Monday, February 27, 2012

5-AZA and the 100% peak-shift in the IFC figures of Lombardi et al. 2009

Oh-- and thank GAWD someone else is saying something about that goddamn 100% peak-shift in the damn flow data:
For example, it is usual to find in any viral infection that infected and uninfected cells exist in equilibrium as two discrete populations. In flow cytometric analysis, this is represented by a bimodal peak. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) taken from CFS patients was shown in the paper as a single peak, indicating that almost every cell was infected with XMRV.
Which is also explainable by treating cells with 5-AZA and inducing a low-but-global expression of cross-reactive ERVs, which was also probably done in Ruscettis lab because I doubt the WPI bought a flow cytometer.

Posted by: ERV | October 12, 2011 9:52 AM
But one has to rule out that a flow cytometer at the University of Nevada, Reno, was used.

So let's see all figures from Lombardi 20009 that were done by intracellular flow cytometry (IFC):
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 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. 

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 S5A: 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. 

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’ PBMC and 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.

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.
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).

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