the article

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In a message dated 8/3/00 10:56:36 PM Eastern Daylight Time, SMILING GAIL

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<< In a message dated 8/3/00 10:35:07 PM Eastern Daylight Time,

ckc@... writes:

<< Down Syndrome/Autism - dual diagnosis

by Cyndi Ringoen B.A., B.S., copyright 1999

>>

Ok, what am I missing here? It makes sense to me, other than stopping the

stimming. That's impossible unless there is someone with them constantly. I

read it twice and didn't find anything that upset me. This is what we did

with Seth and saw a great improvement in him. I am guilty of letting him

have his shoe when he is in a stressful situation. Someone will have to

spell it out for me I guess! LOL

Gail, Mom to; Seth-4, jo-8, Becky-9, -23, Jen-24, Grandma to

Errick-4

and wife to -my hero

>>

[Guest guest]

/ www.sciencexpress.org / 8 October 2009 / Page 1 /

10.1126/science.1179052

Chronic fatigue syndrome (CFS) is a debilitating disease

of unknown etiology that is estimated to affect 17 million

people worldwide. Studying peripheral blood

mononuclear cells (PBMCs) from CFS patients, we

identified DNA from a human gammaretrovirus,

xenotropic murine leukemia virus-related virus (XMRV),

in 68 of 101 patients (67%) compared to 8 of 218 (3.7%)

healthy controls. Cell culture experiments revealed that

patient-derived XMRV is infectious and that both cellassociated

and cell-free transmission of the virus are

possible. Secondary viral infections were established in

uninfected primary lymphocytes and indicator cell lines

following exposure to activated PBMCs, B cells, T cells, or

plasma derived from CFS patients. These findings raise

the possibility that XMRV may be a contributing factor in

the pathogenesis of CFS.

Chronic fatigue syndrome (CFS) is a disorder of unknown

etiology that affects multiple organ systems in the body.

Patients with CFS display abnormalties in immune system

function, often including chronic activation of the innate

immune system and a deficiency in natural killer (NK) cell

activity (1, 2). A number of viruses, including ubiquitous

herpesviruses and enteroviruses have been implicated as

possible environmental triggers of CFS (1). Patients with CFS

often have active ï¢ herpesvirus infections, suggesting an

underlying immune deficiency.

The recent discovery of a gammaretrovirus, XMRV, in the

tumor tissue of a subset of prostate cancer patients prompted

us to test whether XMRV might be associated with CFS.

Both of these disorders, XMRV-positive prostate cancer and

CFS, have been linked to alterations in the antiviral enzyme

RNase L (3–5). Using the Whittemore Institute's

(WPI) national tissue repository, which contains samples

from well-characterized cohorts of CFS, we isolated nucleic

acids from PBMCs and assayed the samples for XMRV gag

sequences by nested PCR (5, 6). Of the 101 CFS samples

analyzed, 68 (67%) contained XMRV gag sequence.

Detection of XMRV was confirmed in 7 of 11 WPI CFS

samples at the Cleveland Clinic by PCR-amplifying and

sequencing segments of XMRV env (352 nt) and gag (736 nt)

in CFS PBMC DNA (Fig. 1A) (6). In contrast, XMRV gag

sequences were detected in 8 of 218 (3.7%) PBMC DNA

specimens from healthy individuals. Of the 11 healthy control

DNA samples analyzed by PCR for both env and gag, only

one sample was positive for gag and none for env (Fig. 1B).

In all positive cases, the XMRV gag and env sequences were

more than 99% similar to those previously reported for

prostate tumor-associated strains of XMRV (VP62, VP35,

and VP42) (fig. S1) (5).

