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Researchers in the Simmons lab chart the embryonic origins and appearance of diverse intestinal cellular compartments, with important implications for intestinal diseases.
An Overview of Targeted Therapies in Acute Myeloid Leukemia.
Acute myeloid leukemia (AML) is the most aggressive adult leukemia, characterized by clonal differentiation arrest of progenitor or precursor hematopoietic cells. Intense preclinical and clinical research has led to regulatory approval of several targeted therapeutics, administered either as single agents or as combination therapies. However, the majority of patients still face a poor prognosis and disease relapse frequently occurs due to selection of therapy-resistant clones. Hence, more effective novel therapies, most likely as innovative, rational combination therapies, are urgently needed. Chromosomal aberrations, gene mutations, and epigenetic alterations drive AML pathogenesis but concurrently provide vulnerabilities to specifically target leukemic cells. Other molecules, either aberrantly active and/or overexpressed in leukemic stem cells, may also be leveraged for therapeutic benefit. This concise review of targeted therapies for AML treatment, which are either approved or are being actively investigated in clinical trials or recent preclinical studies, provides a flavor of the direction of travel, but also highlights the current challenges in AML treatment.
Serologic Response to Borrelia Antigens Varies with Clinical Phenotype in Children and Young Adults with Lyme Disease.
Lyme disease is commonly diagnosed by serologic response to Borrelia burgdorferi and related species, but the relationship between serologic targets and clinical features is unknown. We developed a multiantigen Luminex-based panel and evaluated IgG responses in 527 children 1 to 21 years of age assessed for Lyme disease across 4 Pedi Lyme Net emergency departments, including 127 Lyme cases defined by either an erythema migrans (EM) lesion or positive C6 enzyme immunoassay followed by immunoblotting and 400 patients considered clinical mimics. Of 42 antigens tested, 26 elicited specific reactivity in Lyme patients without marked age-dependent variation. Children with single EM lesions typically lacked Borrelia-specific IgG. By principal-component analysis, children with early disseminated and late Lyme disease clustered separately from clinical mimics and also from each other. Neurological disease and arthritis exhibited distinct serologic responses, with OspC variants overrepresented in neurological disease and p100, BmpA, p58, and p45 overrepresented in arthritis. Machine learning identified a 3-antigen panel (VlsE_Bb, p41_Bb, and OspC_Bafz) that distinguished Lyme disease from clinical mimics with a sensitivity of 86.6% (95% confidence interval [CI], 80.3 to 92.1) and a specificity of 95.5% (95% CI, 93.4 to 97.4). Sensitivity was much lower in early Lyme disease (38.5%; 95% CI, 15.4 to 69.2). Interestingly, 17 children classified as Lyme mimics had a positive 3-antigen panel, suggesting that more comprehensive serologic analysis could help refine Lyme diagnosis. In conclusion, multiplex antigen panels provide a novel approach to understanding the immune response in Lyme disease, potentially helping to facilitate accurate diagnosis and to understand differences between clinical stages.
Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint.
OBJECTIVE: Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. METHODS: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. RESULTS: Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture. CONCLUSIONS: Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.
The neutrotime transcriptional signature defines a single continuum of neutrophils across biological compartments.
Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.
Neutrophil transit time and localization within the megakaryocyte define morphologically distinct forms of emperipolesis.
Neutrophils transit through megakaryocytes in a process termed emperipolesis, but it is unknown whether this interaction is a single type of cell-in-cell interaction or a set of distinct processes. Using a murine in vitro model, we characterized emperipolesis by live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome to enter the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or the perinuclear endoplasmic reticulum. These findings reveal that megakaryocyte emperipolesis reflects at least 2 distinct processes differing in transit time and morphology, fast and slow emperipolesis, suggesting divergent physiologic functions.
A protocol for simultaneous high-sensitivity genotyping and chromatin accessibility profiling in single cells.
Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) resolves the heterogeneity of epigenetic states across cells but does not typically capture exonic mutations, which limits our knowledge of how somatic mutations alter chromatin landscapes. Here, we present a plate-based approach coupling high-sensitivity genotyping of genomic loci with high-content scATAC-seq libraries from the same single cells. We first describe steps for optimization of genotyping primers, followed by detailed guidance on the preparation of both scATAC-seq and single-cell genotyping libraries, fully automated on high-throughput liquid handling platforms. For complete details on the use and execution of this protocol, please refer to Turkalj, Jakobsen et al.1.
Rapid clonal selection within early hematopoietic cell compartments presages outcome to ivosidenib combination therapy.
