SARS-CoV-2 ORF3a expression in brain disrupts the autophagy-lysosomal pathway, impairs sphingolipid homeostasis, and drives neuropathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.

Visualizing Sphingosine-1-Phosphate Receptor 1(S1P1) Signaling During Central Nervous System De- and Remyelination.

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1-5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P - S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed ß-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal ß-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Gene expression changes in Tay-Sachs disease begin early in fetal brain development.

Treatment of monogenic disorders has historically relied on symptomatic management with limited ability to target primary molecular deficits. However, recent advances in gene therapy and related technologies aim to correct these underlying deficiencies, raising the possibility of disease management or even prevention for diseases that can be treated pre-symptomatically. Tay-Sachs disease (TSD) would be one such candidate, however very little is known about the presymptomatic stage of TSD. To better understand the effects of TSD on brain development, we evaluated the transcriptomes of human fetal brain samples with biallelic pathogenic variants in HEXA. We identified dramatic changes in the transcriptome, suggesting a perturbation of normal development. We also observed a shift in the expression of the sphingolipid metabolic pathway away from production of the HEXA substrate, GM2 ganglioside, presumptively to compensate for dysfunction of the enzyme. However, we do not observe transcriptomic signatures of end-stage disease, suggesting that developmental perturbations precede neurodegeneration. To our knowledge, this is the first report of the relationship between fetal disease pathology in juvenile onset TSD and the analysis of gene expression in fetal TSD tissues. This study highlights the need to better understand the "pre-symptomatic" stage of disease to set realistic expectations for patients receiving early therapeutic intervention.

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane-bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.

Poly(I:C) promotes neurotoxic amyloid ß accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid ß (Aß) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aß-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aß degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aß toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aß was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aß1-42. Altogether, these results suggest that viral infections induce Aß neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aß degradation.

Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron.

BACKGROUND: In transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. RESULTS: LIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DISCUSSION: The difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels.

Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.

HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation.

Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, the immunological functions of the ApoM-S1P complex are unknown. Here we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P1 receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM (Apom(-/-)) had increased proliferation of Lin(-) Sca-1(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S1P1 suppressed LSK and CLP cell proliferation in vivo. ApoM was stably associated with bone marrow CLPs, which showed active S1P1 signalling in vivo. Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom(-/-) mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-S1P1 signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

Preliminary evaluation of a new flow cytometry method for the routine hematology workflow.

Background In a generalist laboratory, the integration of the data obtained from hematology analyzers (HAs) with those from multiparametric flow cytometry (FMC) could increase the specificity and sensitivity of first level screening to identify the pathological samples. The aim of this study was to perform a preliminary evaluation of a new simple hybrid method (HM). The method was obtained by integration between HAs reagents into FCM, with a basic monoclonal antibodies panel for the leukocytes differential count. Methods Eighty-one peripheral blood samples, collected in K3EDTA tubes, were analyzed by XN-module, and CyFlow Space System, using both standard MoAbs and HM method analysis, and with the optical microscopy (OM). Within-run imprecision was carried out using normal samples, the carryover was evaluated, data comparison was performed with Passing-Bablok regression and Bland-Altman plots. Results The within-run imprecision of HM methods ranged between 1.4% for neutrophils (NE) and 10.1% for monocytes (MO) always equal or lower to the OM. The comparison between HM methods vs. OM shows Passing-Bablok regression slopes comprised between 0.83 for lymphocyte (LY) and 1.14 for MO, whilst the intercepts ranged between -0.18 for NE and 0.25 for LY. Bland-Altman relative bias was comprised between -12.43% for NE, and 19.77% for eosinophils. In all 11 pathological samples the agreement between the methods was 100%. Conclusions The new hybrid method generates a leukocytes differential count suitable for routine clinical use and it is also useful for identifying morphological abnormalities with a reduction in cost and improvement of screening for first level hematology workflow.

After haemin treatment intracellular non-haem iron increases prior to haem oxygenase-1 induction: A study in human monocytic cell line THP-1.

BACKGROUND: Iron overload is a major health concern for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, in turn, inducing cell death. We previously demonstrated that haemin-induced cell death in human monocytic THP-1 cells is consistent with ferroptosis, an iron-dependent cell death regulation mechanism. However, direct measurement of iron after haemin treatment has not yet been conducted. In this study, we measured intracellular non-haem iron concentration and haem oxygenase levels after haemin treatment. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and the cell lysate was prepared. Non-haem iron concentration of the cell lysate was measured using the Nitroso-PSAP method. Expression of haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) was quantified by western blotting. RESULTS: We measured intracellular non-haem iron and the expression of haem oxygenases post-haemin treatment. Concentration of non-haem iron post-haemin treatment increased dependently with time and dose. HO-1 expression was detected 4 h after haemin treatment, whereas HO-2 expression was constitutive. DISCUSSION: Increase in non-haem iron prior to induction of HO-1 expression suggests the involvement of HO-2 in haem-induced cytotoxicity. (184 words).

