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BackgroundREGN-COV2, a monoclonal antibody cocktail drug against the SARS-COV-2 virus, has proven to be effective in preventing the development of severe COVID-19 and is increasingly being administered in outpatient and home settings. Adverse events such as fever and decreased oxygen saturation may occur after administration of REGN-COV2, and although these symptoms are generally mild and transient, predicting the occurrence of these adverse events is useful in developing a monitoring plan for patients.MethodsWe performed a retrospective analysis of 76 patients who received REGN-CoV2 between August and September 2021. We collected information on fever, decreased oxygen saturation requiring oxygen supplementation, and other adverse events from medical records. Patients were divided into two subgroups: those who presented with fever or oxygen desaturation and those who did not, and underlying medical conditions and laboratory data were compared between each group. The parameters that exhibited significant differences were further tested using Fisher’s exact test to evaluate whether appropriate thresholds could be set to distinguish the incidence group from the non-incidence group.FindingsOf the 76 patients, 47 had fever of 38.5°C or higher within 24 hours after administration, and 27 of these patients had a body temperature of 37.5°C or lower before administration. Oxygen was required in 17 cases, 7 of which required oxygen more than 24 hours after administration of REGN-COV2, and additional treatment such as dexamethasone was given as the disease progressed to moderate. Among the parameters analyzed, lymphocyte count and IFNλ3 showed significant differences between the fever and non-fever groups. This was also the case in the comparison excluding patients who had fever before administration. There was also a significant difference in ferritin and CRP between the oxygen required and non-required groups. In addition to IFNλ3, ferritin, and CRP, there was a significant difference in LDH between the group that required additional treatment and the group that did not. When lymphocytes count 5.0 was used as the cutoff, sensitivity 72%, specificity 76%, odds ratio 8.220 (2.857 to 22.22; pInterpretationsTransient fever and decreased oxygen saturation are common adverse events after REGN-CoV2 administration, and their occurrence correlated with the severity factor of COVID-19 itself. Evaluation of these items at the time of administration is useful not only for predicting the severity of COVID-19 but also for the development of adverse events in patients receiving REGN-CoV2.

Rationale Siglec-8 is a surface receptor predominantly expressed on human eosinophils where its ligation induces reactive oxygen species (ROS) formation and cell death. Since Siglec-8 has intracellular tyrosine-based motifs, we hypothesized that Src family kinases (SFKs) are involved in ROS formation and cell death induced by Siglec-8 engagement. Methods Human peripheral blood eosinophils were purified and incubated with anti-Siglec-8 monoclonal antibodies (mAb, agonist), IL-5, and SFK pharmacological inhibitors. We focused on Siglec-8-induced cell death in short-term IL-5-activated cells leading to a regulated necrosis-type cell death. ROS production was determined by dihydrorhodamine (DHR) 123 labeling and flow cytometry, or by chemiluminescence using Amplex red. Activation of SFK was determined using phospholuminex and Western blotting. Results In order to determine cellular localization of ROS production, we measured intra and extracellular ROS. While an ETosis stimulus (calcium ionophore A23187) led to extracellular ROS (ecROS) production, Siglec-8-engagement in short-term IL-5 activated cells led to intracellular ROS (icROS) accumulation. Consistently, inhibition of extracellular ROS by catalase inhibited ETosis, but not IL-5-primed Siglec-8-induced cell death. In order to determine signaling events for Siglec-8, we performed Western blotting and found SFK phosphorylation in lysates from eosinophils stimulated with anti-Siglec-8 mAb ± IL-5. In order to identify which SFKs were involved, we used the phospholuminex assay and found increased levels of phosphorylated Fgr in the cytoplasmic fraction of cells co-stimulated with anti-Siglec-8 and IL-5 for 3 hours compared with cells stimulated with IL-5 alone. To test the involvement of SFKs in ROS production and cell death, we used SFK inhibitors PP2 and dasatinib, both of which completely inhibited eosinophil ROS production and cell death induced by anti-Siglec-8 and IL-5 co-stimulation. Conclusion Siglec-8 engagement in short-term IL-5-activated eosinophils causes icROS production and SKF phosphorylation, and both are essential in mediating Siglec-8-induced cell death.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by structural and/or functional impairment of cilia throughout the whole body. Early diagnosis of PCD is important for the prevention of long‑term sequelae, however early diagnosis is a challenge due to the phenotypic heterogeneity of PCD. In the current study, the patient with PCD was diagnosed at nine years old following several efforts to control intractable airway symptoms. The patient experienced a chronic productive cough beginning in early childhood and had multiple episodes of pneumonia and otitis media with effusion and sinusitis. No situs inversus or other heterotaxias were reported. Serial chest X‑rays exhibited persistent atelectasis and bronchiectasis in the right middle lobe. When the patient was nine years old, electron microscopy of his cilia and genetic analysis were conducted. Electron microscopy of a biopsy specimen from the nasal mucosa indicated loss of the outer dynein arms. Whole‑exome analysis of the genome demonstrated the presence of compound heterozygous mutations in DNAH5: NM_001369.2:c.5983C>T, p.Arg1995X in exon 36 and NM_001369.2:c.9101delG, p.Gly3034ValfsX22 in exon 54; neither of which have been previously reported in the literature in a Japanese patient. Notably, this case is, to the best of our knowledge, the first reported case of PCD caused by the DNAH5 mutation in a Japanese patient.

