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.

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.

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 diet is associated with distinct preclinical pediatric NAFLD phenotypes in juvenile nonhuman primate offspring.

Pediatric NAFLD has distinct and variable pathology, yet causation remains unclear. We have shown that maternal Western-style diet (mWSD) compared with maternal chow diet (CD) consumption in nonhuman primates produces hepatic injury and steatosis in fetal offspring. Here, we define the role of mWSD and postweaning Western-style diet (pwWSD) exposures on molecular mechanisms linked to NAFLD development in a cohort of 3-year-old juvenile nonhuman primates offspring exposed to maternal CD or mWSD followed by CD or Western-style diet after weaning. We used histologic, transcriptomic, and metabolomic analyses to identify hepatic pathways regulating NAFLD. Offspring exposed to mWSD showed increased hepatic periportal collagen deposition but unchanged hepatic triglyceride levels and body weight. mWSD was associated with a downregulation of gene expression pathways underlying HNF4α activity and protein, and downregulation of antioxidant signaling, mitochondrial biogenesis, and PPAR signaling pathways. In offspring exposed to both mWSD and pwWSD, liver RNA profiles showed upregulation of pathways promoting fibrosis and endoplasmic reticulum stress and increased BiP protein expression with pwWSD. pwWSD increased acylcarnitines and decreased anti-inflammatory fatty acids, which was more pronounced when coupled with mWSD exposure. Further, mWSD shifted liver metabolites towards decreased purine catabolism in favor of synthesis, suggesting a mitochondrial DNA repair response. Our findings demonstrate that 3-year-old offspring exposed to mWSD but weaned to a CD have periportal collagen deposition, with transcriptional and metabolic pathways underlying hepatic oxidative stress, compromised mitochondrial lipid sensing, and decreased antioxidant response. Exposure to pwWSD worsens these phenotypes, triggers endoplasmic reticulum stress, and increases fibrosis. Overall, mWSD exposure is associated with altered expression of candidate genes and metabolites related to NAFLD that persist in juvenile offspring preceding clinical presentation of NAFLD.

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.

Maternal Western diet exposure increases periportal fibrosis beginning in utero in nonhuman primate offspring.

Maternal obesity affects nearly one-third of pregnancies and is a major risk factor for nonalcoholic fatty liver disease (NAFLD) in adolescent offspring, yet the mechanisms behind NAFLD remain poorly understood. Here, we demonstrate that nonhuman primate fetuses exposed to maternal Western-style diet (WSD) displayed increased fibrillar collagen deposition in the liver periportal region, with increased ACTA2 and TIMP1 staining, indicating localized hepatic stellate cell (HSC) and myofibroblast activation. This collagen deposition pattern persisted in 1-year-old offspring, despite weaning to a control diet (CD). Maternal WSD exposure increased the frequency of DCs and reduced memory CD4+ T cells in fetal liver without affecting systemic or hepatic inflammatory cytokines. Switching obese dams from WSD to CD before conception or supplementation of the WSD with resveratrol decreased fetal hepatic collagen deposition and reduced markers of portal triad fibrosis, oxidative stress, and fetal hypoxemia. These results demonstrate that HSCs and myofibroblasts are sensitive to maternal WSD-associated oxidative stress in the fetal liver, which is accompanied by increased periportal collagen deposition, indicative of early fibrogenesis beginning in utero. Alleviating maternal WSD-driven oxidative stress in the fetal liver holds promise for halting steatosis and fibrosis and preventing developmental programming of NAFLD.

Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport.

Secretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.

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.

Early Microbes Modify Immune System Development and Metabolic Homeostasis-The "Restaurant" Hypothesis Revisited.

