Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive disease caused by loss‐of‐function variants in the ADA2 gene. DADA2 typically presents in childhood and is characterized by vasculopathy, stroke, inflammation, immunodeficiency, as well as hematologic manifestations. ADA2 protein is predominantly present in stimulated monocytes, dendritic cells, and macrophages. To elucidate molecular mechanisms in DADA2, CD14+ monocytes from 14 patients and 6 healthy donors were analyzed using single‐cell RNA sequencing (scRNA‐seq). Monocytes were purified by positive selection based on CD14 expression. Subpopulations were imputed from their transcriptomes. Based on scRNA‐seq, monocytes could be classified as classical, intermediate, and nonclassical. Further, we used gene pathway analytics to interpret patterns of up‐ and down‐regulated gene transcription. In DADA2, the frequency of nonclassical monocytes was higher compared with that of healthy donors, and M1 macrophage markers were up‐regulated in patients. By comparing gene expression of each monocyte subtype between patients and healthy donors, we identified upregulated immune response pathways, including IFNα/β and IFNγ signaling, in all monocyte subtypes. Distinctively, the TNFR2 noncanonical NF‐κB pathway was up‐regulated only in nonclassical monocytes. Patients’ plasma showed increased IFNγ and TNFα levels. Our results suggest that elevated IFNγ activates cell signaling, leading to differentiation into M1 macrophages from monocytes and release of TNFα. Immune responses and more general response to stimuli pathways were up‐regulated in DADA2 monocytes, and protein synthesis pathways were down‐regulated, perhaps as stress responses. Our identification of novel aberrant immune pathways has implications for therapeutic approaches in DADA2 (registered at clinicaltrials.gov NCT00071045).
T-cell large granular lymphocyte leukemia (T-LGLL) is a lymphoproliferative disease and bone marrow failure syndrome which responds to immunosuppressive therapies. We show single-cell TCR coupled with RNA sequencing of CD3+ T cells from 13 patients, sampled before and after alemtuzumab treatments. Effector memory T cells and loss of T cell receptor (TCR) repertoire diversity are prevalent in T-LGLL. Shared TCRA and TCRB clonotypes are absent. Deregulation of cell survival and apoptosis gene programs, and marked downregulation of apoptosis genes in CD8+ clones, are prominent features of T-LGLL cells. Apoptosis genes are upregulated after alemtuzumab treatment, especially in responders than non-responders; baseline expression levels of apoptosis genes are predictive of hematologic response. Alemtuzumab does not attenuate TCR clonality, and TCR diversity is further skewed after treatment. Inferences made from analysis of single cell data inform understanding of the pathophysiologic mechanisms of clonal expansion and persistence in T-LGLL.
IMPORTANCEOlmsted syndrome is a rare and disabling genodermatosis for which no successful treatment is currently available. OBJECTIVE To evaluate the clinical response to the mammalian target of rapamycin (mTOR) inhibitor sirolimus and/or the epidermal growth factor receptor (EGFR) inhibitor erlotinib among patients with Olmsted syndrome. DESIGN, SETTING, AND PARTICIPANTSThis case series focused on 4 children with treatment-refractory Olmsted syndrome. These children received treatments (initiated in 2017 and 2018) at the outpatient dermatology clinic at the Children'
Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes, including systemic infections, lymphedema, cytopenias, and myeloid neoplasms. Genotype–phenotype correlation is not well understood mechanistically in GATA2 deficiency. We performed whole transcriptome sequencing of single hematopoietic stem and progenitor cells from 8 patients, who had pathogenic GATA2 mutations and myelodysplasia. Mapping patients’ cells onto normal hematopoiesis, we observed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. HSPCs of patients exhibited distinct patterns of gene expression and coexpression compared with counterparts from healthy donors. Distinct lineages showed differently altered transcriptional profiles. Stem cells in patients had dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. The prominent deficiency in lympho-myeloid lineages in GATA2 deficiency appeared at least partly due to the expression of aberrant gene programs in stem cells prior to lineage commitment. We computationally imputed cells with chromosomal abnormalities and determined their gene expression; DNA repair genes were downregulated in trisomy 8 cells, potentially rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetic abnormalities showed defects in genes related to multilineage differentiation and cell cycle. Single-cell RNA sequencing is powerful in resolving transcriptomes of cell subpopulations despite a paucity of cells in marrow failure. Our study discloses previously uncharacterized transcriptome signatures of stem cells and progenitors in GATA2 deficiency, providing a broad perspective of potential mechanisms by which germline mutations modulate early hematopoiesis in a human disease. This trial was registered at www.clinicaltrials.gov as NCT01905826, NCT01861106, and NCT00001620.