Pulmonary veno-occlusive disease (PVOD) is a rare chronic lung disease that is difficult to diagnose due to non-specific clinical findings. Little is known about the pathogenesis of PVOD. Reported herein is the case of an 11-year-old girl who initially presented with 'bat-wing' shadows on chest radiography. This finding, coupled with prominent hemosiderosis in bronchoalveolar lavage fluid, initially led to a misdiagnosis of idiopathic pulmonary hemosiderosis. Oral prednisolone dramatically improved signs and symptoms initially, but her condition then gradually deteriorated during maintenance therapy with corticosteroids and other immunosuppressants. PVOD was suspected but not confirmed owing to a lack of hallmark radiographic findings and contraindications for lung biopsy. Three years later, while arranging for lung transplantation, the patient experienced sudden onset of fatal massive pulmonary edema. PVOD was confirmed at autopsy. This case provides insights regarding an unfamiliar presentation of PVOD and may help physicians to avoid diagnostic pitfalls.

Hemoglobin (Hb) Bristol-Alesha is caused by a GTG --ATG mutation at codon 67 in the Hb beta chain, resulting in abnormal beta globin chains with mutated molecules from normal beta67 valine (Val) to beta67 methionine (Met) or beta67 aspartate (Asp). We describe a Japanese child with this rare hemoglobinopathy and a very unstable Hb molecule phenotype. The diagnosis of hemolytic anemia was made when the patient was 6 months of age. Development of marked splenomegaly necessitated red blood cell transfusions twice a month. After splenectomy when the patient was 4 years of age, laboratory findings of hemolytic anemia became more prominent. Specific abnormal Hb molecules initially were not detected, and the alpha/beta globin synthesis ratio was abnormal at 2.22. After splenectomy, we identified the presence of abnormal beta-globin chains with a beta67Val:beta67Met:beta67Asp molecule ratio of 74:11:15. We speculate that the high fraction of the beta67Met molecule in this patient, compared with that in previously reported cases, caused extreme Hb instability, which resulted in thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.

