Reposted here is the study "Lombardi et al. 2009" as well as parts of the "Supporting Online Material". This is work in progress. If you want to add something, leave me a comment!
Please note:
- I kept the order of the text of the study, but added line breaks and added images from the study where I saw them fit
- Added parts of the "Supporting Online Material"
- Added "Mikovits' Slide of Shame"
- All emphasis, highlights, color and comments are mine
- Comments are in square brackets
- Colors denote possible origin of research results:
WPI/Mikovits
NCI/Ruscetti
CC/Silverman
Nested PCR: WPI (PCR for all samples)
Single-round PCR and sequencing: CC/Silverman (PCR for only a few samples)
Western Blots, Slide of Shame: WPI? NCI?
Western Blots: WPI? NCI?
IFC: possibly NCI/Ruscetti (not ruled out: UniN, Reno)
Antibodies (used for IFC and WB)
Cell Lines (not colored: HCD-57 used as positive controls)
Discussion of CFS, results of other studies, posed questions and other idle talk
Material with (for me) unknown association remains in black
Todo: Show cell lines in different color – Done- Todo: Make another blog-post for comments by Ruscetti/Mikovits
- Todo: Make another blog-post for Addendum
- Todo: Add patient numbers for all slides
Some remarks:
- If we do not count the one sentence in the opening remarks and the two sentences in the closing remarks of the study, then we see that the main result of this study ("67% XMRV positive by Nested PCR") are reported unbelievably short:
Using the Whittemore Peterson 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.
… In contrast, XMRV gag sequences were detected in 8 of 218 (3.7%) PBMC DNA specimens from healthy individuals.
That's all there is to the main result! It's only three sentences in total, with the core of the main result being one(!) sentence only! One sentence! Again, these are the main results of this study and no supporting material is supplied, no figures, no tables, neither in the study itself nor in the supporting online materials! At the same time several figures and tables with lots of text are supplied for the other results!
- The study and the "supporting online material" were most likely written by different persons. See naming-style of figures: "A." versus "(A)"
- The study and the "Addendum" were most likely written by different persons. "PBMCs" versus "PMCs"
- Figure 4A: not referenced in the study
- Figure S2: Probably exported as a JPG complete with text (or possibly scanned) and then added as JPG
- Figure S5A: Infectious XMRV in CFS patients’ "PBMCs" versus "T-cell cultures"
- Figure 4B: Inverted WB. Different (digital?) machine, from different lab then the other WBs (scan of gel)?
- All IFC figures look similar, except IFC figure S4B
- I am having real problems wrapping my head around the different non-PCR methods employed. To be clear: I (rudimentary) understand most of the concepts, but the text of the study jumbles all together and makes it really hard to see were one method ends, were another method starts and which methods were independent of each other. Was the EM independent of the WBs and the IFCs for antibodies or for viral protein? Or did it use the same cultures? Or did some use the same culture? And would a contaminated culture cause trouble for all?
- Why/which were there "patient PBMC DNA specimens stored at the NCI (Frederick, MD) since 2007"?
- No discussion how samples were selected which were sent to Silverman (Remember: Silverman received only a few samples and tested most of the samples positive by single round PCR)
- No mention of the single round PCR supposedly done at the WPI to select samples for Silverman (as discussed in the much later published Addendum).
Let the games begin:
This article has been retracted
This article has been retracted
Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome
Vincent C. Lombardi, 1* Francis W. Ruscetti, 2* Jaydip Das Gupta, 3 Max A. Pfost, 1 Kathryn S. Hagen, 1 Daniel L. Peterson, 1 Sandra K. Ruscetti, 4 Rachel K. Bagni, 5 Cari Petrow-Sadowski, 6 Bert Gold, 2 Michael Dean, 2 Robert H. Silverman, 3 Judy A. Mikovits 1†
1 Whittemore Peterson Institute, Reno, NV 89557, USA.
2 Laboratory of Experimental Immunology, National Cancer Institute- Frederick, Frederick, MD 21701, USA.
3 Department of Cancer Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44106, USA.
4 Laboratory of Cancer Prevention, National Cancer Institute-Frederick, Frederick, MD 21701, USA.
5 Advanced Technology Program, National Cancer Institute-Frederick, Frederick, MD 21701, USA.
6 Basic 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@wpinstitute.org
Received 14 July 2009; accepted 31 August 2009
Published online 8 October 2009; 10.1126/science.1179052
Include this information when citing this paper.
[[ Abstract and introduction ]]
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 cell-associated 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 β 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).
[[ The "Nested PCR" ]]
[[ Patients: +68 / -33 / Σ101 ]]
[[ Controls: +8 / -210 / Σ218 ]]
[[ Called "cDNA nested PCR" in the Addendum ]]
[[ The "Single Round PCR" and Sequencing ]]
[[ Patients: +7 / -4 / Σ11 ]]
[[ Controls: +1 / -10 / Σ11 ]]
Using the Whittemore Peterson 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).
Fig. 1A and Fig. 1B. XMRV sequences in PBMC DNA from CFS patients.
Single round PCR for gag, env and gapdh sequences in PBMCs of (A) CFS patients and (B) 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.
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 cancer-derived 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 (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.
[Who wrote this sentence?]