The developing infant gut microbiome affects metabolism, maturation of the gastrointestinal tract, immune system function, and brain development. Initial seeding of the neonatal microbiota occurs through maternal and environmental contact. Maternal diet, antibiotic use, and cesarean section alter the offspring microbiota composition, at least temporarily. Nutrients are thought to regulate initial perinatal microbial colonization, a paradigm known as the "Restaurant" hypothesis. This hypothesis proposes that early nutritional stresses alter both the initial colonizing bacteria and the development of signaling pathways controlled by microbial mediators. These stresses fine-tune the immune system and metabolic homeostasis in early life, potentially setting the stage for long-term metabolic and immune health. Dysbiosis, an imbalance or a maladaptation in the microbiota, can be caused by several factors including dietary alterations and antibiotics. Dysbiosis can alter biological processes in the gut and in tissues and organs throughout the body. Misregulated development and activity of both the innate and adaptive immune systems, driven by early dysbiosis, could have long-lasting pathologic consequences such as increased autoimmunity, increased adiposity, and non-alcoholic fatty liver disease (NAFLD). This review will focus on factors during pregnancy and the neonatal period that impact a neonate's gut microbiome, as well as the mechanisms and possible links from early infancy that can drive increased risk for diseases including obesity and NAFLD. The complex pathways that connect diet, the microbiota, immune system development, and metabolism, particularly in early life, present exciting new frontiers for biomedical research.

Acquired haemophilia: an easy diagnosis to miss in a patient taking warfarin.

Acquired Haemophilia (AH) is an autoimmune bleeding disorder, which despite being rare, can be fatal. It occurs in patients with previously normal haemostasis who spontaneously develop IgG autoantibodies against factor VIII. Unlike congenital haemophilia, it manifests as spontaneous bleeding into skin and soft tissues. The presentation can be masked in patients who are receiving warfarin where the bleeding is often attributed to warfarin therapy, as in the case described in this report. Consideration of AH is important in patients taking anticoagulants, when coagulopathy and bleeding fails to correct with usual measures.

The anticoagulation of calf thrombosis (ACT) project: results from the randomized controlled external pilot trial.

BACKGROUND: There is currently little evidence defining the clinical importance of detecting and treating isolated distal DVT (IDDVT). International guidelines vary regarding diagnostic and therapeutic advice. The potential benefits of anticoagulation are unquantified. We sought to evaluate the feasibility of a randomized controlled study within a modern framework and provide a primary outcome point estimate. METHODS: In this open-label, external pilot randomized controlled trial, consecutive, symptomatic, ambulatory patients with IDDVT were approached for inclusion. Participants were allocated to receive either therapeutic anticoagulation or conservative management. Patients underwent blinded color-duplex imaging at 7 and 21 days and follow-up at 3 months. Principal feasibility outcomes included recruitment rate and attrition. The principal clinical outcome was a composite including proximal propagation, pulmonary embolism, death attributable to VTE disease, or major bleeding. Analysis was by intention to treat. RESULTS: In total, 93 patients with IDDVT were screened, and 70 of those eligible (88.6%) were recruited. All patients but one were followed-up by direct contact after 90 days. Allocation crossover occurred in 15 patients (21.4%). The principal clinical outcome occurred in four of 35 of those conservatively treated (11.4%) and zero of 35 in the anticoagulated group (absolute risk reduction, 11.4%; 95% CI, -1.5 to 26.7, P = .11, number needed to treat of nine). There were no major bleeding episodes. CONCLUSIONS: We have established the feasibility of definitive study regarding the value of therapeutic anticoagulation in IDDVT and provide an approximate point estimate for serious complications with a contemporary conservative strategy. TRIAL REGISTRY: Current Controlled Trials; No.: ISRCTN75175695; URL: www.controlled-trials.com.

Single whole-leg compression ultrasound for exclusion of deep vein thrombosis in symptomatic ambulatory patients: a prospective observational cohort study.

