It is well known that the normalization step of microarray data makes a difference in the downstream analysis. All normalization methods rely on certain assumptions, so differences in results can be ...traced to different sensitivities to violation of the assumptions. Illustrating the lack of robustness, in a striking spike-in experiment all existing normalization methods fail because of an imbalance between up- and down-regulated genes. This means it is still important to develop a normalization method that is robust against violation of the standard assumptions
We develop a new algorithm based on identification of the least-variant set (LVS) of genes across the arrays. The array-to-array variation is evaluated in the robust linear model fit of pre-normalized probe-level data. The genes are then used as a reference set for a non-linear normalization. The method is applicable to any existing expression summaries, such as MAS5 or RMA.
We show that LVS normalization outperforms other normalization methods when the standard assumptions are not satisfied. In the complex spike-in study, LVS performs similarly to the ideal (in practice unknown) housekeeping-gene normalization. An R package called lvs is available in http://www.meb.ki.se/~yudpaw.
Patients with brain tumor or pancreatic cancer exhibit the poorest prognosis, while immune fitness and cellular immune exhaustion impacts their survival immensely. This work identifies differences in ...the immune reactivity to the common human pathogens cytomegalovirus (CMV) and Epstein-Barr virus (EBV) between patients with brain tumor in comparison to those with pancreatic cancer and healthy individuals.
We characterized the humoral and cellular immune responses of patients with brain tumor or pancreatic cancer to cytomegalovirus structural protein pp65 (CMV-pp65) as well as Epstein-Barr nuclear antigen-1 (EBNA-1) by whole-blood assay and ELISA.
Anti-CMV-pp65 plasma immunoglobulin gamma (IgG) titers were significantly lower in patients with brain tumor compared to healthy donors and patients with pancreatic cancer. Among the responding patients with GBM, those with a weak anti-CMV IgG response also had a decreased median overall survival (p = 0.017, 667 vs 419 days) while patients with brain tumor showed a generally suppressed anti-CMV immune-reactivity. Patients with brain tumor exhibited a significantly lower interferon gamma (IFNγ) response to EBNA-1 and CMV-pp65 compared to patients with pancreatic cancer or healthy donors. This antigen-specific response was further amplified in patients with brain tumor upon conditioning of whole blood with IL-2/IL-15/IL-21. Exclusively in this setting, among the responding patients with GBM, those exhibiting a EBV-specific cellular immune response above the median also displayed an increased median overall survival pattern compared to weak responders (753 vs 370 days, p < 0.001).
This report provides (i) a fast and easy assay using common viral antigens and cytokine stimulation to screen for immune fitness/exhaustion of patients with brain tumor in comparison to pancreatic cancer and healthy individuals and (ii) EBV/CMV-induced IFNγ production as a potential marker of survival in patients with brain tumor.
Abstract Background Bacille Calmette-Guérin (BCG) is the world’s most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy and in autoimmune diseases due to its ...immunomodulatory properties. Up to now, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. Methods We tested sera of BCG-vaccinated healthy adults on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained after 56, 112 and 252 days of vaccination, respectively. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. Results IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all tested study subjects, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (IL2Rβ, FGFR1), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to TGFβR1 and in agreement with the peptide microarray findings; immunohistochemical analysis of TB infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to IL-4R showed increased reactivity 112 days post vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on 112 and 252 days as compared to day 56 post BCG vaccination, compared to day 56. Conclusions BCG vaccination modifies the host’s immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG vaccinated individuals.
Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute ...lymphoblastic leukemia (ALL) performed during the years 2005–2021 (n = 138, including 51 children), aiming to identify the optimal use of lineage‐specific recipient‐donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3+ bone marrow cells sampled at least 30 days before a relapse. Relapse could be predicted with a sensitivity of 73% and a specificity of 83%. Results were similar for children but with a higher recipient chimerism cutoff. Additionally, adults that had at least one chimerism value <0.12% in CD3+ peripheral blood cells within the first 60 days after HSCT had 89% probability of being relapse‐free after 2‐years compared to 64%. Results were similar for children but again necessitating a higher chimerism cutoff. These results suggest that high‐sensitive lineage‐specific chimerism analysis can be used for (1) early ALL relapse prediction by longitudinal chimerism monitoring in CD3+ bone marrow cells and (2) relapse risk stratification by analyzing CD3+ blood cells early post‐HSCT.
Summary
A high content peptide microarray containing the entire influenza A virus A/California/08/2009(H1N1) proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the ...haemagglutinin from the A/South Carolina/1/1918(H1N1) strain, was used to gauge serum IgG epitope signatures before and after Pandemrix® vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu‐specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251–265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor‐binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine.
Highlights • CMV, EBV and ESAT-6 – specific IFN-γ responses in TB patients with pulmonary TB at the time point of diagnosis correlate with cure and survival during anti-TB therapy. • Future studies ...concerning i) CVM/EBV-vectored vaccines are recommended, ii) gauging CMV / EBV responses as prognostic markers to identify patients at high risk to succumb to TB disease and to identify time points for Host-Directed Therapies (HDTs).
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