[[ IFC, WB and Abs Overview ]]
[[ Intracellular Flow Cytometry, Western Blot and Antibodies ]]
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 [what 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).
[Please note the usage of XMRV VP62 plasmid as a spiked control for WB]
[Please note that Fig. S3 was probably made from a JPG]
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.
[[ "IFC for Viral Proteins" ]]
[[ Flow cytometry for viral proteins ]]
[[ PBMCs activated with PHA and IL-2 ]]
[[ patients: +19 / -11 / Σ30 ]]
[[ controls: +16 / -0 / Σ16 ]]
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).
Fig. 2A. 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.
The majority [how many?] 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).
Figure S4A. 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.
These results were confirmed by Western blots (Fig. 2B and C) (6) using Abs to SFFV Env, mouse xenotropic MLV and MLV p30 Gag.
Fig. 2B. Expression of XMRV Proteins in PBMCs from CFS patients.
(B) 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.
Samples from five healthy donors exhibited no expression of XMRV proteins (Fig. 2C).
Fig. 2C. Expression of XMRV Proteins in PBMCs from CFS patients.
(C) Lysates of activated PBMCs from healthy 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). [Mikovits' Slide of Shame] Lanes 8: SFFV-infected HCD-57 cells. At left: molecular weight markers in kD
[Once more, that is Mikovits' Slide of Shame]
[And here are texts from the lanes]
| Lane | Lombardi 2009 | Original | IACFS/ME 2011 |
| 1 | Normal | Normal T 3/11 | Normal |
| 2 | Normal | Normal T 3/21 | 2905 PBMC |
| 3 | 1235 | 2905 5 AZA 4/11 | 2905 PBMC + 5-AZA |
| 4 | Normal | PBMC 4/11 | Normal |
| 5 | Normal | PBMC 4/10 | 1674 |
| 6 | 1236 | 1674 5 AZA 4/11 | 1674 + 5-AZA |
| 7 | Normal | PBMC 4/15 | Normal |
| 8 | SFFV-infected HCD-57 | HCD.57 SFFVP | SFFV-infected HCD-57 |
The frequencies of CFS cases vs. healthy controls that were positive and negative for XMRV sequences were used to calculate a Pearson χ² value of 154 (two-tailed P value of 8.1 × 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.
[[Which PBMCs? T-cells and B-cells]]
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).
Fig. 2D. Expression of XMRV Proteins in PBMCs from CFS patients.
(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.
Figure S4A. 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.
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).
Figure S4B. Expression of XMRV proteins in PBMC from CFS patients.
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.
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.
Figure S4C. Expression of XMRV proteins in PBMC from CFS patients.
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.
[[ Viral Protein ]]
[[ Activated PBMCs, co-cultured LNCaP ]]
We next investigated whether the viral proteins detected in PBMCs from CFS patients [how many?] 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 co-culture 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).
Fig. 3A. 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.
Figure S5A: Infectious XMRV in CFS patients’ PBMCs ….
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.
[[ Electron microscopy (EM) ]]
[[ from LNCaP co-culture ]]
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).
Fig. 3B. Infectious XMRV in PBMCs from CFS patients. (B) Transmission electron micrograph of LNCaP cells infected by incubation with an activated T cell culture from a CFS patient.
Fig. 3C. Infectious XMRV in PBMCs from CFS patients. (C) Transmission electron micrograph of virus particles released by infected LNCaP cells.
[[ Figure 4A is not referenced in the study! ]]
Fig. 4A. 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.
[[ "Infectious Agent in Plasma" ]]
[[ Viral Transmission: LNCaP cells incubated with plasma ]]
[[ patients: +10 / -2 / Σ12 ]]
[[ controls: +0 / -12 / Σ12 ]]
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).
Figure S5B: Infectious XMRV in CFS patients’ … plasma.
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.
[[ "Cell-Free Transmission" ]]
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).
Fig. 4B. Infectious XMRV and antibodies to XMRV in CFS patient plasma.
(B) 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).
Fig. 4C. Infectious XMRV and antibodies to XMRV in CFS patient plasma.
(C) 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.
Together, these results suggest that both cell-associated and cell-free transmission of CFS-associated XMRV are possible.
[[ The "Antibodies in Plasma" ]]
[[ Flow cytometry for detection of antiviral antibodies in CFS plasma ]]
[[ Patients: +9 / -9 / Σ9 ]]
[[ Controls: +0 / -7 / Σ7 ]]
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).
Fig. 4D. Infectious XMRV and antibodies to XMRV in CFS patient plasma.
(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.
Figure S6A: 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.
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).
Figure S6B: Presence of antibodies in CFS plasma that recognize the cell surface of SFFV Env expressing BAF3ER 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).
These results are consistent with the hypothesis that CFS patients mount a specific immune response to XMRV.
[[ Discussion of results ]]
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 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.
So much for the study itself.
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 of 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 52oC annealing, for 35 cycles. For second round PCR, annealing was at 54oC 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.
[Why were there samples stored at the NCI since 2007?]
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 37oC. 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 22oC 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 4oC 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 4oC. 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 4oC. 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 4oC, 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.
[RNASEL]
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.
[Why does the SOM specify "CFS plasma" here?]
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 4oC 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 4oC 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 4oC 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.
End of transmission – for now.