International guidance has recently recommended serial proximal compression ultrasound (CUS) as first line imaging for suspected deep vein thrombosis (DVT). Single whole-leg CUS is a routine alternative diagnostic strategy that can reduce repeated attendances and identify alternative pathology. We conducted a prospective observational cohort study. Consecutive ambulatory, adult patients with suspected DVT and negative or inconclusive whole-leg CUS had anticoagulation withheld and were followed for 3 months. The primary outcome was a predefined clinically relevant adverse event rate. Secondary outcomes included technical failure, alternative diagnoses and all cause mortality. 212 patients agreed to participate and completed follow up. One patient was subsequently diagnosed with an isolated distal DVT. The adverse event rate was thus 1/212, 0·47% (95% confidence interval [CI] 0·08-2·62). Technical imaging failure occurred in 11·3% of cases (95% CI 7·7-16·3). Several potential predictors of an inconclusive result were identified on multivariate analysis. 150 (70·8%) patients were provided with a documented alternative diagnosis. Patients who have anticoagulation withheld following a negative or inconclusive whole-leg CUS for suspected DVT have a low rate of adverse events. Technical failure remains an issue: several factors were significantly associated with inconclusive results and may warrant an alternative diagnostic approach.

Anticoagulation in chronic kidney disease patients-the practical aspects.

There is an increasing awareness about the risks of arterial and venous thromboembolism (TE) in hospital patients and general public which has led to consideration of thrombosis prevention measures in earnest. Early recognition of the symptoms of TE disease has led to timely administration of antiplatelet and anticoagulant drugs, translating to better outcome in many of these patients. In this respect, patients with chronic kidney disease (CKD) represent a special group. They indeed represent a high-risk group for thrombosis both in the cardiovascular territory and also in the venous circulation. At the same time, abnormalities in the platelet membranes put them at risk of bleeding which is significantly more than other patients with chronic diseases. Anticoagulation may be ideal to prevent the former, but the co-existing bleeding risk and also that the commonly used drugs for inhibiting coagulation are eliminated by renal pathways pose additional problems. In this review, we try to explain the complex thrombotic-haemorrhagic state of chronic kidney disease patients, and practical considerations for the management of anticoagulation in them with a focus on heparins.

The Anticoagulation of Calf Thrombosis (ACT) project: study protocol for a randomized controlled trial.

BACKGROUND: Half of all lower limb deep vein thrombi (DVT) in symptomatic ambulatory patients are located in the distal (calf) veins. While proximal disease warrants therapeutic anticoagulation to reduce the associated risks, distal DVT often goes untreated. However, a proportion of untreated distal disease will undoubtedly propagate or embolize. Concern also exists that untreated disease could lead to long-term post thrombotic changes. Currently, it is not possible to predict which distal thrombi will develop such complications. Whether these potential risks outweigh those associated with unrestricted anticoagulation remains unclear. The Anticoagulation of Calf Thrombosis (ACT) trial aims to compare therapeutic anticoagulation against conservative management for patients with acute symptomatic distal deep vein thrombosis. METHODS: ACT is a pragmatic, open-label, randomized controlled trial. Adult patients diagnosed with acute distal DVT will be allocated to either therapeutic anticoagulation or conservative management. All patients will undergo 3 months of clinical and assessor blinded sonographic follow-up, followed by 2-year final review. The project will commence initially as an external pilot study, recruiting over a 16-month period at a single center to assess feasibility measures and clinical event rates. Primary outcome measures will assess feasibility endpoints. Secondary clinical outcomes will be collected to gather accurate data for the design of a definitive clinical trial and will include: (1) a composite endpoint combining thrombus propagation to the popliteal vein or above, development of symptomatic pulmonary embolism or sudden death attributable to venous thromboembolic disease; (2) the incidence of major and minor bleeding episodes; (3) the incidence of post-thrombotic leg syndrome at 2 years using a validated screening tool; and (4) the incidence of venous thromboembolism (VTE) recurrence at 2 years. DISCUSSION: The ACT trial will explore the feasibility of comparing therapeutic anticoagulation to conservative management in acute distal DVT, within a modern cohort. We also aim to provide contemporary data on clot propagation, bleeding rates and long-term outcomes within both groups. These results will inform the conduct of a definitive study if feasibility is established.