Morphological and optical properties of human immature platelet-enriched population produced in immunodeficient mice.

Ultrastructure analysis of immature platelets is difficult because of the lack of a suitable marker and their relatively low concentration in total platelets. We investigated the morphological and optical properties of human immature platelets produced and enriched in immunodeficient mice via human CD34-positive cell administration. Immunodeficient mice were injected with human CD34-positive cells and administered eltrombopag orally for 14 days (eltro-mice). Some of these mice were maintained for 2-3 months (steady-state-mice). Platelets were double-stained with a human CD41 antibody and a nuclear staining dye (Sysmex hematology analyzer XN series reagent), and then analyzed by flowcytometry FCM to identify human immature platelets. Human CD41-positive cells were isolated from citrated blood by magnetic cell sorting with human CD41 antibody, and examined using electron microscopy. Flow cytometric analysis with the XN reagent demonstrated that peripheral blood from eltro-mice had a higher percentage of immature platelet fraction in human platelets than that from steady-state-mice. The geometric mean of XN reagent fluorescence for human platelets, divided with that for mouse platelets, revealed that the ratios in eltro-mice were significantly higher than those in steady-state-mice, thus indicating that immature platelets were highly enriched in eltro-mice. Scanning and transmission electron microscopy revealed that human citrated platelets isolated from eltro-mice tended to be larger (n = 15, p = 0.276) and contained more mitochondria than those isolated from steady-state-mice (n = 10, p = 0.0002). Therefore, an increased number of mitochondria, rather than platelet size, is a distinctive feature of immature platelets.

Haemin-induced cell death in human monocytic cells is consistent with ferroptosis.

BACKGROUND: Iron overload is a major issue for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, and the haemin in turn induces cell death and the generation of reactive oxygen species (ROS) in both murine macrophages and human monocytic THP-1 cells. This haemin-induced cell death process has been shown to be iron-dependent. Thus, we hypothesized that haemin-induced THP-1 cell death is a result of ferroptosis, an iron-dependent mechanism of cell death regulation. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and haemin-induced cell death and ROS generation were assessed using flow cytometry. RESULTS: Haemin-induced THP-1 cell death showed a necrosis pattern, and treatment with iron chelators suppressed both haemin-induced cell death and ROS generation. Treatment with ferrostatin-1, a ferroptosis inhibitor, suppressed haemin-induced cell death without affecting ROS generation, whereas erastin, a ferroptosis inducer, enhanced both haemin-induced cell death and ROS generation. DISCUSSION: Our findings support haemin-induced cell death as an example of ferroptosis. Therefore, ferroptosis inhibitors may be useful for the treatment or prevention of transfusion iron overload.

Imaging S1P1 activation in vivo.

Sphingosine-1-phosphate receptor 1 (S1P1) is a G protein-coupled receptor that is activated by the sphingolipid ligand sphingosine-1-phosphate (S1P). S1P1 is widely expressed across tissues and, when activated, has broad functions in the immune, vascular and nervous systems. In several diseases in which inflammation plays a critical role, S1P1 activation has been found to be involved in pathogenesis. However, the details of S1P1 activation in vivo under different physiologic conditions are not well understood. Here we describe how a new in vivo methodology to identify S1P1 activation has helped increase understanding of the manner in which this signaling molecule functions both in homeostasis and during inflammation.

Iron-chelating agent, deferasirox, inhibits neutrophil activation and extracellular trap formation.

Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 µmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 µmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

[The Utility of XE-2 100 Analyzer's NEUT-X and NEUT-Y Parameters for Detecting Neutrophil Dysplasia in Myelodysplastic Syndromes].

Morphological abnormalities of blood cells are the typical characteristics of myelodysplastic syndromes (MDS). Recently, the usefulness of multiparameter automatic hematology analyzer XE-2100 for detecting neutrophil dysplasia has been reported by using parameters of NEUT-X and NEUT-Y, reflecting neutrophil cytoplasmic granularity and the cellular content of nucleic acid and protein, respectively. We evaluated the utility of these parameters by analyzing the blood samples of fifty MDS patients consulting Kakogawa West Municipal Hospital between Jan, 2010 and Jun, 2014, as well as 100 persons undergoing medical examinations during the same period as controls. Neutrophil granulation level was classified as normal, hypo-granular, or agranular under microscopic observation, and degranulation index was calculated using the following formula. The relationship between NEUT-X, NEUT-Y values and degranulation index was studied as below. Degranulation index = agranular (%) x 2+ hypo-granular (%) x 1 + normal (%) x 0 Neut-X values of MDS patients were 1,350 (mean), 1,345 (median), and NEUT-Y values, 430 (mean) and 432 (median). The NEUT-X and NEUT-Y values of control patients were 1,350, 1,349, 446 and 445, respectively. Correlation efficiency between degranulation index and NEUT-X or NEUT-Y were r = 0.62 or 0.52, respectively. Relationship between NEUT-X and NEUT-Y for all patients was r = 0.90. All the 10 patients showing NEUT-X lower than 1,315 and NEUT-Y lower than 400 simultaneously were MDS. Hence, we conclude that NEUT-X and NEUT-Y information is useful for quantitative evaluation of neutrophil morphological abnormalities.

