RESUMO
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.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Astrócitos , Insulisina , Neprilisina , Viroses , Animais , Ratos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/virologia , Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Astrócitos/virologia , Insulisina/metabolismo , Neprilisina/metabolismo , Receptor 3 Toll-Like/antagonistas & inibidores , Poli I-C/farmacologia , Viroses/complicaçõesRESUMO
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.
Assuntos
Sobrecarga de Ferro , Ferro , Morte Celular , Ferritinas , Hemina/farmacologia , Humanos , Ferro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transferrina/metabolismo , Transferrina/farmacologiaRESUMO
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).
Assuntos
Heme Oxigenase-1/biossíntese , Hemina/farmacologia , Espaço Intracelular/metabolismo , Ferro/metabolismo , Monócitos/enzimologia , Morte Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células THP-1 , Fatores de TempoRESUMO
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.
Assuntos
Hemina/farmacologia , Quelantes de Ferro/uso terapêutico , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cicloexilaminas/farmacologia , Humanos , Quelantes de Ferro/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Fenilenodiaminas/farmacologia , Espécies Reativas de Oxigênio/sangue , Espécies Reativas de Oxigênio/metabolismo , Células THP-1RESUMO
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.
Assuntos
Benzoatos/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Triazóis/farmacologia , Células Cultivadas , Deferasirox , Relação Dose-Resposta a Droga , Armadilhas Extracelulares/metabolismo , Humanos , Ativação de Neutrófilo/fisiologia , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Contagem de Leucócitos , Síndromes Mielodisplásicas/patologia , Neutrófilos/patologia , Adolescente , Adulto , Feminino , Humanos , Contagem de Leucócitos/instrumentação , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Hemólise , Neutrófilos/metabolismo , Membrana Nuclear/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Feminino , Heme , Humanos , Masculino , Microscopia Confocal , Neutrófilos/patologia , Membrana Nuclear/patologia , Fatores de Tempo , Reação TransfusionalRESUMO
Impairment of macrophage phagocytosis is a major cause of chronic inflammation. Bisphosphonates (BPs) are widely used as anti-osteoclastic agents. The effects of BPs on monocyte-macrophage lineage cells are being increasingly reported; however, the detailed effects of BPs on macrophage phagocytic activity are still unclear. We examined the effects of four BPs: clodronate as a non-nitrogen containing BP (non-N-BP), and pamidronate, alendronate, and zoledronate as nitrogen-containing BP(N-BP), on macrophage phagocytic activity. The uptake of high fluorescence-labeled polystyrene beads by the human monocytic cell line THP-1 was investigated by flow cytometry. All three N-BPs suppressed the phagocytosis of macrophages more potently than the non-N-BP, clodronate. Pamidronate and zoledronate were more potent than alendronate. BP induced the apoptosis of THP-1. Pamidronate and zoledronate induced apoptosis more effectively than clodronate. The method described to observe phagocytosis was simple and quantitative, and might be useful in screening for the effects of drugs, such as N-BP and non-N-BP, on phagocytic activity.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Citometria de Fluxo/métodos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Alendronato/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Corantes Fluorescentes , Humanos , Imidazóis/farmacologia , Macrófagos/patologia , Pamidronato , Ácido ZoledrônicoRESUMO
OBJECTIVE: Reactive oxygen species (ROS) production by neutrophils induces pulmonary endothelial cell damage and results in acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits the ROS production and neutrophil extracellular trap (NET) formation induced by phorbol myristate acetate and formylmethionylleucylphenylalanine in vitro. In the present study, we investigated the effects of DFS in vivo using a mouse model of lipopolysaccharide (LPS)-induced ALI. METHODS: After DFS administration for 7 days, ALI was induced in mice by LPS via intratracheal administration. RESULTS: LPS treatment induced neutrophil invasion in the lung tissues, along with NET formation and a significant increase in the quantity of double-stranded DNA in the bronchoalveolar lavage fluid, while pre-administered DFS inhibited these phenomena. However, alteration of neutrophil morphology in the cytoplasm in terms of shape and vacuolization was not inhibited by the pre-administration of DFS, possibly through ROS production. CONCLUSIONS: DFS suppressed neutrophil invasion into lung tissues and reduced the double-stranded DNA content released by the neutrophils. These results suggest that DFS can potentially be used to prevent diseases related to neutrophil activation including ALI, thrombosis, and vascular endothelial dysfunction.