Sequences of full-length XMRV genomes from two CFS

patients and a partial genome from a third patient were

generated (table S1). CFS XMRV strains 1106 and 1178 each

differed by six nucleotides (nt) from the reference prostate

cancer strain XMRV VP62 (EF185282), and with the

exception of one nt, the variant nucleotides mapped to

different locations within the XMRV genome, suggesting

independent infections. By comparison, prostate cancerderived

XMRV strains VP35 and VP42 differed from VP62

by 13 and 10 nt, respectively. Thus, the complete XMRV

genomes in CFS patients are > 99% identical in sequence to

those detected in patients with prostate cancer. To exclude the

possibility that we were detecting a murine leukemia virus

Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients

with Chronic

Fatigue Syndrome

C. Lombardi,1* Francis W. Ruscetti,2* Jaydip Das Gupta,3 Max A.

Pfost,1 S. Hagen,1 L. ,1

K. Ruscetti,4 K. Bagni,5 Cari Petrow-Sadowski,6 Bert

Gold,2 Dean,2 H. Silverman,3 Judy A.

Mikovits1†

1Whittemore Institute, Reno, NV 89557, USA. 2Laboratory of

Experimental Immunology, National Cancer Institute-

Frederick, Frederick, MD 21701, USA. 3Department of Cancer Biology, The

Lerner Research Institute, The Cleveland Clinic

Foundation, Cleveland, OH 44106, USA. 4Laboratory of Cancer Prevention,

National Cancer Institute-Frederick, Frederick, MD

21701, USA. 5Advanced Technology Program, National Cancer

Institute-Frederick, Frederick, MD 21701, USA. 6Basic

Research Program, Scientific Applications International Corporation,

National Cancer Institute-Frederick, Frederick, MD

21701, USA.

*These authors contributed equally to this work.

†To whom correspondence should be addressed. E-mail:

judym@...

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10.1126/science.1179052

(MLV) laboratory contaminant, we determined the

phylogenetic relationship between endogenous (nonecotropic)

MLV sequences, XMRV sequences, and sequences from CFS

patients 1104, 1106 and 1178 (fig. S2). XMRV sequences

from the CFS patients clustered with the XMRV sequences

from prostate cancer cases and formed a distinct branch from

nonecotropic MLVs common in inbred mouse strains. Thus,

the virus detected in the CFS patients' blood samples is

unlikely to be a contaminant.

To determine whether XMRV proteins were expressed in

PBMCs from CFS patients, we developed intracellular flow

cytometry (IFC) and Western blot (WB) assays, using

antibodies (Abs) with novel viral specificities. These

antibodies included among others: (i) rat monoclonal

antibody (mAb) to the spleen focus-forming virus (SFFV)

envelope (Env), which reacts with all polytropic and

xenotropic MLVs (7), (ii) goat antisera to whole mouse NZB

xenotropic MLV; and (iii) a rat mAb to MLV p30 Gag (8).

All of these Abs detected the human VP62 XMRV strain

grown in human Raji, LNCaP and Sup-T1 cells (fig. S3) (5).

IFC of activated lymphocytes (6, 9) revealed that 19 of 30

PBMC samples from CFS patients reacted with the anti-MLV

p30 Gag mAb (Fig. 2A). The majority of the 19 positive

samples also reacted with antisera to other purified MLV

proteins (fig. S4A). In contrast, 16 healthy control PBMC

cultures tested negative (Fig. 2A, fig. S4A). These results

were confirmed by Western blots (Fig. 2B and C) (6) using

Abs to SFFV Env, mouse xenotropic MLV and MLV p30

Gag. Samples from five healthy donors exhibited no

expression of XMRV proteins (Fig. 2C). The frequencies of

CFS cases vs. healthy controls that were positive and negative

for XMRV sequences were used to calculate a Pearson ï£2

value of 154 (two-tailed P value of 8.1 ï‚´ 10–35). These data

yield an odds ratio of 54.1 (95% confidence interval of 23.8-

122), suggesting a non-random association with XMRV and

CFS patients.

To determine which types of lymphocytes in blood express

XMRV, we isolated B and T cells from one patient's PBMCs

(6). Using mAb to MLV p30 Gag and IFC, we found that

both activated T and B cells were infected with XMRV (Fig.

2D, fig. S4A). Furthermore, using mAb to SFFV Env, we

found that > 95% of the cells in a B-cell line developed from

another patient were positive for XMRV Env (Fig. S4B).