We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8-9 g/dl, his reticulocyte counts were 300-500 x 109/l, and his total bilirubin level was 25.65-42.75 micro mol/l. The patient suffered two episodes of respiratory infection-associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3.0 years, his developmental milestones (developmental quotients measured using the Tsumori-Inage methods) score was 49% (normal 74-131%), and his height was below average by -2.0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low ( C) PGK1 gene mutation. This mutation results in the Ala-353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

While studying Ikaros proteins in childhood acute myeloid leukemia (AML), Ikaros isoform 6 (Ik6) expression was detected in 7 of 10 cases of M4 and M5 leukemia, but in none of the remaining French-American-British subtypes (M2, 8 cases; M7, 6 cases). The spliced Ikaros isoforms 4 to 8 (Ik4-8) suppress the function of full-length Ik1 or Ik2 in a dominant-negative manner, owing to their reduced numbers of DNA binding sites. Thus, dominant-negative Ikaros isoforms may inhibit the normal transcriptional regulation of hematopoietic cell development. To clarify the function of Ik6 in developing blood cells, this isoform was transiently transfected into an Ik2+, interleukin-3 (IL-3)–dependent 32D murine myeloid precursor cell line and studied the expression of Bcl-2 family proteins in relation to in vitro cell growth, using a tetracycline-inducible TREx system. The possibility of aberrant cell regulation due to Ikaros functional changes was examined by cotransfecting both Ik2 and Ik6 into Ikaros/Aiolos/Helios triple-negative Cos-7 cells. The results demonstrated IL-3–independent growth by Ik6-transfected 32D clones coincident with up-regulation of the antiapoptotic protein Bcl-XL. Up-regulation of Bcl-XL, but not of other Bcl-2 family proteins, was associated with the suppression of functional Ik2 by Ik6 in a dominant-negative fashion. Thus, the pathogenesis of myelomonocytic/monocytic AML may involve aberrant regulation of apoptosis due to unscheduled expression of the Ik6 isoform.

Background Sialic acid–binding immunoglobulin-like lectin (Siglec)-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8–mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity. Objective In this report we investigate the mechanism of Siglec-8–mediated cell death in activated eosinophils. Methods Human peripheral blood eosinophils were treated with agonistic anti–Siglec-8 antibody and IL-5, and cell death was determined by using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence. Results Costimulation with anti–Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti–Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti–Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in cells stimulated with IL-5 alone; anti–Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti–Siglec-8/IL-5–induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, an ROS inhibitor prevented the anti–Siglec-8/IL-5–induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti–Siglec-8 and was sufficient to induce enhanced cell death in IL-5–treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation. Conclusions In activated eosinophils ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5–induced ERK phosphorylation, which results in a novel mode of biochemically regulated eosinophil cell death.

Pulmonary veno-occlusive disease (PVOD) is a rare chronic lung disease that is difficult to diagnose due to non-specific clinical findings. Little is known about the pathogenesis of PVOD. Reported herein is the case of an 11-year-old girl who initially presented with 'bat-wing' shadows on chest radiography. This finding, coupled with prominent hemosiderosis in bronchoalveolar lavage fluid, initially led to a misdiagnosis of idiopathic pulmonary hemosiderosis. Oral prednisolone dramatically improved signs and symptoms initially, but her condition then gradually deteriorated during maintenance therapy with corticosteroids and other immunosuppressants. PVOD was suspected but not confirmed owing to a lack of hallmark radiographic findings and contraindications for lung biopsy. Three years later, while arranging for lung transplantation, the patient experienced sudden onset of fatal massive pulmonary edema. PVOD was confirmed at autopsy. This case provides insights regarding an unfamiliar presentation of PVOD and may help physicians to avoid diagnostic pitfalls.

An 11-year-old girl presented with fever and a large cervical lymphadenopathy. Indicators of inflammation were remarkable: she had extremely high levels of serum interleukin-6 (IL-6) (398 pg/ml) in addition to hypergammaglobulinemia and hypoalbuminemia. Computed tomography (CT) revealed swollen systemic lymph nodes. Two weeks after the onset of symptoms she developed polyarthralgia. Biopsy of the cervical lymph node revealed massive infiltration of plasma cells without hyaline vascular changes. She was diagnosed with systemic juvenile idiopathic arthritis (JIA). The patient's symptoms and hypercytokinemia disappeared soon after corticosteroid treatment was started. This case demonstrates that overproduction of IL-6 is common to systemic JIA and multicentric Castleman's disease.

