in those with infectious neuro-immune diseases. They create lifelong infection in their hosts by integrating into the genome of their victims. Thirty years of murine gammaretroviral research provide compelling evidence that these viruses cause immune deficiencies, neurological disease and cancer in mammals and are therefore possible contributors to human neuro-immune diseases such as CFS. However, good scientific work is difficult and takes time. These ongoing studies deserve to receive a fair and impartial evaluation in the peer-review process. The critical question which remains is not simply whether gammaretroviruses play a role in CFS or cancer but in how many other human diseases? Therefore, we feel this is an extremely premature action which is not in the best interest of the scientific community or human health and again we respectfully request that you allow the scientific process to run its course unhindered by bias.

Thank you for your thoughtful consideration of this matter.

Sincerely yours,

Dr. Judy A Mikovits
 
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https://laikaspoetnik.wordpress.com...ence-of-xmrv-in-chronic-fatigue-syndrome-cfs/

The PCR conditions are badly reported in the WPI paper, published in Science[1]. As a matter of fact I wonder how it ever came through the review.

Unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.

The sensitivity of the PCR is not shown (nor discussed).

No positive control is mentioned. The negative controls were just vials without added DNA.

Although the PCR is near the detection limit, only first round products are shown (without confirmation of the identity of the product). The positive bands are really strong, whereas you expect them to be weak (near the detection limit after two rounds). This is suggestive of contamination.

PBMC have been used as a source and that is fine, but one of WPI’s open letters/news items (Feb 18), in response to the first UK study, says the following:

point 7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells. (emphasis mine)

But carefully reading the methods, mentioned in the “supporting material” I only read:

The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 ºC for further culture and analysis or resuspended in TRIzol (…) and stored at -80 ºC for DNA and RNA extraction and analysis. (emphasis mine)

Either …. or. Seems clear to me that the PBMC were not cultured for PCR, at least not in the experiments described in the science paper.

How can one accuse other scientists of not “duplicating” the results if the methods are so poorly described and the authors don’t adhere to it themselves??

Strikingly only those PCR-reactions are shown, performed by the Cleveland Clinic (using one round), not the actual PCR-data performed by WPI. That is really odd.

It is also not clear whether the results obtained by the various tests were consistent.

Suzanne D. Vernon, PhD, Scientific Director of the CFIDS Association of America (charitable organization dedicated to CFS) has digged deeper into the topic. This is what she wrote [9]:

Of the 101 CFS subjects reported in the paper,results for the various assays are shown for only 32 CFS subjects. Of the 32 CFS subjects whose results for any of the tests are displayed, 12 CFS subjects were positive for XMRV on more than one assay. The other 20 CFS subjects were documented as positive by just one testing method. Using information from a public presentation at the federal CFS Advisory Committee, four of the 12 CFS subjects (WPI 1118, 1150, 1199 and 1125) included in the Science paper were also reported to have cancer – either lymphoma, mantle cell lymphoma or myelodysplasia. The presentation reported that 17 WPI repository CFS subjects with cancer had tested positive for XMRV. So how well are these CFS cases characterized, really?

Summary and Conclusion

In a recent publication in Science, Lombardi and co-authors from the WPI reported the detection of XMRV-related, a novel retrovirus that was first identified in prostate cancer samples.

Their main finding, presence of XMRV in peripheral blood cells could not be replicated by 3 other studies, even under sensitive PCR conditions.

The original Science study has severe flaws, discussed above. For one thing WPI doesn’t seem to adhere to the PCR to test XMRV any longer.

It is still possible that XMRV is present in amounts at or near the detection limit. But it is equally possible that the finding is an artifact (the paper being so inaccurate and incomplete). And even if XMRV was reproducible present in CFS patients, causality is still not proven and it is way too far to offer patients “diagnostic tests” and retroviral treatment.

Perhaps the most worrisome part of it all is the non-scientific attitude of WPI-employees towards colleague-scientists, their continuous communication via press releases. And the way they try to directly reach patients, who -i can’t blame them-, are fed up with people not taking them serious and who are longing for a better diagnosis and most of all a better treatment. But this is not the way.
 

Hustler

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Kina
I have addressed and torn apart all your questions and posts on this. I answered everything with my posts previously about your criticisms. Just what kind of impression are u trying to give?
Take a holiday.
Chill out,a little.
You sound like you need it.
Ask the cdc for a ticket to the Caribbean !
I m sure they can do you that favour !
Have a cocktail !
Put your feet up !
And bill it on the cdc s accounts !!!
 
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