Monitoring Signal Transduction after Kidney Transplantation

The introduction of calcineurin inhibitors (CNIs) and mycophenolic acid (MPA) to the transplantation clinic improved the outcomes of kidney transplantation. Therapeutic drug monitoring of these immunosuppressive drugs is currently based on a pharmacokinetic approach that measures whole-blood (pre-dose) drug concentrations. Unfortunately, these measurements cannot accurately predict clinical outcomes after kidney transplantation, such as rejection, and have a poor correlation with long-term outcomes after transplantation. Pharmacodynamic monitoring is an alternative and possibly complimentary way for therapeutic drug monitoring that measures the biological effects of the drug at its target site. The research described in this thesis assessed novel techniques for pharmacodynamic monitoring of immunosuppressive therapy after kidney transplantation. Two promising methods are the phospho-specific flow cytometry assay and the intracellular monitoring of NFATc1 amplification. Phospho-specific flow cytometry measures the phosphorylation of signaling molecules involved in T cell activation, such as p-p38MAPK and p-ERK and p-Akt. The assay can be applied to monitor therapeutic drug effects on both T cells and monocytes. The phospho-specific flow cytometry assay also revealed that the currently prescribed immunosuppressive drugs have only a limited effect on intracellular monocyte activation pathways, demonstrating the need for new drugs that target monocytes after kidney transplantation. Another promising method for PD monitoring of tacrolimus in T cells is the analysis of NFATc1 amplification. Measuring NFATc1 amplification is a specific whole-blood test to monitor the biological effects of tacrolimus in T cells of kidney transplant recipients. Both the phospho-specific flow cytometry assay and the NFATc1 amplification assay can measure the activation of signaling molecules in different T cell subsets. In the future, when both assays are ready for routine use we recommend that these assays should be used in combination with pharmacokinetic measurements

The subtle spreading of sexist norms

Even when overt sexism and prejudice become rarer, social norms that perpetuate inequality are remarkably persistent. The present research lays out one of the subtle ways in which sexist norms may spread through society, by pointing to the role of responses to sexism. We investigate how third parties infer social norms about sexism when observing social interactions. In three studies among male students (Studies 1 and 2) and male and female students (Study 3), we demonstrate that subtle variations in how people respond to a sexist statement can have a substantial influence on inferences third parties make about sexist norms. Specifically, when a sexist statement is made and the conversation continues in a smoothly flowing fashion, third parties infer that this opinion is shared among interaction partners, perceived as appropriate, and that sexism is normative among them. However, when a sexist statement is followed by a brief silence that disrupts the flow of the conversation, observers think that it is contentious and that sexism is neither shared nor normative. Importantly, the effects of the manipulation generalized to the perception of sexist descriptive norms among male students in general. We conclude that social and cultural norms are not just inferred from conversation content, but also from conversational flow

Functional impairment of circulating FcεRI+ monocytes and myeloid dendritic cells in hepatocellular carcinoma and cholangiocarcinoma patients

Background Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response. Methods In this study, we have functionally characterized, by flow cytometry, circulating myeloid dendritic cells (mDCs) and FcεRI+ monocytes in a group of healthy individuals (n = 10) and in a group of patients with HCC (n = 19) and CCA (n = 8), at the time point of the surgical resection (T0) and once the patient had recovered from surgery (T1). Moreover, we proceeded to a more in depth phenotypic characterization of the FcεRI+ monocyte subpopulation. Results A significant decrease in the frequency of TNFα producing FcεRI+ monocytes and mDCs in HCC and CCA patients when compared to the group of healthy individuals was observed, and a close association between FcεRI+ monocytes and mDCs dysfunction was identified. In addition, the phenotypic characteristics of FcεRI+ monocytes from healthy individuals strongly suggest that this population drives to mDCs, which matches with the fact that both populations are functionally affected. Conclusions The frequency and the function of circulating mDCs and FcεRI+ monocytes are affected in both HCC and CCA patients, and FcεRI+ monocytes could represent those fated to become mDCs.publishe

Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes

Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma.

PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A (RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M‒positive patients, compared with 84.9% for 36 RASSF1A-M‒negative patients [P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel

Pharmacodynamic Monitoring of Tacrolimus-based Immunosuppression in CD14+ Monocytes after Kidney Transplantation

Background: Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type–specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. Methods: Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14+ monocytes was quantified by phospho-specific flow cytometry. Results: Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range −28% to 77% for p-p38MAPK and 20%; range −22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range −52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = −0.65; P = 0.01 and rs = −0.58; P = 0.03, respectively). Conclusions: Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type–specific manner

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls ( n = 40) and neuroblastoma patients with localized ( n = 10) and metastatic disease ( n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel ( PHOX2B, TH, CHRNA3) and a cell cycle regulation panel ( E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further

Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma

PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A ( RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M ‒positive patients, compared with 84.9% for 36 RASSF1A-M ‒negative patients [ P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel

Personalized therapy for mycophenolate:Consensus report by the international association of therapeutic drug monitoring and clinical toxicology

When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.</p