XMRV protein expression in CFS patient-derived activated T

and B cells grown for 42 days in culture was confirmed by

Western blots (fig. S4C) using Abs to SFFV Env and

xenotropic MLV.

We next investigated whether the viral proteins detected in

PBMCs from CFS patients represent infectious XMRV.

Activated lymphocytes (6) were co-cultured with LNCaP, a

prostate cancer cell line with defects in both the JAK-STAT

and the RNase L pathways (10, 11) that was previously

shown to be permissive for XMRV infection (12). After coculture

with activated PBMCs from CFS patients, LNCaP

cells expressed XMRV Env and multiple XMRV Gag

proteins by Western blot (Fig. 3A) and IFC (fig. S5A).

Transmission electron microscopy (EM) of the infected

LNCaP cells (Fig. 3B) as well as virus preparations from

these cells (Fig. 3C) revealed 90-100 nm diameter budding

particles consistent with a gamma (type C) retrovirus (13).

We also found that XMRV could be transmitted from CFS

patient plasma to LNCaP cells when we applied a virus

centrifugation protocol to enhance infectivity (6, 14, 15).

Both XMRV gp70 Env and p30 Gag were abundantly

expressed in LNCaP cells incubated with plasma samples

from 10 of 12 CFS patients, whereas no viral protein

expression was detected in LNCaP cells incubated with

plasma samples from 12 healthy donors (Fig. 3A). Likewise,

LNCaP cells incubated with patient plasma tested positive for

XMRV p30 Gag in IFC assays (fig. S5B). We also observed

cell-free transmission of XMRV from the PBMCs of CFS

patients to the T-cell line SupT1 (Fig. 4B) and both primary

and secondary transmission of cell-free virus from the

activated T cells of CFS patients to normal T cell cultures

(Fig.4C). Together, these results suggest that both cellassociated

and cell-free transmission of CFS-associated

XMRV are possible..

We next investigated whether XMRV stimulates an

immune response in CFS patients. For this purpose, we

developed a flow cytometry assay that allowed us to detect

antibodies to XMRV Env by exploiting its close homology to

SFFV Env (16). Plasma from 9 out of 18 CFS patients

infected with XMRV reacted with a mouse B cell line

expressing recombinant SFFV Env (BaF3ER-SFFV-Env) but

not to SFFV Env negative control cells (BaF3ER), analogous

to the binding of the SFFV Env mAb to these cells (Fig. 4D

and S6A). In contrast, plasma from seven healthy donors did

not react (Fig. 4D and fig. S6A). Furthermore, all nine

positive plasma samples from CFS patients but none of the

plasma samples from healthy donors blocked the binding of

the SFFV Env mAb to SFFV Env on the cell surface (fig.

S6B). These results are consistent with the hypothesis that

CFS patients mount a specific immune response to XMRV.

Neurological maladies and immune dysfunction with

inflammatory cytokine and chemokine upregulation are some

of the most commonly reported features associated with CFS.

Several retroviruses, including the MLVs and the primate

retroviruses, HIV and HTLV-1, are associated with

neurological diseases as well as cancer (17). Studies of

retrovirus-induced neurodegeneration in rodent models have

indicated that vascular and inflammatory changes mediated

by cytokines and chemokines precedes the neurological

pathology (18, 19). The presence of infectious XMRV in

lymphocytes may account for some of these observations of

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10.1126/science.1179052

altered immune responsiveness and neurological function in

CFS patients.

In summary, we have discovered a highly significant

association between the XMRV retrovirus and CFS. This

observation raises several important questions. Is XMRV

infection a causal factor in the pathogenesis of CFS or a

passenger virus in the immunosuppressed CFS patient

population? What is the relationship between XMRV

infection status and the presence or absence of other viruses

that are often associated with CFS (e.g., herpesviruses)?

Conceivably these viruses could be cofactors in pathogenesis,

as is the case for HIV-mediated disease, where co-infecting

pathogens play an important role (20). Patients with CFS

have an elevated incidence of cancer (21). Does XMRV

infection alter the risk of cancer development in CFS? As

noted above, XMRV has been detected in prostate tumors

from patients expressing a specific genetic variant of the

RNASEL gene (5). In contrast, in our study of this CFS

cohort, we found that XMRV infection status does not

correlate with the RNASEL genotype (6) (table S2).