A 3-year-old male presented with Chlamydia pneumoniae infection-related hemophagocytic lymphohistiocytosis (HLH). The patient developed an episode of HLH with severe skin eruption following C. pneumoniae pneumonia. Symptoms responded to steroid/cyclosporine A therapy, but the patient slowly lost consciousness and developed systemic flaccid paralysis. He was diagnosed with encephalitis/myelitis by brain and spinal MRI. Neurological symptoms and signs gradually resolved. We thought that the immune response to C. pneumoniae infection triggered the development of HLH, associated with unusual neurological complications. This report describes a novel case of C. pneumoniae-associated HLH and with poliomyelitis like flaccid paralysis.

Ikaros is an essential regulator of lymphocyte differentiation. Mice transgenic for the Ikaros dominant negative (DN) mutation rapidly develop lymphoid malignancies. Various human leukemias have also been reported to express Ikaros DN isoforms, but its role in leukemogenesis is yet to be defined. We demonstrate that overexpressed Ikaros DN (Ik6) prolonged the survival of two different murine pro-B cell lines in cytokine deprived condition, and this was associated with increased expression of Bcl-xl. A survey of the upstream controller(s) of Bcl-xl expression revealed Ik6 overexpression enhanced the phosphorylation of JAK2 and STAT5. Interestingly, the Ik6 expressing cell lines showed reduced expression of B-cell differentiation surface marker CD45R (B220), which is also known as a JAK2 inhibitor. Although further evaluation with human clinical materials are required, these results propose a putative role of Ik6 in the development of B-lineage acute lymphoblastic leukemia, by activating the JAK2-STAT5 pathway and thus stimulating the production of Bcl-xl.

We described the cerebral thrombotic complications developed in 2 adolescent patients treated with L-asparaginase-containing regimens. For determining risk factors, we retrospectively analysed hemostatic markers in 19 pediatric patients with leukemia or lymphoma who were treated with either 1 of the 2 L-asparaginase-containing regimens; 11 were treated with VLP1 and the remaining 8 were treated with the VLAD protocol. The data indicated that low coagulation factors in association with increased plasma D-dimer levels during or post-L-asparaginase administration combined with fresh frozen plasma infusion might have activated coagulation processes in these patients. Careful management is required to prevent such episodes in patients with markedly decreased coagulation factors and increased D-dimer levels following L-asparaginase administration.

The glutathione-S-transferase (GST) polymorphism may affect the outcome of treatment of leukemia because GSTs play an important role in detoxifying the chemotherapeutic agents used to kill leukemia cells. However, results of previous reports have been controversial. This study was undertaken to clarify the influence of GST polymorphism on the outcome of childhood B-precursor acute lymphoblastic leukemia (ALL).Eighty-two patients with childhood B-precursor ALL treated during 1988-1999 with our ALL protocol (median follow-up time 89.5 months, range 31 -169 months) were examined for GST gene patterns. The effect of GSTM1 and GSTT1 deletion genotypes on the clinical features and therapeutic results was analyzed.All patients attained complete remission but 12 had an early relapse (within 30 months of the initiation of treatment). In univariate analysis, early relapse of ALL was correlated significantly with the presence of the t(9;22)(q34;q11) cytogenetic abnormality (p=0.0003), high white blood cell counts (p=0.015) and double null genotype (p=0.027). Multivariate analysis revealed that the GST double null genotype was the only significant independent predictor of early relapse (p=0.018).The simultaneous deletion of both the GSTM1 and GSTT1 genes is more predictive than any other parameter of early relapse of childhood B-precursor ALL.

We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8-9 g/dl, his reticulocyte counts were 300-500 x 109/l, and his total bilirubin level was 25.65-42.75 micro mol/l. The patient suffered two episodes of respiratory infection-associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3.0 years, his developmental milestones (developmental quotients measured using the Tsumori-Inage methods) score was 49% (normal 74-131%), and his height was below average by -2.0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G--C) PGK1 gene mutation. This mutation results in the Ala-353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms. Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation. Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants. Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. Disclosures No relevant conflicts of interest to declare.

BackgroundSiglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.MethodsConsequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.ResultsEngagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.ConclusionsThese data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.

Abstract 2134 Siglec-8 is a membrane protein predominantly expressed on eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-induced cell death is markedly enhanced by the eosinophil activation and survival factor IL-5. Thus, Siglec-8 induces cell death preferentially in activated eosinophils, making it an attractive therapeutic target for eosinophil-mediated diseases. However, the mechanism of this survival factor-enhanced cell death is not known. While Siglec-8 ligation (by anti-Siglec-8 antibody) induces caspase-dependent apoptosis in resting eosinophils, it induces caspase-independent cell death in activated eosinophils. We hypothesize that co-stimulating the Siglec-8 and IL-5 pathways induces a necrotic cell death pathway. By morphologically characterizing human peripheral blood eosinophils as “apoptotic” (i.e., shrunk cells with condensed chromatin) or “necrotic” (i.e., swollen cells, disrupted membrane integrity), we found that anti-Siglec-8 + IL-5 co-stimulation yielded more necrotic eosinophils (P = 0.055, 6 donors) than stimulation with anti-Siglec-8 alone. Additionally, we stained with Annexin V and 7AAD and assessed the percent of Annexin V+ cells that are 7AAD+ as an indicator of increased transition of apoptotic cells to secondary necrosis and/or cells dying primarily by necrosis. We found that anti-Siglec-8 + IL-5 co-stimulated cells had a higher ratio of 7AAD+ cells compared with cells treated with anti-Siglec-8 alone (P < 0.001, 25 experiments with 11 independent donors). This higher 7AAD+ ratio, the morphological characteristics and the caspase-independent cell death of co-stimulated cells suggest that Siglec-8 ligation induces a necrotic form of cell death in IL-5-stimulated eosinophils by activating a specific and distinct biochemical pathway. Our previous studies have shown that reactive oxygen species (ROS) production is involved in Siglec-8-induced cell death in both resting and activated eosinophils. However, we have observed that phosphorylation of ERK1/2 and MEK1 was significantly increased in cells co-stimulated with anti-Siglec-8 + IL-5 compared to cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors U0126 and PD184352 completely blocked anti-Siglec-8 + IL-5-induced cell death; however, intracellular ROS production induced by Siglec-8 ligation was MEK1-independent. In contrast, the ROS inhibitor DPI prevented the anti-Siglec-8 + IL-5-induced enhancement of ERK1/2 phosphorylation and subsequent cell death. Enhanced ROS accumulation in IL-5 treated cells was sufficient to induce enhanced cell death, similar to anti-Siglec-8 treatment. These findings suggest that Siglec-8 ligation leads to ROS-dependent enhancement of IL-5-induced ERK1/2 phosphorylation, which results in enhanced Siglec-8-induced eosinophil cell death. How ERK phosphorylation induces cell death in co-stimulated eosinophils is not known, and ERK's involvement is surprising considering its role in activation of IL-5-stimulated eosinophils. However, recent studies have shown that ERK can be involved in specific types of cell death, namely necroptosis or autophagy, and that spatiotemporal parameters determine whether ERK will induce cell death or activation. Thus, we hypothesized that ERK localization will be altered in eosinophils co-stimulated with anti-Siglec-8 + IL-5 compared with cells treated with IL-5 alone. Western blotting of nuclear and cytoplasmic fractions and immunofluorescence suggest that enhanced ERK1/2 localization and phosphorylation are sustained for at least 2 hours in the nucleus of anti-Siglec-8 + IL-5 co-stimulated cells; cells treated with IL-5 alone have only brief ERK1/2 nuclear localization. The sustained nuclear activation of ERK may explain the change in IL-5 function from eosinophil activation/survival to necrotic death upon Siglec-8 ligation. In summary, ERK is involved in regulating the decision point for eosinophil activation, apoptosis or regulated necrosis. Disclosures: No relevant conflicts of interest to declare.