Acquired resistance to targeted, non-intensive therapies is common in myeloid malignancies. However, the kinetics of selection, the hematopoietic cell compartments where selection occurs, and the molecular mechanisms underlying selection remain open questions. To address this, we studied the kinetics of clonal and transcriptional responses to ivosidenib + venetoclax ± azacitidine combination therapy across hematopoiesis in 8 patients with IDH1-mutant myeloid malignancy. All 8 patients initially responded to treatment but 6 relapsed while 2 remained in sustained remission for >4 years. We performed combined high-sensitivity single-cell (sc) genotyping and scRNA-seq in index-sorted sequential patient samples. In all patients, clonal selection occurred rapidly, within 1-3 treatment cycles. Clonal selection preceded treatment failure by months to years. Relapse was associated with expansion of either clones harboring newly-detected myeloid driver mutations or pre-existing minor clones that underwent differentiation delay upon treatment exposure. In both cases, clonal selection occurred within immature cell populations previously shown to contain leukemic stem cell (LSC) potential. Different genetic alterations within relapse-associated clones converged onto common upregulated transcriptional programs of stemness, branched-chain amino acid catabolism, and genes sensitive to menin inhibition. Importantly, this relapse-associated transcriptional signature was selected within 3 cycles of therapy. In contrast, in both patients remaining in remission, leukemic clones were rapidly eradicated and replaced by clonal and wild-type hematopoiesis. Overall, in patients treated with ivosidenib combination therapy, rapid clonal selection occurs within the first treatment cycles. In those patients destined to relapse, genetically heterogeneous resistant clones are characterized by common transcriptional programs.
Human and mouse neutrophils share core transcriptional programs in both homeostatic and inflamed contexts.
Neutrophils are frequently studied in mouse models, but the extent to which findings translate to humans remains poorly defined. In an integrative analysis of 11 mouse and 13 human datasets, we find a strong correlation of neutrophil gene expression across species. In inflammation, neutrophils display substantial transcriptional diversity but share a core inflammation program. This program includes genes encoding IL-1 family members, CD14, IL-4R, CD69, and PD-L1. Chromatin accessibility of core inflammation genes increases in blood compared to bone marrow and further in tissue. Transcription factor enrichment analysis implicates members of the NF-κB family and AP-1 complex as important drivers, and HoxB8 neutrophils with JunB knockout show a reduced expression of core inflammation genes in resting and activated cells. In independent single-cell validation data, neutrophil activation by type I or type II interferon, G-CSF, and E. coli leads to upregulation in core inflammation genes. In COVID-19 patients, higher expression of core inflammation genes in neutrophils is associated with more severe disease. In vitro treatment with GM-CSF, LPS, and type II interferon induces surface protein upregulation of core inflammation members. Together, we demonstrate transcriptional conservation in neutrophils in homeostasis and identify a core inflammation program shared across heterogeneous inflammatory conditions.
Stabilization of the prevalence of childhood obesity in Switzerland.
BACKGROUND: The prevalence of childhood obesity is increasing rapidly in most industrialised countries, but several countries, including Switzerland, have recently reported a levelling off or even a reversal of this alarming trend. STUDY AIM: Our aim was to evaluate the prevalence of childhood obesity in a national sample of Swiss school children recruited to assess iodine nutrition and with this to reconfirm a recently shown stabilising trend. DESIGN: Using a probability-proportionate-to-size cluster sampling, 6 to 12 year old children (n = 907) were recruited in a study of iodine status in Switzerland. Height and weight were measured and body mass index calculated in all children. RESULTS: The prevalence of overweight detected was 12.5% (SE = 1.6) in boys and 12.8% (1.6) in girls while 6.2% (1.1) of boys and 4.2% (0.9) of girls were classified as obese. The highest prevalence of overweight and obesity were found in the Southern region and in bigger cities (>100000 inhabitants). Compared to the most recent national study in 2007, the prevalence was slightly higher, but the differences were not significant, suggesting negative sampling bias does not strongly affect surveys of paediatric adiposity in Switzerland. CONCLUSION: This present study clearly confirms the stabilisation of the prevalence of childhood overweight and obesity in Switzerland.
Iodine requirements and the risks and benefits of correcting iodine deficiency in populations.