Finally, it is worth noting that 3.7% of the healthy donors

in our study tested positive for XMRV sequences. This

suggests that several million Americans may be infected with

a retrovirus of as yet unknown pathogenic potential.

References and Notes.

1. L. D. Devanur, J. R. Kerr, J. Clin. Virol. 37, 139 (2006).

2. T. L. Whiteside, D. Friberg, Am. J. Me.d 105, 27S (1998).

3. R. J. Suhadolnik et al., J. Interferon Cytokine Res. 17, 377

(1997).

4. G. Casey et al., Nat. Genet. 32, 581 (2002).

5. A. Urisman et al., PLoS Pathog. 2, 211 (2006).

6. Materials and methods are available as supporting material

on Science Online.

7. Wolff, R. Koller, S. Ruscetti, J. Virol. 43, 472 (1982)

8. B. Chesebro et al., Virology 127, 134 (1983).

9. K.A and F. W. Ruscetti, Adv. Immunol. 31,137

(1981).

10. G. Dunn, K. Sheehan, L. Old, R. Schreiber, Cancer Res.

65, 3447 (2005)

11. Y. Xiang et al., Cancer Res. 63, 6795 (2003).

12. B. Dong et al., Proc. Natl. Acad. Sci. U.S.A. 104, 1655

(2007).

13. B. J. Poiesz et al., Proc. Natl. Acad. Sci. U.S.A. 77, 7415

(1980).

14. G. R. Pietroboni, G. B. Harnett, M. R. Bucens, J. Virol.

Methods 24, 85 (1989).

15. S. M. Yoo et al., J. Virol. Methods 154, 160 (2008).

16. L. Wolff, E. Scolnick, S. Ruscetti, Proc. Natl. Acad. Sci.

U.S.A. 80, 4718 (1983)

17. C. Power., Trends Neurosci. 24, 162 (2001).

18. X. Li, C., Hanson. J. Cmarik, S. Ruscetti J. Virol. 83,

4912 (2009).

19. K.E. ., B Chesebro Curr. Top. Microbiol.

Immunol. 303, 67 (2006).

20. A. Lisco., C. Vanpouille., L. Margolis Curr. HIV/AIDS

Rep. 6, 5(2009)

21. P. H. Levine et al., Cancer Res. 52, 5516s (1992).

22. We thank D. Bertolette, Y. Huang, C. Hanson and J.

Troxler for expert technical assistance; K. Nagashima for

electron microscopy; and C. Ware and K. Hunter for

thoughtful discussions. Funded by the Whittemore

Institute and the Whittemore Family Foundation;

the National Cancer Institute; the National Institutes of

Health (under contract HHSN26120080001E); and grants

to R.H.S. from NCI/NIH (CA104943), the U.S.

Department of Defense Prostate Cancer Research Program

W81XWH-07-1338, V Foundation for Cancer Research,

Charlotte Geyer Foundation, and Mal and Lea Bank. The

content of this publication does not reflect the views or

policies of the Department of Health and Human Services,

nor does mention of trade names, commercial products, or

organizations imply endorsement by the U.S. Government.

R.H.S. may receive royalty payments in the future from

Abbott Laboratories, Inc. GenBank accession numbers are

as follows: WPI-1130, GQ483508; WPI-1138, GQ483509;

WPI-1169, GQ483510; WPI-1178, GQ497343; WPI-1106,

GQ497344; WPI-1104, GQ497345.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1179052/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 and S2

References

14 July 2009; accepted 31 August 2009

Published online 8 October 2009; 10.1126/science.1179052

Include this information when citing this paper

Fig. 1. XMRV sequences in PBMC DNA from CFS patients.

Single round PCR for gag, env and gapdh sequences in

PBMCs of (A) CFS patients and ( healthy controls. The

positions of the amplicons are indicated and DNA markers

(ladder) are shown. Representative results from one group of

20 patients are shown.