Autophagy regulates sphingolipid levels in the liver.

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

Heme-related molecules induce rapid production of neutrophil extracellular traps.

BACKGROUND: Pulmonary endothelial cell damages caused by neutrophil overactivation could result in acute lung injuries including transfusion-related acute lung injury (TRALI). We previously reported that heme-related molecules derived from hemolysis induced the production of reactive oxygen species from neutrophils. Recently, neutrophil extracellular traps (NETs) have been demonstrated to associate with the onset of TRALI. STUDY DESIGN AND METHODS: In this study, neutrophils' morphologic changes induced by the heme-related molecule hemin were confirmed to be NETs via confocal laser scanning microscopy and electron microscopy (EM). Additionally, concentrations of hemin in red blood cell (RBC) components were measured via enzyme-linked immunosorbent assay and possible contribution of these molecules to the onset of TRALI was discussed. RESULTS: SYTOX green staining observation via confocal laser scanning microscopy revealed that neutrophil morphology changed rapidly upon addition of hemin. The nuclei began to be enlarged and become segmented after 5 minutes, and NET-like structures were released from neutrophils after 15 minutes. In EM observation, NET-like structures appeared after 10 minutes and the nucleoplasm was partially separated from the nuclear membrane, which were consistent with the features of NET formation. These structures stained positively for both myeloperoxidase and histone H3 antibodies. CONCLUSION: Thus, our results suggest that hemin induced NETs in 15 minutes, a quicker reaction than NET induction by phorbol myristate acetate requiring 3 hours. Moreover, since RBC components, especially those with long-term storage, contained sufficient hemin concentration to induce NETs, special attention to hemolysis of stored RBC components is important.

Non-activated T and B lymphocytes become morphologically distinguishable after detergent treatment.

T and B lymphocytes are difficult to distinguish morphologically even with electron microscopy, and antibodies are generally used to make the distinction. A specific reagent, consisting of nonionic and cationic detergents, is used for leukocyte differentiation using the Sysmex automated blood analyzer. This reagent increases cell membrane porosity and enables the introduction of fluorescent dye into leukocytes. In this study, we investigated the effect of this specific detergent on the morphology of T and B lymphocytes. T and B lymphocytes were obtained by density gradient centrifugation and magnetic cell sorting, with a minimum of 90% isolation efficiency. T and B lymphocytes were then treated with the specific detergent and fluorescent dye, and their distribution was analyzed based on side scatter and fluorescence intensity using general-purpose flow cytometry (FCM). Fluorescent images were observed using a confocal laser scanning microscope (CLSM), cellular inner structures using a transmission electron microscopy (TEM), and cell surfaces using a scanning electron microscope (SEM). The ratio of cholesterol to total lipid in cell membranes of B and T lymphocytes was measured using a fluorescent assay kit. The distribution of fluorescence intensity was different between T and B lymphocyte clusters, according to the FCM analysis. CLSM observations revealed that the fluorescent dye mainly stained cytoplasmic organelles. FCM, TEM, and SEM observations revealed that B lymphocytes are more likely to lose surface antigens and intracellular organelles than T lymphocytes, which allows the visual distinction between T and B lymphocytes. The ratio of cholesterol to total lipid in T lymphocyte membranes had tendency higher than that in B lymphocyte membranes. In this study, we demonstrate that cells with differences in cell membrane cholesterol amounts, such as B and T lymphocytes could be identified using an inexpensive detergent, as an alternative to costly antibodies.

Flow cytometric detection and microscopic observation of activated eosinophils in peripheral blood.

Eosinophils possess highly electron-dense granules with crystal-like structures and are characterized as high side scatter (SSC) areas by flow cytometry analysis. Eosinophils with low SSC features have been noted in extremely rare cases; however, the underlying cause remains unclear. Eosinophils in the low SSC area were analyzed using microscopy. A transmission electron microscope revealed the loss of crystal-like structures in granules with low electron density and piecemeal degranulation, which was undetectable by May-Grünwald-Giemsa staining. Based on the results of flow cytometry, May-Grünwald-Giemsa staining and transmission electron microscopy, SSC values could help potentially detect crystal-like structures and piecemeal degranulation eosinophils.