Cell-free DNA levels are increased in acute graft-versus-host disease.

BACKGROUND: Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown. OBJECTIVE: The aim of this study is to investigate the role of cfDNA as a marker of aGvHD. METHODS: We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction. RESULTS: In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells. CONCLUSION: We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.

Clinical usefulness of drug-laboratory test interaction alerts: a multicentre survey.

OBJECTIVES: Knowledge of possible drug-laboratory test interactions (DLTIs) is important for the interpretation of laboratory test results. Failure to recognize these interactions may lead to misinterpretation, a delayed or erroneous diagnosis, or unnecessary extra diagnostic tests or therapy, which may harm patients. The aim of this multicentre survey was to evaluate the clinical value of DLTI alerts. METHODS: A survey was designed with six predefined clinical cases selected from the clinical laboratory practice with a potential DLTI. Physicians from several departments, including internal medicine, cardiology, intensive care, surgery and geriatrics in six participating hospitals were recruited to fill in the survey. The survey addressed their knowledge of DLTIs, motivation to receive an alert and opinion on the potential influence on medical decision making. RESULTS: A total of 210 physicians completed the survey. Of these respondents 93% had a positive attitude towards receiving DLTI alerts; however, the reported value differed per case and per respondent's background. In each clinical case, medical decision making was influenced as a consequence of the reported DLTI message (ranging from 3 to 45% of respondents per case). CONCLUSIONS: In this multicentre survey, most physicians stated DLTI messages to be useful in laboratory test interpretation. Medical decision making was influenced by reporting DLTI alerts in each case. Alerts should be adjusted according to the needs and preferences of the receiving physicians.

Mesenchymal Stromal Cells Stimulate the Proliferation and IL-22 Production of Group 3 Innate Lymphoid Cells.

Infusion of mesenchymal stromal cells (MSCs) is a promising and increasingly applied therapy for patients who suffer from a variety of inflammatory diseases, including graft-versus-host disease (GvHD), a common and life-threatening complication after allogeneic hematopoietic stem cell transplantation. The therapeutic effect of MSCs is mainly ascribed to their ability to suppress T cells and to support tissue repair. However, clinical response rates in patients with GvHD are limited to 50%, and the determinants for MSC responsiveness are unknown. We recently reported that high frequencies of activated group 3 innate lymphoid cells (ILC3s) before and after allogeneic hematopoietic stem cell transplantation were associated with a lower risk of GvHD. This may be related to IL-22 production by ILC3s, a cytokine important for intestinal epithelial cell homeostasis. In this study, we investigated whether ILC3s may contribute to the therapeutic effect of MSCs by studying the interaction between MSCs and ILC3s in vitro. ILC3s isolated from human tonsils were cocultured with human bone marrow-derived MSCs for 5 d in the presence of IL-2. Coculture with MSCs enhanced the proliferation and IL-22 production of ILC3s. Reciprocally, ILC3s promoted ICAM-1 and VCAM-1 expression on MSCs. For both directions, the activation was mainly mediated by cell-cell contact and by MSC-derived IL-7 and likely by aryl hydrocarbon receptor ligands. Thus, in addition to inhibiting the proliferation of alloreactive T cells, MSCs also promote the expansion and IL-22 production of ILC3s, which may contribute to healthy homeostasis and wound repair in the treatment of various inflammatory conditions in the intestine, including GvHD.

Nuclear shape, protrusive behaviour and in vivo retention of human bone marrow mesenchymal stromal cells is controlled by Lamin-A/C expression.

Culture expanded mesenchymal stromal cells (MSCs) are being extensively studied for therapeutic applications, including treatment of graft-versus-host disease, osteogenesis imperfecta and for enhancing engraftment of hematopoietic stem cells after transplantation. Thus far, clinical trials have shown that the therapeutic efficiency of MSCs is variable, which may in part be due to inefficient cell migration. Here we demonstrate that human MSCs display remarkable low migratory behaviour compared to other mesodermal-derived primary human cell types. We reveal that specifically in MSCs the nucleus is irregularly shaped and nuclear lamina are prone to wrinkling. In addition, we show that expression of Lamin A/C is relatively high in MSCs. We further demonstrate that in vitro MSC migration through confined pores is limited by their nuclei, a property that might correlate to the therapeutic inefficiency of administered MSC in vivo. Silencing expression of Lamin A/C in MSCs improves nuclear envelope morphology, promotes the protrusive activity of MSCs through confined pores and enhances their retention in the lung after intravenous administration in vivo. Our findings suggest that the intrinsic nuclear lamina properties of MSCs underlie their limited capacity to migrate, and that strategies that target the nuclear lamina might alter MSC-based cellular therapies.

