There is evidence of reduced SARS-CoV-2 vaccine effectiveness in patients with hematological malignancies. We hypothesized that tumor and treatment-related immunosuppression can be depicted in peripheral blood, and that immune profiling prior to vaccination can help predict immunogenicity. We performed a comprehensive immunological characterization of 83 hematological patients before vaccination and measured IgM, IgG, and IgA antibody response to four viral antigens at day +7 after second-dose COVID-19 vaccination using multidimensional and computational flow cytometry. Health care practitioners of similar age were the control group (n = 102). Forty-four out of 59 immune cell types were significantly altered in patients; those with monoclonal gammopathies showed greater immunosuppression than patients with B-cell disorders and Hodgkin lymphoma. Immune dysregulation emerged before treatment, peaked while on-therapy, and did not return to normalcy after stopping treatment. We identified an immunotype that was significantly associated with poor antibody response and uncovered that the frequency of neutrophils, classical monocytes, CD4, and CD8 effector memory CD127low T cells, as well as naive CD21+ and IgM+D+ memory B cells, were independently associated with immunogenicity. Thus, we provide novel immune biomarkers to predict COVID-19 vaccine effectiveness in hematological patients, which are complementary to treatment-related factors and may help tailoring possible vaccine boosters.

The Spanish Hematic Derivatives Group, consisting of 26 biobanks, was established in 2011. We describe here the viability results of our publically available standard operating procedure to freeze and thaw peripheral blood mononuclear cells (PBMCs). Our protocol maximizes PBMC viability while avoiding where possible interbiobank and intrabiobank assay variability.

Analytical proteomics has experienced exponential progress in the last decade and can be expected to lead research studies on diagnostic and therapeutic biomarkers in the near future. Because the development of this type of analysis requires the use of a large number of human samples with a minimum of quality requirements, our objective was to identify appropriate indicators for quality control of plasma samples stored in biobanks for research in proteomics. To accomplish this, plasma samples from 100 healthy donors were obtained and processed according to the pre-analytical variables of: a) time delay for the first centrifugation of the original blood sample (4 or 24 h) and b) number of freeze/thaw cycles (1, 2 or 3) of the processed plasma samples. The analyses of samples were performed by different and complementary methods such as SPE MALDI-TOF, DICE, shotgun (iTRAQ, nLC MALDI TOF/TOF) and targeted nLC MS/MS proteomic techniques (SRM). In general, because the distribution of proteins in all samples was found to be very similar, the results shown that delayed processing of blood samples and the number of freeze/thaw cycles has little or no effect on the integrity of proteins in the plasma samples. Significance: The results of the present work indicate that blood proteins in plasma are broadly insensitive to such preanalytical variables as delayed processing or freeze/thaw cycles when analyzed at the peptide level. Although there are other studies related to protein stability of clinical samples with similar results, what is remarkable about our work is the large number of plasma samples examined and that our analyses assessed protein integrity by combining a wide set of complementary proteomic approaches performed at different proteomic platform participating laboratories that all yielded similar results. We believe our study is the most comprehensive performed to date to determine the changes in proteins induced by delayed sample processing and plasma freeze/thaw cycles. (C) 2016 Elsevier B.V. All rights reserved.

G protein-coupled receptor kinase 2 (GRK2) has recently emerged as a negative modulator of insulin signaling. GRK2 downregulation improves insulin sensitivity and prevents systemic insulin resistance. Cardiac GRK2 levels are increased in human heart failure, while genetically inhibiting GRK2 leads to cardioprotection in mice. However, the molecular basis underlying the deleterious effects of GRK2 up-regulation and the beneficial effects of its inhibition in the heart are not fully understood. Therefore, we have explored the interconnections among a systemic insulin resistant status, GRK2 dosage and cardiac insulin sensitivity in adult (9 month-old) animals. GRK2(+/-) mice display enhanced cardiac insulin sensitivity and mild heart hypertrophy with preserved systolic function. Cardiac gene expression is reprogrammed in these animals, with increased expression of genes related to physiological hypertrophy, while the expression of genes related to pathological hypertrophy or to diabetes/obesity co-morbidities is repressed. Notably, we find that cardiac GRK2 levels increase in situations where insulin resistance develops, such as in ob/ob mice or after high fat diet feeding. Our data suggest that GRK2 downregulation/inhibition can help maintain cardiac function in the face of co-morbidities such as insulin resistance, diabetes or obesity by sustaining insulin sensitivity and promoting a gene expression reprogramming that confers cardioprotection.