Iodine deficiency has multiple adverse effects on growth and development due to inadequate thyroid hormone production that are termed the iodine deficiency disorders (IDD). IDD remains the most common cause of preventable mental impairment worldwide. IDD assessment methods include urinary iodine concentration, goiter, thyroglobulin and newborn thyrotropin. In nearly all iodine-deficient countries, the best strategy to control IDD is salt iodization, one of the most cost-effective ways to contribute to economic and social development. When salt iodization is not possible, iodine supplements can be targeted to vulnerable groups. Introduction of iodized salt to regions of chronic IDD may transiently increase the incidence of thyroid disorders, and programs should include monitoring for both iodine deficiency and excess. Although more data on the epidemiology of thyroid disorders caused by differences in iodine intake are needed, overall, the relatively small risks of iodine excess are far outweighed by the substantial risks of iodine deficiency.
The influence of iron status on iodine utilization and thyroid function.
Despite significant progress, deficiencies of iron and iodine remain major public health problems affecting > or =30% of the global population. These deficiencies often coexist in children. Recent studies have demonstrated that a high prevalence of iron deficiency among children in areas of endemic goiter may reduce the effectiveness of iodized salt programs. These findings argue strongly for improving iron status in areas of overlapping deficiency, not only to combat anemia but also to increase the efficacy of iodine prophylaxis. The dual fortification of salt with iodine and iron may prove to be an effective and sustainable method to accomplish these important goals.
Comparison of manual and automated ELISA methods for serum ferritin analysis.
Serum ferritin concentration is a sensitive measure of body iron stores. The aim of this study was to compare the performance of two commercially available enzyme-linked immunoassays (ELISAs) for serum ferritin: a widely used manual assay kit (Spectro Ferritin MT), and a new fully automated assay (Immulite). We analyzed serum samples from Moroccan school-aged children (n=51) from a rural area with a high prevalence of iron deficiency anemia (IDA). Four replicates of each sample were analyzed using both assays. For the manual method, the interassay repeatability was 24%, 22%, and 11%, and intraassay precision was 18.3%, 9.2%, and 9.1% at increasing serum ferritin concentrations. Using the automated assay, the interassay repeatability was 7%, 6%, and 6%, and intraassay precision was 1.5%, 5.4%, and 5.5% at increasing serum ferritin concentrations. The two assays were well correlated (y=1.16x+1.83; r=0.98). However, the limits of agreement (LOAs) were wide, particularly at low concentrations. A comparison of the assay results with recommended cutoffs for serum ferritin generated sharply different estimates of the prevalence of iron deficiency (ID) in the sample. We conclude that the automated assay has several potential advantages compared to the manual method, including better precision, less operator dependence, and faster sample through-put.
The effects of iron fortification on the gut microbiota in African children: a randomized controlled trial in Cote d'Ivoire.
BACKGROUND: Iron is essential for the growth and virulence of many pathogenic enterobacteria, whereas beneficial barrier bacteria, such as lactobacilli, do not require iron. Thus, increasing colonic iron could select gut microbiota for humans that are unfavorable to the host. OBJECTIVE: The objective was to determine the effect of iron fortification on gut microbiota and gut inflammation in African children. DESIGN: In a 6-mo, randomized, double-blind, controlled trial, 6-14-y-old Ivorian children (n = 139) received iron-fortified biscuits, which contained 20 mg Fe/d, 4 times/wk as electrolytic iron or nonfortifoed biscuits. We measured changes in hemoglobin concentrations, inflammation, iron status, helminths, diarrhea, fecal calprotectin concentrations, and microbiota diversity and composition (n = 60) and the prevalence of selected enteropathogens. RESULTS: At baseline, there were greater numbers of fecal enterobacteria than of lactobacilli and bifidobacteria (P < 0.02). Iron fortification was ineffective; there were no differences in iron status, anemia, or hookworm prevalence at 6 mo. The fecal microbiota was modified by iron fortification as shown by a significant increase in profile dissimilarity (P < 0.0001) in the iron group as compared with the control group. There was a significant increase in the number of enterobacteria (P < 0.005) and a decrease in lactobacilli (P < 0.0001) in the iron group after 6 mo. In the iron group, there was an increase in the mean fecal calprotectin concentration (P < 0.01), which is a marker of gut inflammation, that correlated with the increase in fecal enterobacteria (P < 0.05). CONCLUSIONS: Anemic African children carry an unfavorable ratio of fecal enterobacteria to bifidobacteria and lactobacilli, which is increased by iron fortification. Thus, iron fortification in this population produces a potentially more pathogenic gut microbiota profile, and this profile is associated with increased gut inflammation. This trial was registered at controlled-trials.com as ISRCTN21782274.