Here goes the three posters of Irsi Caixa Spanish retrovirologist team, the ones that make the link with EBV and XMRV, and if I am not mistaken, I also understand that they DID find XMRV in CFS patients:
Detection of XMRV sequences in EBV-transformed B cell lines
J. Blanco1, J. Carrillo1, E. Garcia1, J. Areal2, B. Clotet1, C. Cabrera11
IrsiCaixa Foundation, Retrovirology Lab, Barcelona, Spain;
2HUGTIP, Urology Dept, Barcelona, Spain
Background: XMRV infection has been found in humans linked with prostate cancer and chronic fatigue syndrome (CFS). XMRV is able to infect a wide range of cells and has been found in a variety of cell types both in Vitro and in vivo, including B cells and other immune cell types.
Materials and methods:
In order to determine the presence of XMRV sequences in B cells, we have screened 21 B cell lines available in our laboratory. These cells were generated by EBV immortalization of PBMC obtained from 11 CFS affected individuals (fulfilling both Fukuda and Canadian criteria), 5 healthy donors, 4 HIV infected individuals and 1 prostate cancer patient.
DNA was extracted from dry cell pellets and XMRV sequences were amplified using a real-time PCR covering a 150bp pol sequence and using a nested approach in both gag and Env genes.
Results:
Envelope amplification yielded positive bands in 4 out of 21 individuals tested, 3 CFS affected individuals and 1 healthy donor.
However, gag amplification yielded only 3 positive samples (1 CFS affected individual, 1 healthy donor and one HIV+ patient).
In contrast, Real-time PCR of Pol fragment detected 7 positives samples in 14 individuals tested (4 SFC , 2 donors and 1 HIV+ individuals).
To confirm the presence of XMRV sequences we performed sequence analyses of gag and env amplicons. The analysis of the three available gag sequences confirmed the XMRV characteristic 24-nt deletion, which is not found in any known exogenous MuLV. Sequences were 100% identical to reported XMRV sequences. Furthermore, envelope sequences were also homologous to previously described XMRV sequences. In this case, sequence variability was low or absent. Interestingly, most of changes observed corresponded to G to A mutations that were accumulated in one positive sample.
Conclusions: Despite the discrepancies observed in the different PCR approaches using gag, pol or env sequences, our data suggest that EBV transformed B cell lines harbor XMRV specific sequences, and therefore this cell type may represent a reservoir for XMRV contributing to its potential pathogenesis.
Pathogenesis
Xenotropic murine leukemia virus-related infection of human lymphoid tissue ex vivo
C. Cabrera1, M. Curriu1, J. Carrillo1, M. Massanella1, E. Garcia1, B. Clotet1, C. Carrato2, J. Blanco11
Fundacio irsiCaixa, Retrovirology Laboratory, Badalona, Spain;
2Hospital Universitari Germans Trias i Pujol,
Departamento de Anatoma Patolgica, Badalona, Spain
Background: The gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV) has been recently associated with prostate cancer and chronic fatigue syndrome. In patients with both diseases, the virus has been found in a variety of cell types, including T and B cells. Moreover, in XMRV-infected rhesus macaques there is evidence of viral replication in lymphoid organs, suggesting that lymphocytes are a primary target for XMRV. Histocultures of tonsils support productive infection with various lymphotropic viruses, including HIV and HHV-6.
In this study, ex vivo lymphoid tissue was used to investigate the pathogenic mechanisms of XMRV
Materials and methods: Human tonsils from 2 healthy individuals undergoing tonsillectomy were collected in PBS and cultured in small pieces (2mm3) over gelfoam soaked in RPMI 1640 medium. Small tissue blocks were left uninfected or were infected ex vivo with a XMRV viral stock obtained from a 22Rv1 cell culture supernatant. Culture medium was replaced every 3 days. After 14 days in culture, uninfected and infected tissues were mechanically homogenized and cells were isolated. Viral infection was evaluated at different times in the cells migrating out of the tissue and at day 14 in tissue cells, by analyzing the presence of viral DNA by PCR. In addition, tissue cells were immunophenotyped and analyzed by flow cytometry
Results: At day 7 post-infection cells migrating out of the tissue were positive for XMRV DNA. After 14 days of culture, tissue cells were also highly positive, confirming that XRMV (as other viruses) was able to infect human tonsil tissue fragments in the absence of exogenous stimulation. Despite this apparent efficient infection, cells showed similar percentages of T and B cells in uninfected and infected tissues. XMRV infection did not modify the percentage of CD3 (76 and 75% in XMRV+ and XMRV- tissue, respectively), CD4 (53% vs 52%), CD8 (39% vs 40%) or CD19 cells (3% vs 1%). A deeper analysis of T cell subsets showed that XMRV infection did not modify the nave/memory cell ratio, as evaluated by CD45RO staining, or immune activation markers, as evaluated by the expression of HLA-DR and CD38 in both CD4 and CD8 T cells
Conclusions: The development of in vitro models to study XMRV pathogenicity is essential for understanding its role in human diseases. Our data show that histoculture of human lymphoid tissue is a suitable model for the analysis of XMRV pathogenesis. In this system we observed a infection by XMRV although this infection did not result in depletion of T or B cells nor an immune activation, suggesting the absence of evident depleting effect of XMRV in these cells in lymphoid tissue.