Fig. 2. Expression of XMRV Proteins in PBMCs from CFS

patients. (A) PBMCs were activated with PHA and IL-2,

reacted with a mAb to MLV p30 Gag and analyzed by IFC.

( Lysates of activated PBMCs from CFS patients (lanes 1-

5) were analyzed by Western blots with rat anti-SFFV Env

mAb (top panel), goat anti-xenotropic MLV (middle panel) or

goat anti-MLV p30 Gag (bottom panel). Lane 7: lysate from

SFFV-infected HCD-57 cells. At left: molecular weight

markers in kD. © Lysates of activated PBMCs from healthy

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10.1126/science.1179052

donors (lanes 1, 2, 4, 5, and 7) or from CFS patients (lanes 3

and 6) were analyzed by Western blots using rat anti-SFFV

Env mAb (top panel) or goat anti-MLV p30 Gag (bottom

panel). Lanes 8: SFFV-infected HCD-57 cells. At left:

molecular weight markers in kD (D) CD4+ T cells (left) or

CD19+ B cells (right) were purified, activated and examined

by flow cytometry for XMRV Gag using an anti-MLV p30

Gag mAb.

Fig. 3. Infectious XMRV in PBMCs from CFS patients. (A)

Lysates of LNCaP cells co-cultured with PBMCs from CFS

patients (lanes 1, 3, and 5) or healthy donors (lanes 2 and 4)

were analyzed by Western blots with rat anti-SFFV Env mAb

(top panel) or goat anti-xenotropic MLV (bottom panel).

Lane 6: uninfected LNCaP; lane 7: SFFV-infected HCD-57

cells. At left: molecular weight markers in kD. (

Transmission electron micrograph of LNCaP cells infected by

incubation with an activated T cell culture from a CFS

patient. © Transmission electron micrograph of virus

particles released by infected LNCaP cells.

Fig. 4. Infectious XMRV and antibodies to XMRV in CFS

patient plasma. (A) Plasma from CFS patients (lanes 1-6)

were incubated with LNCaP cells and lysates prepared after

six passages. Viral protein expression was detected by

Western blots with rat anti-SFFV Env mAb (top panel) or

goat anti-MLV p30 Gag (bottom panel). Lane 7: uninfected

LNCaP; lane 8: SFFV-infected HCD-57 cells. At left:

molecular weight markers in kD. ( Cell-free transmission

of XMRV to the SupT1 cell line was demonstrated using

transwell co-culture with patient PBMCs followed by nested

gag PCR. Lane 1: MW marker. Lane 2: SupT1 co-cultured

with Raji. Lanes 3-7: SupT1 co-cultured with CFS patient

PBMCs. Lane 8: No template control (NTC). © Normal T

cells were exposed to cell-free supernatants obtained from T

cells (lanes 1,5,6) or B cells (lane 4) from CFS patients.

Lanes 7 and 8 are secondary infections of normal activated T

cells. Initially, uninfected primary T cells were exposed to

supernatants from patients WPI-1220 (lane 7) and WPI-1221

(lane 8) PBMCs. Lanes 2 and 3: uninfected T cells; Lane 9:

SFFV-infected HCD-57 cells. Viral protein expression was

detected by Western blot using a rat anti-SFFV Env mAb. At

left: molecular weight markers in kD. (D). Plasma samples

from a CFS patient or from a healthy control as well as SFFV

Env mAb or control were reacted with BaF3ER cells (top) or

BaF3ER cells expressing recombinant SFFV Env (bottom)

and analyzed by flow cytometry.

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[Guest guest]

I am so sorry about your sister. What a tough battle. It sounds like she had a great spirit.

The article

, "" <pkmoisechartermi (DOT) net>, "joann weston" <westomjohotmail>, kplacrosscomcast (DOT) net

Date: Sunday, October 11, 2009, 10:23 AM

Here's a link to the article about fitness and breast cancer I mentioned last week. I'm actually really pleased with how it came out. Someone at church had already gotten the paper and brought the article to me this morning too. She seemed really touched by it which was very nice but I'm especially hoping it will help other people recover their own confidence (after any kind of illness or setback) using fitness as an awesome tool for whole body, mind and spirit health.