Background: Immune aplastic anemia (SAA) disproportionally affects children and young adults. Immunosuppression (IST) remains the treatment of choice for patients less than 40 years of age without a fully human leukocyte antigen (HLA) matched sibling. In the pediatric population (aged <18 years), from NIH historic studies, horse antithymocyte globulin (h-ATG) and cyclosporine(CSA) regimes have produced good results, with an overall response rate (ORR) of 74% at 6 months, a relapse rate of 33% at 10 years, and an overall survival (OS) of 80% at 10 years (Scheinberg P, J Pediatrics 2008), generally better outcomes than in adults. Eltrombopag (EPAG) is an oral thrombopoietin-receptor agonist that has been shown to significantly improve response rates when combined with h-ATG and CSA for treatment-naïve patients (Townsley D, NEJM 2017). We report here a pediatric subgroup analysis of our clinical trial (NCT01623167) to assess whether the addition of EPAG to IST improved ORR in children with SAA. Methods: Between 2012 and 2018 a total of 39 patients <18 years of age with SAA were treated with front line EPAG in combination with h-ATG and CSA. We assessed ORR at 6 months, relapse rate, rate of clonal evolution, and OS. Additionally, we monitored for the development of clonal evolution and for the emergence of somatic clones. Germline testing for inherited marrow failure was performed in all patients. Response was defined by blood counts no longer satisfying criteria for SAA. Outcomes were compared to two treatment-naive cohorts: adult patients aged 18 years and older who received EPAG in combination with immunosuppression (adult EPAG group), and a historical cohort (from 1989 to 2010) of 87 pediatric patients aged <18 years who were treated with immunosuppression (pediatric IST group). The pediatric IST group included all patients treated with a h-ATG based regimen including h-ATG with CSA, and h-ATG with CSA and additional immunosuppression (either mycophenolate mofetil or sirolimus). A historical adult group (Adult IST group) of 322 patients treated from 1989 to 2010 with the same IST regimens as the pediatric IST group was also used as a comparator. Analyses were based on intention-to-treat. Results: Median age in the pediatric EPAG group was 15 years and in the pediatric IST group 11 years. Thirty-two patients (of an initial 39) were evaluable for response at 6 months. Seven patients (18%) were taken off study prior to 6 months to undertake bone marrow transplant, due to either failure to respond by 3 months or clinical urgency in count recovery. Overall, 28 (72%) of all treated patients achieved a response at 6 months, by comparison, responses were observed in 74% of the pediatric IST group (p=0.836) and in 83% in the adult EPAG group (p=0.143). In contrast, patients in the adult EPAG group had a significant difference in 6 month ORR compared with the adult IST group (83% versus 58%, p= <0.0001). The PK analysis of EPAG suggested comparable exposure in pediatric (6-17 years) and adult population. Of the 28 patients who achieved a response at 6 months, 43% relapsed, compared to 28% in the pediatric IST group (p=0.252). Median time to relapse in the pediatric EPAG group was 565 days from IST. There was no significant difference in survival between the pediatric EPAG group and either the pediatric IST group or the adult EPAG group. Clonal evolution occurred in 3 (8%) of patients (monosomy 7, del 5q, and translocation 5:12); 7% of patients evolved in the pediatric IST group. Somatic mutation testing was performed in 36 patients at baseline and was negative in all. At the 6 month timepoint, 5 patients had developed new somatic mutations (2 ASXL1, 2 ATRX, 1 BCOR), three clones appeared in responders and two in non-responders. Germline testing did not reveal any pathogenic variants. Conclusion: Eltrombopag added to immunosuppression did not improve ORR at 6 months in pediatric patients with SAA compared to our historical cohort of pediatric IST patients. In contrast, adults had much improved responses with EPAG. Some patients were taken off study prior to 6 month evaluation (18% compared to 6% in the pediatric IST group) to pursue BMT, likely reflecting recent improvements in outcomes and donor availability. In our study the addition of eltrombopag to IST did not provide any obvious therapeutic benefit to pediatric patients with severe aplastic anemia. This research was supported in part by the Intramural Research Program of the NIH and NHLBI. Disclosures No relevant conflicts of interest to declare.
Achieving bone fracture union after trauma represents a major challenge for the orthopedic surgeon. Fracture non-healing has a multifactorial etiology and there are many risk factors for non-fusion. Environmental factors such as wound contamination, infection, and open fractures can contribute to non-healing, as can patient specific factors such as poor vascular status and improper immunologic response to fracture. Nitric oxide (NO) is a small, neutral, hydrophobic, highly reactive free radical that can diffuse across local cell membranes and exert paracrine functions in the vascular wall. This molecule plays a role in many biologic pathways, and participates in wound healing through decontamination, mediating inflammation, angiogenesis, and tissue remodeling. Additionally, NO is thought to play a role in fighting wound infection by mitigating growth of both Gram negative and Gram positive pathogens. Herein, we discuss recent developments in NO delivery mechanisms and potential implications for patients with bone fractures. NO donors are functional groups that store and release NO, independent of the enzymatic actions of NOS. Donor molecules include organic nitrates/nitrites, metal-NO complexes, and low molecular weight NO donors such as NONOates. Numerous advancements have also been made in developing mechanisms for localized nanomaterial delivery of nitric oxide to bone. NO-releasing aerogels, sol- gel derived nanomaterials, dendrimers, NO-releasing micelles, and core cross linked star (CCS) polymers are all discussed as potential avenues of NO delivery to bone. As a further target for improved fracture healing, 3d bone scaffolds have been developed to include potential for nanoparticulated NO release. These advancements are discussed in detail, and their potential therapeutic advantages are explored. This review aims to provide valuable insight for translational researchers who wish to improve the armamentarium of the feature trauma surgeon through use of NO mediated augmentation of bone healing.
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