Humanized hemato-lymphoid system mice

Over the last decades, incrementally improved xenograft mouse models, supporting the engraftment and development of a human hemato-lymphoid system, have been developed and now represent an important research tool in the field. The most significant contributions made by means of humanized mice are the identification of normal and leukemic hematopoietic stem cells, the characterization of the human hematopoietic hierarchy, and their use as preclinical therapy models for malignant hematopoietic disorders. Successful xenotransplantation depends on three major factors: tolerance by the mouse host, correct spatial location, and appropriately cross-reactive support and interaction factors such as cytokines and major histocompatibility complex molecules. Each of these can be modified. Experimental approaches include the genetic modification of mice to faithfully express human support factors as non-cross-reactive cytokines, to create free niche space, the co-transplantation of human mesenchymal stem cells, the implantation of humanized ossicles or other stroma, and the implantation of human thymic tissue. Besides the source of hematopoietic cells, the conditioning regimen and the route of transplantation also significantly affect human hematopoietic development in vivo. We review here the achievements, most recent developments, and the remaining challenges in the generation of pre-clinically-predictive systems for human hematology and immunology, closely resembling the human situation in a xenogeneic mouse environment

Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

Neutrophils are the most abundant innate immune cells and the first line of defense against many pathogenic microbes, including the human fungal pathogen Candida albicans. Among the neutrophils’ arsenal of effector functions, neutrophil extracellular traps (NETs) are thought to be of particular importance for trapping and killing the large fungal filaments by means of their web-like structures that consist of chromatin fibers decorated with proteolytic enzymes and host defense proteins. Peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histones in activated neutrophils correlates with chromatin decondensation and extrusion and is widely accepted to act as an integral process of NET induction (NETosis). However, the requirement of PAD4-mediated histone citrullination for NET release during C. albicans infection remains unclear. In this study, we show that although PAD4-dependent neutrophil histone citrullination is readily induced by C. albicans, PAD4 is dispensable for NETosis in response to the fungus and other common NET-inducing stimuli. Moreover, PAD4 is not required for antifungal immunity during mucosal and systemic C. albicans infection. Our results demonstrate that PAD4 is dispensable for C. albicans-induced NETosis, and they highlight the limitations of using histone citrullination as a marker for NETs and PAD4−/− mice as a model of NET-deficiency

Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic ("don't eat me") CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6C$^{hi}$ monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more "pro-phagocytic," suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages

PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy.

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard frontline drug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly(ADP-ribose) polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos" in acute myeloid leukemia (AML) cell lines (n = 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n = 10/10 tested), and primary cell samples from patients with AML (n = 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus -negative patient groups (hazard ratio [HR] = 0.28-0.37, p = 0.002-0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses

Bone marrow haematopoiesis in patients with COVID-19

AIMS Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection broadly affects organ homeostasis, including the haematopoietic system. Autopsy studies are a crucial tool for investigation of organ-specific pathologies. Here we perform an in-depth analysis of the impact of severe coronavirus disease 2019 (COVID-19) on bone marrow haematopoiesis in correlation with clinical and laboratory parameters. METHODS AND RESULTS Twenty-eight autopsy cases and five controls from two academic centres were included in the study. We performed a comprehensive analysis of bone marrow pathology and microenvironment features with clinical and laboratory parameters and assessed SARS-CoV-2 infection of the bone marrow by quantitative polymerase chain reaction (qPCR) analysis. In COVID-19 patients, bone marrow specimens showed a left-shifted myelopoiesis (19 of 28, 64%), increased myeloid-erythroid ratio (eight of 28, 28%), increased megakaryopoiesis (six of 28, 21%) and lymphocytosis (four of 28, 14%). Strikingly, a high proportion of COVID-19 specimens showed erythrophagocytosis (15 of 28, 54%) and the presence of siderophages (11 of 15, 73%) compared to control cases (none of five, 0%). Clinically, erythrophagocytosis correlated with lower haemoglobin levels and was more frequently observed in patients from the second wave. Analysis of the immune environment showed a strong increase in CD68+ macrophages (16 of 28, 57%) and a borderline lymphocytosis (five of 28, 18%). The stromal microenvironment showed oedema (two of 28, 7%) and severe capillary congestion (one of 28, 4%) in isolated cases. No stromal fibrosis or microvascular thrombosis was found. While all cases had confirmed positive testing of SARS-CoV-2 in the respiratory system, SARS-CoV-2 was not detected in the bone marrow by high-sensitivity PCR, suggesting that SARS-CoV-2 does not commonly replicate in the haematopoietic microenvironment. CONCLUSIONS SARS-CoV-2 infection indirectly impacts the haematological compartment and the bone marrow immune environment. Erythrophagocytosis is frequent and associated with lower haemoglobin levels in patients with severe COVID-19

Calreticulin mutations affect its chaperone function and perturb the glycoproteome

Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations. Keywords: CP: Cancer; CP: Molecular biology; calreticulin; chaperone; glycoprotein; limited proteolysis-coupled mass spectrometry; myeloperoxidase; myeloproliferative neoplasm; protein folding; proteome

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets. Keywords: calreticulin; epitope mapping; frameshift mutations; myeloproliferative neoplasms; peptide antibodies

Ruxolitinib in patients with polycythemia vera resistant and/or intolerant to hydroxyurea:European observational study

Background: Hydroxyurea (HU) is a commonly used first-line treatment in patients with polycythemia vera (PV). However, approximately 15%–24% of PV patients report intolerance and resistance to HU. Methods: This phase IV, European, real-world, observational study assessed the efficacy and safety of ruxolitinib in PV patients who were resistant and/or intolerant to HU, with a 24-month follow-up. The primary objective was to describe the profile and disease burden of PV patients. Results: In the 350 enrolled patients, 70% were &gt;60 years old. Most patients (59.4%) had received ≥1 phlebotomy in the 12 months prior to the first dose of ruxolitinib. Overall, 68.2% of patients achieved hematocrit control with 92.3% patients having hematocrit &lt;45% and 35.4% achieved hematologic remission at month 24. 85.1% of patients had no phlebotomies during the study. Treatment-related adverse events were reported in 54.3% of patients and the most common event was anemia (22.6%). Of the 10 reported deaths, two were suspected to be study drug-related. Conclusion: This study demonstrates that ruxolitinib treatment in PV maintains durable hematocrit control with a decrease in the number of phlebotomies in the majority of patients and was generally well tolerated.</p

The sympathomimetic agonist mirabegron did not lower JAK2-V617F allele burden, but restored nestin-positive cells and reduced reticulin fibrosis in patients with myeloproliferative neoplasms: results of phase II study SAKK 33/14

The β-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a β-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F positive MPN with a mutant allele burden >20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary endpoint, reduction of the JAK2-V617F allele burden ≥50%, was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematological improvement. As a side study, bone marrow biopsies were evaluated in 20 patients.We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (p<0.0001) and a slight decrease of reticulin fibrosis from a median grade of 1.0 (interquartile range 0-3) to 0.5 (interquartile range 0-2) (p=0.01) between start and end of mirabegron treatment. Despite the fact that the primary endpoint of reducing JAK2-V617F allele burden was not reached, the observed effects on nestin+ MSCs and reticulin fibrosis is encouraging and shows that mirabegron can modify the microenvironment where the JAK2-mutant stem cells are maintained