[Guest guest]

Thanki you for your kind words -

From: nancydewolf@ sbcglobal. net <nancydewolf@ sbcglobal. net>Subject: The article, "" <pkmoisechartermi (DOT) net>, "joann weston" <westomjohotmail (DOT) com>, kplacrosscomcast (DOT) netDate: Sunday, October 11, 2009, 10:23 AM

Here's a link to the article about fitness and breast cancer I mentioned last week. I'm actually really pleased with how it came out. Someone at church had already gotten the paper and brought the article to me this morning too. She seemed really touched by it which was very nice but I'm especially hoping it will help other people recover their own confidence (after any kind of illness or setback) using fitness as an awesome tool for whole body, mind and spirit health.

[Guest guest]

I am also very sorry.  I must have missed this original post    It sounds like she lived life to the fullest up until the end.  Good for her.  Hugs to you.DarcyOn Mon, Oct 12, 2009 at 4:41 PM, <snowlily1960@...> wrote:

 

I am so sorry about your sister. What a tough battle. It sounds like she had a great spirit.

The article

, " " <pkmoise@...>, " joann weston " <westomjo@...>, kplacross@...

Date: Sunday, October 11, 2009, 10:23 AM

 

Here's a link to the article about fitness and breast cancer I mentioned last week. I'm actually really pleased with how it came out. Someone at church had already gotten the paper and brought the article to me this morning too. She seemed really touched by it which was very nice but I'm especially hoping it will help other people recover their own confidence (after any kind of illness or setback) using fitness as an awesome tool for whole body, mind and spirit health.

 

 

 

[Guest guest]

Such kind words, thank you -

From: nancydewolf@ sbcglobal. net <nancydewolf@ sbcglobal. net>Subject: The article, "" <pkmoisechartermi (DOT) net>, "joann weston" <westomjohotmail (DOT) com>, kplacrosscomcast (DOT) netDate: Sunday, October 11, 2009, 10:23 AM

Here's a link to the article about fitness and breast cancer I mentioned last week. I'm actually really pleased with how it came out. Someone at church had already gotten the paper and brought the article to me this morning too. She seemed really touched by it which was very nice but I'm especially hoping it will help other people recover their own confidence (after any kind of illness or setback) using fitness as an awesome tool for whole body, mind and spirit health.

[Guest guest]

,

I finally got around to reading the article. Like others said, great job!

(It's also nice to see your face now too!) Thanks for sharing your story. My

mom is now a 5-year survivor of breast cancer, so I appreciate your struggle.

I had no idea you had been through so much or that you did so much to overcome

it--especially the 60-mile walk. You're a better man than I am!

>

> Oops! I forgot to include the link, sorry about that! Here it is:

>

>

http://www.freep.com/article/20091011/FEATURES08/910110317/1033/Features08/Showi\

ng-cancer-who-s-boss-

>

>

> The article

>

>

> Here's a link to the article about fitness and breast cancer I mentioned

last week. I'm actually really pleased with how it came out. Someone at church

had already gotten the paper and brought the article to me this morning too. She

seemed really touched by it which was very nice but I'm especially hoping it

will help other people recover their own confidence (after any kind of illness

or setback) using fitness as an awesome tool for whole body, mind and spirit

health.

>

>

>

[Guest guest]

Yeah ! Great article and cute picture!!! How is your recovery faring??

a

> >

> > Oops! I forgot to include the link, sorry about that! Here it is:

> >

> >

http://www.freep.com/article/20091011/FEATURES08/910110317/1033/Features08/Showi\

ng-cancer-who-s-boss-

> >

> >

> > The article

> >

> >

> > Here's a link to the article about fitness and breast cancer I mentioned

last week. I'm actually really pleased with how it came out. Someone at church

had already gotten the paper and brought the article to me this morning too. She

seemed really touched by it which was very nice but I'm especially hoping it

will help other people recover their own confidence (after any kind of illness

or setback) using fitness as an awesome tool for whole body, mind and spirit

health.

> >

> >

> >

>