Interferon-Gamma Impairs Maintenance and Alters Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells.

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.

Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates.

Here, we characterize a subset of ILC3s that express Neuropilin1 (NRP1) and are present in lymphoid tissues, but not in the peripheral blood or skin. NRP1+ group 3 innate lymphoid cells (ILC3s) display in vitro lymphoid tissue inducer (LTi) activity. In agreement with this, NRP1+ ILC3s are mainly located in proximity to high endothelial venules (HEVs) and express cell surface molecules involved in lymphocyte migration in secondary lymphoid tissues via HEVs. NRP1 was also expressed on mouse fetal LTi cells, indicating that NRP1 is a conserved marker for LTi cells. Human NRP1+ ILC3s are primed cells because they express CD45RO and produce higher amounts of cytokines than NRP1- cells, which express CD45RA. The NRP1 ligand vascular endothelial growth factor A (VEGF-A) served as a chemotactic factor for NRP1+ ILC3s. NRP1+ ILC3s are present in lung tissues from smokers and patients with chronic obstructive pulmonary disease, suggesting a role in angiogenesis and/or the initiation of ectopic pulmonary lymphoid aggregates.

The Potential of Mesenchymal Stromal Cells as Treatment for Severe Steroid-Refractory Acute Graft-Versus-Host Disease: A Critical Review of the Literature.

BACKGROUND: Acute graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic stem cell transplantation which causes high morbidity and mortality among patients who do not respond to steroid treatment. Mesenchymal stromal cells (MSCs) have immune modulatory abilities and earned their place in the treatment of GvHD after a pediatric patient remarkably recovered from steroid-refractory acute GvHD with MSC salvage therapy. Large, prospective clinical trials evaluating the potency of MSCs have however not been published. METHODS: To evaluate the therapeutic potential of MSCs in the treatment of steroid-refractory acute GvHD, we conducted a systematic literature search. We included all studies that focused on MSC treatment of adult allogeneic hematopoietic stem cell transplantation recipients with grades III to IV steroid-refractory acute GvHD and were transparent about their methods and patient selection criteria. RESULTS: From a total of 255 articles, 9 articles met the quality criteria for this study. The proportion of patients achieving complete resolution of all symptoms (complete response, CR) varied between 8% and 83%. Four of the 9 studies reported CR rates above 50%. The GvHD grade at the time of treatment was identified as a predictor of clinical response. Interestingly, complete response but not partial response to MSCs was associated with overall survival. No serious side effects of MSC therapy were reported. CONCLUSIONS: MSC treatment does improve the outcome in steroid-refractory acute GvHD patients but well-designed, prospective randomized clinical trials are needed to confirm the potential of MSCs as salvage therapy for steroid-refractory GvHD and to identify those patients that will benefit most.

Organ-specific migration of mesenchymal stromal cells: Who, when, where and why?

Mesenchymal stromal cells (MSC) represent a type of multipotent cells that can differentiate to various mesenchymal lineages. MSC can be isolated from different tissues and require ex vivo expansion to exert their regenerative and immunosuppressive function for various clinical applications. The efficacy of these MSC-based therapies at least partly depends on migration and specific homing towards the site where the cells are needed. MSC express a wide variety of integrins, chemokine- and growth factor receptors, though culture-expansion dramatically alters their migratory and engraftment potential. However, it has become clear that tissue damage and/or inflammation can enhance the efficacy of MSC homing. In this review, we focus on the migratory potential of MSC to target organs, including bone marrow, bone, spleen & lymph nodes, intestine and heart, and the underlying molecular mechanisms in various preclinical and clinical settings. Better understanding of directed MSC migration will offer new perspectives to modulate MSC expansion and/or clinical protocols to improve their efficacy upon transplantation.