Hyperaldosteronim is associated with left ventricular (LV) hypertrophy (LVH) and fibrosis. Cardiotrophin (CT)-1 is a cytokine that induces myocardial remodeling. We investigated whether CT-1 mediates aldosterone (Aldo)-induced myocardial remodeling in two experimental models. Wistar rats were treated with Aldo-salt (1 mg·kg(-1)·day(-1)) with or without spironolactone (200 mg·kg(-1)·day(-1)) for 3 wk. Wild-type (WT) and CT-1-null mice were infused with Aldo (1 mg·kg(-1)·day(-1)) for 3 wk. Hemodynamic parameters were analyzed. LVH, fibrosis, inflammation, and CT-1 expression were evaluated in both experimental models by histopathological analysis, RT-PCR, Western blot analysis, and ELISA. Hypertensive Aldo-treated rats exhibited increased LV end-diastolic pressure and -dP/dt compared with controls. The cardiac index, LV cross-sectional area and wall thickness, cardiomyocyte size, collagen deposition, and inflammation were increased in Aldo-salt-treated rats. Myocardial expression of molecular markers assessing LVH and fibrosis as well as CT-l levels were also augmented by Aldo-salt. Spironolactone treatment reversed all the above effects. CT-1 correlated positively with hemodynamic, histological, and molecular parameters showing myocardial remodeling. In WT and CT-1-null mice, Aldo infusion did not modify blood pressure. Whereas Aldo treatment induced LVH, fibrosis, and inflammation in WT mice, the mineralocorticoid did not provoke cardiac remodeling in CT-1-null mice. In conclusion, in experimental hyperaldosteronism, the increase in CT-1 expression was associated with parameters showing LVH and fibrosis. CT-1-null mice were resistant to Aldo-induced LVH and fibrosis. These data suggest a key role for CT-1 in cardiac remodeling induced by Aldo independent of changes in blood pressure levels.

Aims: To investigate cardiotrophin-1 (CT-1) effects and regulation in vascular smooth muscle cells (VSMCs) in vitro and in aortic tunica media ex vivo in normotensive Wistar rats and spontaneously hypertensive rats (SHRs). Methods and Results: CT-1 expression was quantified by real-time reverse-transcription PCR and western blotting. CT-1-activated intracellular pathways were assessed by western bloting analysis. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ki67 immunodetection, and cell hypertrophy by planimetry. Extracellular matrix components were quantified by real-time reverse-transcription PCR and western blot, and metalloproteinases activities by zymography. VSMCs from Wistar rats and SHRs expressed spontaneously CT-1 at the mRNA and the protein level, with a two-fold more increase in SHRs. CT-1 phosphorylated p42/44 mitogen-activated protein kinase, p38 mitogen-activated protein kinase, Akt and Stat-3 in both strains. CT-1 stimulated VSMCs proliferation and hypertrophy in both strains, with an enhanced stimulation in SHRs. CT-1 increased the secretion of collagen type I and fibronectin in VSMCs and aortic tunica media of Wistar rats and SHRs, with greater magnitude in SHRs. In SHRs VSMCs in vitro and ex vivo, CT-1 increased the secretion of collagen type III and elastin and the expression of tissue inhibitors of metalloproteinases, without altering metalloproteinase activity. These effects were blocked by CT-1 receptor antibodies. Aldosterone treatment increased CT-1 expression in VSMCs and aortic tunica media from both strains, with a greater magnitude in SHRs. Conclusion: CT-1 induces VSMCs proliferation, hypertrophy and extracellular matrix production, and is upregulated in hypertension and by aldosterone. CT-1 may represent a new target of vascular wall remodeling in hypertension.