Biomarkers of Acute Graft-Versus-Host Disease: Surface Antigens and Micro Rnas in Extracellular Vesicles

Introduction Biomarkers could be crucial to identify patients at high-risk of acute Graft-vs.-Host Disease (aGVHD). Given their involvement in inflammation, Extracellular Vesicles (EVs) may become attractive biomarkers. Moreover, EVs are non-invasively extracted from body fluids. In a preliminary study, we significantly correlated CD146, CD31 and CD140a expression on EVs membranes with the onset of aGVHD (Lia G. Leukemia 2017). Objectives We designed a prospective study to further characterize EVs by their surface antigens and by their content in MicroRNAs. Methods EVs are extracted from serum samples at given time-points (pre-transplant, on day 0, 3, 7, 14, 21, 28, 35, 45 and then monthly up to 1 year) by a protamine-based precipitation method and analyzed by flow-cytometry (Guava EasyCyte Flow Cytometer) for the expression of 13 membrane proteins (CD44, CD138, CD146, KRT18, CD120a, CD8, CD30, CD106, CD25, CD31, CD144, CD86, and CD140a). MicroRNAs (miR100, miR92b, miR155, miR194) are extracted from EVs at pre- transplant and on day 0, 7, 14, 28, and quantified by real time PCR as relative quantification compared to healthy donors after cDNA Reverse Transcription. Logistic Regression Analysis is performed for each marker. Results Thirty-five transplant patients with hematological diseases have so far been enrolled. Seventeen/35 patients (49%) developed grade II-IV aGVHD. Our preliminary findings show that CD146 (melanoma cell adhesion molecule, MCAM-1) and CD44 (homing-associated cell adhesion molecule, H-CAM) were associated with an increased risk of aGVHD (Odds Ratio (OR) 4.3, p=0.008; OR 2.1, p=0.039), whereas CD31 (platelet endothelial cell adhesion molecule, PECAM-1) level was associated with a decreased risk of aGVHD (OR 0.31, p=0.001). Moreover, increased risk of aGVHD was significantly correlated with levels of miR100 (OR 4.66, p=0.004), miR194 (OR 2.2, p=0.01) and miR155 (OR 3.56, p=0.035). Of note, biomarkers associated with aGVHD showed a constant consensual change in signal levels before aGVHD onset (Figure 1). Conclusions An association of 3 EVs membrane antigens and onset of aGVHD was observed. Of note, CD146, CD44 and CD31 belong to the Cell Adhesion Molecule Family and are critical for endothelium and immune cells interactions. The functional role of miR-194 in GVHD pathogenesis remains to be determined while JAK/STAT and TGFβ pathways were shown to be involved in other studies (Gimondi S Exp Hematol, 2016). MiR-100 has been reported to regulate inflammatory neovascularization during GvHD (Leonhardt F, Blood 2013) while miR-155 drives donor T cell expansion and tissue infiltration (Zitzer N, J Immunol 2018, Ranganathan P Blood 2012)

Long-Term Thymic Function and Reconstitution of the T Cell Compartment after T Cell-Replete Haplo-Identical Allografting

INTRODUCTION Post-transplant cyclophosphamide (PTCY) has expanded the application of haploidentical stem cell transplantation (haplo-HSCT). Thymic function may play a pivotal role in long-term clinical outcomes. OBJECTIVES To evaluate the kinetics of long-term immune thymus-dependent reconstitution after PTCY haplo-HSCT. METHODS Twenty-nine patients (median age 53) underwent T-cell replete haplo-HSCT with PTCY. Blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after transplantation. Analyses of CD4 and CD8 T-cell subsets by flow-cytometry were correlated by generalized linear models with Real-Time PCR quantification of signal joint T-cell receptor excision DNA circles (sjTRECs), specific marker of naive T-cells thymopoiesis. A) Naive; b) central; c) memory; and d) revertant CD4 and CD8 T-cells were defined as follows: a) CD45RA+CD62L+; b) CD45RO+CD62L+; c) CD45RO+CD27-; and d) CD45RA+/45RO+, respectively. SjTRECs real-time PCR was performed on genomic DNA (100 ng) extracted from sorted CD4 and CD8 T-cells. RESULTS Following PTCY induced T-cell depletion, a constant gradual increase in absolute numbers of all CD4 and CD8 T cell subsets and of sjTRECs copies from the first month up to two years post-transplant was observed ( Figure 1 ). Overall, at two years, CD4 and CD8 T-cell levels and sjTRECs levels were however lower than those observed in healthy donors. sjTRECs kinetics was associated with the increase in naive T-cells (overall, p CONCLUSIONS Active thymic function despite age-dependent involution, substantially contributes to T-cell reconstitution after haplo-HSCT. Chronic GVHD and older age are however significantly correlated with lower thymic activity. Overall, lower production of sjTRECs after haplo-HSCT as compared after HLA identical sibling HSCT may partly be due to a higher degree of "mismatching" of MHC molecules during thymic re-education

Prognostic or predictive value of circulating cytokines and angiogenic factors for initial treatment of multiple myeloma in the GIMEMA MM0305 randomized controlled trial

Abstract Background Several new drugs are approved for treatment of patients with multiple myeloma (MM), but no validated biomarkers are available for the prediction of a clinical outcome. We aimed to establish whether pretreatment blood and bone marrow plasma concentrations of major cytokines and angiogenic factors (CAFs) of patients from a phase 3 trial of a MM treatment could have a prognostic and predictive value in terms of response to therapy and progression-free and overall survival and whether these patients could be stratified for their prognosis. Methods Blood and bone marrow plasma levels of Ang-2, FGF-2, HGF, VEGF, PDGF-β, IL-8, TNF-α, TIMP-1, and TIMP-2 were determined at diagnosis in MM patients enrolled in the GIMEMA MM0305 randomized controlled trial by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with the type of therapy and patients’ characteristics and with a group of non-MM patients as controls. Results No significant differences were detected between the blood and bone marrow plasma levels of angiogenic cytokines. A cutoff for each CAF was established. The therapeutic response of patients with blood plasma levels of CAFs lower than the cutoff was better than the response of those with higher levels in terms of percentage of responding patients and quality of response. Conclusion FGF-2, HGF, VEGF, and PDGF-β plasma levels at diagnosis have predictive significance for response to treatment. The stratification of patients based on the levels of CAFs at diagnosis and their variations after therapy is useful to characterize different risk groups concerning outcome and response to therapy. Trial registration Clinical trial information can be found at the following link: NCT0106317

Analysis of shear transfer and gap opening in timber–concrete composite members with notched connections

In timber–concrete composite members with notched connections, the notches act as the shear connections between the timber and the concrete part, and have to carry the shear flow necessary for composite action. The shear transfer through the notches generates shear and tensile stresses in both parts of the composite member, which may lead to brittle failure and to an abrupt collapse of the structure. Although simplified design formulas already exist, some structural aspects are still not clear, and a reliable design model is missing. This paper summarizes current design approaches and presents analytical models to understand the shear-carrying mechanism, to estimate the shear stresses acting in the timber and concrete, and to predict failure. The analysis concentrates on three problems: the shearing-off failure of the timber close to the notch, the shear failure of the concrete, and the influence of the shear flow on the gap opening between the timber and concrete. Parts of the model calculations could be compared to experimental observations. The conclusions of this paper contribute to improving current design approaches.ISSN:1359-5997ISSN:1871-687

Impact of the 2019 European Guidelines on Diabetes in Clinical Practice: Real and Simulated Analyses of Lipid Goals

Background: Recent European guidelines on diabetes, prediabetes, and cardiovascular disease developed for the European Society of Cardiology (ESC) in collaboration with the European Association for the Study of Diabetes (EASD) significantly changed some concepts on risk stratification, lipid goals, and recommendations for the use of lipid-lowering drugs. The objectives of this work were to describe the lipid-lowering treatment prescribed for patients with diabetes and to determine the percentage of patients that achieved the lipid goals recommended by the 2019 ESC/EASD Guidelines on Diabetes in real and simulated scenarios. Methods: A multicenter, cross-sectional study was performed. Subjects >18 years with type 2 diabetes were included. The recommendations of the 2019 ESC/EASD Guidelines were followed. The real and simulated (ideal setting using adequate doses of statins ± ezetimibe) scenarios were analyzed. Results: Overall, 528 patients were included. In total, 62.5% of patients received statins (17.1% high intensity). Most patients were stratified as “very high risk” (54.2%) or “high risk” (43.4%). Only 13.3% achieved the double lipid goal (LDL-C and non-HDL-C goals according to the risk categories). In the simulation analysis, the proportion of subjects that did not reach the therapeutic objective decreased in all risk strata, although a considerable proportion of subjects persisted outside the target. Conclusion: The difficulty of achieving lipid goals in diabetic patients was considerable when applying the new guidelines. The situation would improve if we optimized treatment, but the prescription of new lipid-lowering drugs could be limited by their high cost