Isolating mononuclear cells from fetal bone and liver for metabolic, functional, and immunophenotypic analyses in nonhuman primates.

Studying fetal hematopoiesis is challenging as hematopoiesis transitions from the liver to bone marrow. Obtaining human samples is not possible, and small animal models may not provide sufficient biological material. Here, we present a protocol for isolating hematopoietic cells from the nonhuman primate fetal liver and bone. We describe steps for using cells from the same fetus for fluorescence lifetime imaging microscopy to measure metabolism, assessing cellular function, and flow cytometry for immunophenotyping at the single-cell level. For complete details on the use and execution of this protocol, please refer to Nash et al. (2023).1.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Maternal Western-style diet remodels the transcriptional landscape of fetal hematopoietic stem and progenitor cells in rhesus macaques.

Maternal obesity adversely impacts the in utero metabolic environment, but its effect on fetal hematopoiesis remains incompletely understood. During late development, the fetal bone marrow (FBM) becomes the major site where macrophages and B lymphocytes are produced via differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we analyzed the transcriptional landscape of FBM HSPCs at single-cell resolution in fetal macaques exposed to a maternal high-fat Western-style diet (WSD) or a low-fat control diet. We demonstrate that maternal WSD induces a proinflammatory response in FBM HSPCs and fetal macrophages. In addition, maternal WSD consumption suppresses the expression of B cell development genes and decreases the frequency of FBM B cells. Finally, maternal WSD leads to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/- mice. Collectively, these data demonstrate for the first time that maternal WSD impairs fetal HSPC differentiation and function in a translationally relevant nonhuman primate model.

A level-headed approach to measuring direct oral anticoagulants: A 2-year retrospective analysis of DOAC levels from a tertiary UK centre.

INTRODUCTION: Direct oral anticoagulants (DOAC) are commonly prescribed and measuring drug levels may be useful in a number of contexts. However, data on DOAC level measurement and their clinical utility in real-world studies are limited. METHODS: We carried out a 2-year retrospective cohort study of DOAC levels measured at our institution. RESULTS: One hundred and sixty-nine levels measured in 113 patients were included in the final analysis. Our patients had a median age of 69.9. AF was the commonest indication for anticoagulation. Median turnaround time for inpatient levels was 92 minutes. Median FXa inhibitor levels within 6 hours of last dose and following one half-life were similar to those described previously. However, the range of levels was wider than expected. Importantly, some levels remained in an on therapy range even after 3 half-lives. There was no correlation between dabigatran level and time from last dose. The reason for request varied with setting; 23 outpatient levels were to monitor drug efficacy, whereas 54 and 43 inpatient levels were collected in the context of bleeding and emergency surgery respectively. 60.3% of levels had an impact on clinical decision making. CONCLUSION: Our real-world study demonstrates that DOAC levels can be performed in a timely manner to influence clinical decision making. In addition, it suggests there is a wide variation in levels such that it can be difficult to predict in the real world. Overall, this supports the wider use of DOAC levels to help guide clinicians in managing patients taking these drugs.

Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years.

Essential thrombocythemia (ET) is heterogeneous with respect to natural history, X-chromosome inactivation patterns (XCIPs), and presence of the V617F mutation in Janus kinase 2 (JAK2). We studied 111 patients with ET; 39% were JAK2 mutant positive, and clone size (percentage mutant JAK2) was concordant with XCIP when constitutive T-cell patterns were taken into account. JAK2 mutant clones were present in both clonal and polyclonal cases as determined by XCIP, and the former had higher mutant JAK2 levels (median 26% versus 16%; P=.001). No change was observed in serial XCIP analysis of 14 polyclonal patients over a median follow-up of 61 months. Furthermore, 18 of 19 mutant-positive patients showed no significant change in mutant JAK2 level over a median follow-up of 47 months. These results suggest that, in many cases of ET, a small stable clone containing a JAK2 mutation can be maintained as a subpopulation for many years.

No mutations in the GATA-1 gene detected in patients with acquired essential thrombocythemia.

Mutations in the GATA-1 gene have been identified in patients with familial macrothrombocytopenia and Down's syndrome patients with a transient myeloproliferative disorder and/or acute megakaryoblastic leukemia. We screened this gene in 46 patients with essential thrombocythemia and identified only a common single nucleotide polymorphism that is unlikely to be of pathological significance.