Insights into the behaviour of systems biology models from dynamic sensitivity and identifiability analysis: a case study of an NF-kB signaling pathway

Mathematical modelling offers a variety of useful techniques to help in understanding the intrinsic behaviour of complex signal transduction networks. From the system engineering point of view, the dynamics of metabolic and signal transduction models can always be described by nonlinear ordinary differential equations (ODEs) following mass balance principles. Based on the state-space formulation, many methods from the area of automatic control can conveniently be applied to the modelling, analysis and design of cell networks. In the present study, dynamic sensitivity analysis is performed on a model of the IB-NF-B signal pathway system. Univariate analysis of the Euclidean-form overall sensitivities shows that only 8 out of the 64 parameters in the model have major influence on the nuclear NF-B oscillations. The sensitivity matrix is then used to address correlation analysis, identifiability assessment and measurement set selection within the framework of least squares estimation and multivariate analysis. It is shown that certain pairs of parameters are exactly or highly correlated to each other in terms of their effects on the measured variables. The experimental design strategy provides guidance on which proteins should best be considered for measurement such that the unknown parameters can be estimated with the best statistical precision. The whole analysis scheme we describe provides efficient parameter estimation techniques for complex cell networks

Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration

RWE in Europe Paper III: A Roadmap for RWE

Real world evidence (RWE) has been touted as a remedy for current market access issues, facilitating quicker approvals and increased odds of reimbursement at a good price. It is therefore an attractive avenue for pursuit for manufacturers today. This paper, the third in a series looking at the use of RWE in Europe, outlines the discussions held between key opinion leaders in pricing and reimbursement across a number of European countries at a roundtable-style meeting. The aim of the meeting was to develop a 3-year roadmap, and resulting action plan, of initiatives for the enhanced use of RWE in decision-making in the pharmaceutical industry. Following a series of brainstorming sessions across the areas of commissioning and access, clinical evidence and patients and outcomes, contributors were asked to prioritise the importance of a refined set of initiatives identified in these brainstorming sessions to develop the three-year road map. Finally, four key points from the roadmap were identified for initial action: actively engage in early dialogue with payers on RWE needs; consensus exercise on RWD/E in clinical decisions, develop a definition of patient reported/relevant outcomes and develop a model approach for the collection of patient reported/relevant outcomes data. These action points are seen as the most imperative steps for enhancing the role of RWE. If its use is to become more common addressing these steps, as quickly and efficiently as possible, will be vital for all stakeholders in the pharmaceutical arena

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials

Opportunities and challenges for real-world studies on chronic inflammatory joint diseases through data enrichment and collaboration between national registers : the Nordic example

RMD Open 2018;4:e000655. doi:10.1136/ rmdopen-2018-000655Peer reviewe

Standards of genetic testing in the diagnosis and prognostication of systemic mastocytosis in 2022: Recommendations of the EU-US cooperative group

Mastocytosis comprises rare heterogeneous diseases characterized by an increased accumulation of abnormal mast cells in various organs/tissues. The pathogenesis of mastocytosis is strongly linked to the presence of KIT-activating mutations. In systemic mastocytosis (SM), the most frequent mutation encountered is KIT p.D816V, whose presence constitutes one of the minor diagnostic criteria. Different techniques are used to search and quantify the KIT p.D816V mutant; however, allele-specific quantitative PCR and droplet digital PCR are today the most sensitive. The analysis of the KIT p.D816V allele burden has undeniable interest for diagnostic, prognostic, and therapeutic monitoring. The analysis of non–mast cell hematological compartments in SM is similarly important because KIT p.D816V multilineage involvement is associated with a worse prognosis. In addition, in advanced forms of SM, mutations in genes other than KIT are frequently identified and affect negatively disease outcome and response to therapy. Thus, combined quantitative and sensitive analysis of KIT mutations and next-generation sequencing of other recurrently involved myeloid genes make it possible to better characterize the extent of the affected cellular compartments and additional molecular aberrations, providing a more detailed overview of the complex mutational landscape of SM, in relation with the clinical heterogeneity of the disease. In this article, we report the latest recommendations of the EU-US Cooperative Group presented in September 2020 in Vienna during an international working conference, on the techniques we consider standard to detect and quantify the KIT p.D816V mutant in SM and additional myeloid mutations found in SM subtypes.D.D.M., J.J.L., and M.C.C. were supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. P.V. was supported by the Austrian Science Fund (FWF) (grant nos. F4704-B20 and P32470-B)

Standards of pathology in the diagnosis of systemic mastocytosis: recommendations of the EU-US cooperative group

Pathology plays a central role in the diagnosis of systemic mastocytosis (SM), its delineation from other neoplasms and reactive conditions, and in monitoring of SM under therapy. The morphologic hallmark of SM is the accumulation of spindle-shaped, hypogranulated mast cells (MCs) in bone marrow (BM) and other extracutaneous tissues. Four of the 5 World Health Organization–defined diagnostic criteria (ie, compact MC aggregates [=major criterion]; atypical MC morphology; activating KIT point mutations; aberrant expression of CD25 and/or CD2 and/or CD30 in MCs [=minor criteria]) can be addressed by the pathologist. The final classification of SM variants as either BM mastocytosis, indolent SM, smoldering SM, aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), or MC leukemia (MCL) has important prognostic significance and requires the integration of certain morphological, clinical, radiological, and biochemical data, referred to as B- and C-findings. Substantial diagnostic challenges may be posed to the pathologist and clinician especially in the so-called advanced SM variants, that is, ASM, MCL, and SM-AHN. In this article, updated recommendations of the EU-US Cooperative Group regarding standards of pathology in the diagnosis of SM, presented during the year 2020 Working Conference held in September in Vienna, are reported.T. I. George was supported by the ARUP Institute for Clinical and Experimental Pathology. K. Hartmann was supported by the Swiss National Science Foundation, grant number 310030_207705. D. D. Metcalfe, J. J. Lyons, and M. Carter were supported by the Division of Intramural Research, National Institutes of Allergic and Infectious Diseases, National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not represent the official views of the NIH. P. Valent was supported by the Austrian Science Funds (FWF), projects F4701-B20 and F4704-B20

Human P2Y11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4+ and CD8+ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4+ and CD8+ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y11 receptor. In a recombinant expression system, the coexpression of the human P2Y11 receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y11 receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y11 receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y11 receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation