Biology subjects

Verhaegh, W.

Publications and source records attributed to Verhaegh, W..

3 recordsLinked to original sources

bioRxiv2021

Measuring host immune response status by simultaneous quantitative measurement of activity of signal transduction pathways that coordinate functional activity of immune cells from innate and adaptive immune system

For many diseases, including cancer, viral infections such as COVID-19, bacterial infections, and auto-immune diseases, the immune response is a major determinant of progression, response to therapy, and clinical outcome. Innate and adaptive immune response are controlled by coordinated activity of multiple immune cell types. The functional activity state of immune cells is determined by cellular signal transduction pathways (STPs). A novel mRNA-based signaling pathway assay platform has been developed to quantitatively measure relevant STP activities in all types of immune cells and mixed immune cell samples for experimental and diagnostic purposes. We generated a STP activity profile, termed Immune-Pathway Activity Profile (I-PAP), for a variety of immune cell types in resting and activated state, and provide a first example for use in patient samples. MethodsThe technology to measure STP activity has been described for androgen and estrogen receptor, PI3K, MAPK, TGF{beta}, Notch, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathways. STP activity was measured on Affymetrix expression microarray data from preclinical studies containing public data from different types of immune cells, resting/naive or immune-activated in vitro, to establish I-PAPs. Subsequently data from a clinical study on rheumatoid arthritis were analyzed. ResultsI-PAPs of naive/resting and immune-activated CD4+ and CD8+ T cells, T helper cells, B cells, NK cells, monocytes, macrophages, and dendritic cells were established and in agreement with known experimental immunobiology. In whole blood samples of rheumatoid arthritis patients TGF{beta} pathway activity was increased; JAK-STAT3 pathway activity was selectively increased in female patients. In naive CD4+ Tregs TGF{beta} pathway activity was increased, while in memory T effector cells JAK-STAT3 pathway activity tended to increase, suggesting that these immune cell types contributed to whole blood analysis results. ConclusionSTP assay technology (currently being converted to qPCR-based assays) makes it possible to directly measure functional activity of cells of the innate and adaptive immune response enabling quantitative assessment of the immune response of an individual patient. Envisioned utility lies in (1) prediction and monitoring of response to immunomodulatory treatments for a variety of immune-mediated diseases, including RA; (2) uncovering novel treatment targets; (3) improvement and standardization of in vitro immunology research and drug development.

immunology↗

bioRxiv2021

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways. MethodsWe previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data. ResultsCulture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGF{beta}, and NF{kappa}B pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells. ConclusionResults support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGF{beta}, Notch, and NF{kappa}B, and low Wnt pathway activity scores. Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

developmental biology↗

bioRxiv2020

Quantitative measurement of activity of JAK-STAT signaling pathways in blood samples and immune cells to predict innate and adaptive cellular immune response to viral infection and accelerate vaccine development.

The host immune response determines the clinical course of a viral infection, for example in case of COVID-19 infection. The effectiveness of vaccination also depends on the induced immune response. Currently there is no method to measure the cellular immune response in blood samples. The functional activity of cells of innate and adaptive immune system is determined by coordinated activity of signaling pathways, especially the JAK-STAT pathways. Using a previously described approach we developed mRNA-based tests to measure activity of these signaling pathways, and show that they can be used to measure in a quantitative manner the cellular innate and adaptive immune response to a viral infection or vaccine in whole blood, PBMC, and specific immune cell type samples. Pathway activity level and range in healthy individuals was established, enabling interpretation of a pathway activity score on a patient sample without the need for a reference sample. Evidence is presented that the pathway activity analysis may also be useful for in vitro vaccine development and assessment of vaccine immunogenicity. Other envisioned applications lie in development of immunomodulatory drugs and drug response prediction and monitoring. Tests are expected to be of value in the COVID-19 crisis. In addition to the described Affymetrix microarray-based pathway tests for measuring host immune response, qPCR-based versions are in development; the latter can in principle be performed within three hours in routine hospital labs.

immunology↗