Diabetic foot ulcers (DFUs) are among the most frequent complications of diabetes with significant morbidity and mortality. Diabetes can trigger neutrophils to undergo histone citrullination by protein arginine deiminase 4 (encoded by Padi4 in mice) and release neutrophil extracellular traps (NETs). The specific mechanism of NETs-mediated wound healing impairment in diabetes remains unknown. In this study, we show neutrophils are more susceptible to NETosis in diabetic wound environments. Via in vitro experiments and in vivo models of wound healing using wide-type and Padi4 -/- mice, we demonstrate NETs can induce the activation of PAK2 via the membrane receptor TLR-9. Then PAK2 phosphorylates the intracellular protein Merlin/NF2 to inhibit the Hippo-YAP pathway. YAP binds to transcription factor SMAD2 and translocates from the cytoplasm into the nucleus to promote endothelial-to-mesenchymal transition (EndMT), which ultimately impedes angiogenesis and delays wound healing. Suppression of the Merlin/YAP/SMAD2 pathway can attenuate NET-induced EndMT. Inhibition of NETosis accelerates wound healing by reducing EndMT and promoting angiogenesis. Cumulatively, these data suggest NETosis delays diabetic wound healing by inducing EndMT via the Hippo-YAP pathway. Increased understanding of the molecular mechanism that regulates NETosis and EndMT will be of considerable value for providing cellular targets amenable to therapeutic intervention for DFUs.
© The author(s).

Granulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3). Granulocyte integrity and detection of protein markers were optimized so that rare granulocyte populations could be deeply characterized by single cell mass cytometry. For complete details on the use and execution of this protocol, please refer to Vivanco Gonzalez et al. (2020).
© 2022 The Authors.

Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is challenging to diagnose due to frequent myelofibrosis (MF) and a low percentage of blast cells. In the present study, clinical characteristics and experimental observations in 9 adult patients diagnosed with AMKL, who were recruited by the Sino-U.S. Shanghai Leukemia Co-operative Group, were analyzed in order to summarize the diagnostic experience and provide recommendations on diagnosing AMKL. All the patients were diagnosed according to the 2008 World Health Organization diagnostic criteria. The mean age of the patients with AMKL was 59 years (range, 53-68 years). A total of 8 patients had different degrees of anemia, and 2 patients had <5% marrow blasts present in the bone marrow; however, the percentage of positive cells with cluster of differentiation (CD)41 and CD61 expression was >20%, as demonstrated by flow cytometry. A total of 6 patients were positive for platelet-specific antigens, as indicated by immunocytochemistry. Furthermore, 7 patients presented with moderate or marked MF, as demonstrated by a bone marrow biopsy. Karyotypic analysis indicated that 6 patients had abnormal karyotypes. Only 1 patient exhibited the Janus kinase 2V617F mutation. Treatment efficiency was notably poor, with a median survival time of 6.0 months (range, 1.1-24.0 months). In conclusion, the diagnosis of AMKL requires a combination of the results of bone marrow smears and bone marrow biopsy, immunophenotype or immunohistochemistry. We recommend that routine immunophenotypic analysis should include the CD41 and CD61 markers for diagnosing acute leukemia when bone marrow morphology does not indicate the diagnosis.

The aim of the present study was to investigate the clinical characteristics and prognosis of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL) relapse. A total of 390 pediatric patients diagnosed as B-ALL and receiving regular chemotherapy in Jining First People's Hospital from August 2010 to May 2016 were selected. The clinical characteristics, therapeutic response and prognosis were compared between the two groups. There were significant differences in the comparisons of age, leukocyte count in the initial diagnosis and glucocorticoid sensitive test between B-cell ALL (B-ALL) relapse group and non-relapse group; the minimal residual disease (MRD) levels of pediatric patients in the two groups at 33 days and 12 weeks were significantly different. The 3-year event-free survival (EFS) rates of pediatric patients with early, medium and late B-ALL relapse were 12.5±7.8%, 33.1±9.8% and 63.6±6.1%, respectively, and the prognosis of late relapse was significantly better than that of early relapse (P<0.001). The 3-year EFS rates of pediatric patients with bone marrow relapse in standard risk group, intermediate risk group and high risk group were 29.1±6.9, 31.3±6.5 and 28.3±6.3%, respectively; there were no statistically significant differences (P=0.387, P>0.05). Pediatric patients with B-ALL relapse are characterized by higher onset age (≥10 years old), high leukocyte count and hormone insensitivity. Dynamic monitoring of MRD level in B-ALL pediatric patients can predict the relapse.