Altered B, T and NK cell phenotype in chronic fatigue syndrome (CFS) individuals.
M. Curriu1, M. Massanella1, J. Carrillo1, J. Puig2, J. Rigau1, B. Clotet1, C. Cabrera1, J. Blanco11
Fundaci IrsiCaixa, Retrovirology laboratory, Badalona, Spain;
2Hospital Universitari Germans Trias i Pujol, Fundaci Lluita contra la SIDA, Badalona, Spain
Background: Several studies have reported controversial results on the dysfunction of the immune system in Chronic Fatigue Syndrome (CFS) affected individuals. Since the recently described Xenotropic murine leukemia virus- related virus (XMRV) can infect cells from the immune system (B, T or NK cells), we designed different panels of antibodies to deeply characterize the phenotype of B, T and NK lymphocytes populations in patients with CFS.
Material and methods: We obtained blood samples from 12 individuals affected from CFS (fulfilling both Fukuda and Canadian criteria) and 15 healthy donors (HD). B, T and NK cell populations were phenotyped in fresh blood samples and analyzed by multicolour flow cytometry. Fresh PBMCs were obtained to characterize spontaneous ex vivo apoptosis and NK cytotoxic activity against EGFP-K562 cells, both analyzed by flow cytometry.
Results: No changes in the percentage or absolute numbers were observed in principal populations of B (CD19+), T (CD3+, CD3+CD4+, CD3+CD8+) or NK (CD56+ CD16+) cells. However, significant differences were observed in various subsets of these populations.B lymphocytes from CFS individuals showed a decrease in marginal-zone marker CD1c, especially in memory IgG population. The percentage of memory IgG+ cells, effector memory CD38high IgG+ cells and plasmatic B cells (CD38high/CD27high) was also reduced in CFS individuals. In contrast, CFS individuals showed an increase in transitional B cells (IgD+CD38highCD5+CD10+), which present lower spontaneous ex-vivo apoptosis. T-cells from CFS individuals presented increased CD25 levels mainly within CD8+ population.
Proliferation marker Ki67 was significantly diminished in CFS CD4 T-cells and a trend was also observed in CD8 T-cells. In addition, individuals with CFS showed increased CD5 levels within CD8 T-cells which could suggest an anergic state. No signs of altered senescence (CD57+ T cells) or activation (CD38+, HLA-DR+ or double positive cells) were observed in CD4 or CD8 subsets, although, CD8 T cells from CFS individuals showed higher expression of FAS and PD-1 and a slightly higher spontaneous ex-vivo apoptosis. NK cells showed a significant decrease in CD57+ expression and expressed higher levels of CD69 and activating NK Cytotoxic Receptors (NCR) NKp30, NKp44 and NKp46. Nevertheless, this altered phenotype did not impact function, as we did not observe differences in NK cytotoxic activity in CFS patients compared with HD.
Conclusions: CFS patients showed qualitative but not quantitative alterations in all major immune cell types. B cells presented a phenotype partially similar to some autoimmune disorders or viral infections. Interestingly, an anergic phenotype observed in T cells from CFS individuals could be related with an impaired control of viral infections and could explain the lack of increased activation and senescence observed in our patients. Finally, altered NK phenotype did not seem to significantly modify cytotoxic activity. On the whole, the results suggest a global immune dysfunction with an unknown aetiology as potential contributor to the mechanism of CFS.
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