Assuntos
Deferasirox , Armadilhas Extracelulares , Quelantes de Ferro , Pulmão , Pneumonia , Animais , Quelantes , Deferasirox/farmacologia , Inflamação , Ferro , Quelantes de Ferro/farmacologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos , Pneumonia/tratamento farmacológicoRESUMO
Quantitative detection of minimal residual disease has prognostic value for some leukemias. Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARalpha fusion gene from t(15;17). Added to three PML-RARalpha isoforms, alternative spliced forms of PML exons give rise to multiple isoforms even within a single patient. To date, multiple primer pairs for the detection of the various PML-RARalpha transcripts have been designed, potentially generating some nonspecific amplification products. Here, we established a real-time quantitative PCR (RQ-PCR) strategy with a single primer pair using LightCycler (sp-RQ-PCR), which could simultaneously detect three isoforms with equal specificity and sensitivity as well as alternative spliced forms. Results obtained with sp-RQ-PCR for 39 samples from 15 APL patients and 31 non-APL samples were compared with those with TaqMan assay with three primer pairs. In two of the APL samples, PML-RARalpha was detected in the TM, but not in the sp-RQ-PCR or nested PCR. Furthermore, the sp-RQ-PCR showed no positive results for the 31 non-APL samples, whereas the TM identified 13% (4/31) as positive. Electrophoresis detected some artifacts in the TM, which do not correspond to PML-RARalpha. We conclude that our sp-RQ-PCR is specific enough to identify various forms of PML-RARalpha and yields no false-positive results.
Assuntos
Leucemia Promielocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Receptores do Ácido Retinoico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Primers do DNA/genética , Humanos , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Neoplásico/análise , Reprodutibilidade dos TestesRESUMO
Ratios of young platelets or reticulated platelets can be routinely obtained as an immature platelet fraction (IPF) with the XE-2100 automated hematology analyzer (Sysmex, Kobe, Japan). We combined IPF analysis of 31 patients with myelodysplastic syndrome (MDS) with a complete blood count, a bone marrow examination, and a chromosome analysis. The patients with >40 x 10(9)/L platelets were classified as group A, and those with > or =40 x 10(9)/L were placed in group B. The 2 groups were subclassified as A1 or B1 for patients with an IPF of <10% and as A2 or B2 for those with an IPF > or =10%. Categories A1, A2, B1, and B2 comprised 12 patients, 6 patients, 7 patients, and 6 patients, respectively. Patients with a relatively high IPF (>10%) (category A2 or B2) showed distinctive characteristics. Group B2 showed a higher frequency of chromosomal abnormalities than B1 (P = .029), and group A2 tended to show a higher incidence of clinical improvement than A1 (P = .08). IPF determination may be clinically useful for the assessment of prognosis for MDS patients.
Assuntos
Plaquetas/patologia , Síndromes Mielodisplásicas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células Sanguíneas , Exame de Medula Óssea , Aberrações Cromossômicas , Técnicas de Laboratório Clínico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Contagem de Plaquetas , Prognóstico , Adulto JovemRESUMO
Our previous observation of a higher incidence of FLT3-ITD in DR(-) M1/M2 AML than in DR(+) M1/M2 led to an investigation of NPM1 mutation in the same samples, since DR(-) AML and AML with NPM1 mutation share such characteristics as normal karyotype, the absence of CD34, and FLT3-ITD. NPM1 mutation was found in 18 of 26 (69.2%) of DR(-) cases, but not in any of 28 DR(+) cases. FLT3-ITD was noted in 66.7% of the cases with NPM1 mutation. These findings point to DR negativity as another phenotypic feature of AML with NPM1 mutation.
Assuntos
Antígenos HLA-DR/análise , Leucemia Mieloide Aguda/genética , Sequência de Bases , DNA Complementar , Humanos , Leucemia Mieloide Aguda/imunologia , Nucleofosmina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Immature or reticulated platelets are known as a clinical marker of thrombopoiesis. Recently, an automatic method was established to detect reticulated platelets as immature platelet fraction (IPF) by means of hematology analyzer XE-2100. We assessed the effects of IPF detection after chemotherapy for various pediatric malignant disorders of 16 patients. Our results indicate that IPF should be considered a useful marker of imminent platelet recovery so that unnecessary platelet transfusion can be avoided.
Assuntos
Plaquetas/citologia , Transfusão de Plaquetas/métodos , Adolescente , Automação , Plaquetas/metabolismo , Criança , Pré-Escolar , Desenho de Equipamento , Humanos , Lactente , Recém-Nascido , Contagem de Plaquetas , Valores de Referência , Transplante de Células-Tronco , Trombopoese , Fatores de TempoRESUMO
BACKGROUND: Rheumatoid factor (RF) and autoantibodies against cyclic citrullinated peptide (CCP) are markers that might help physicians diagnose rheumatoid arthritis. PURPOSE: To determine whether anti-CCP antibody more accurately identifies patients with rheumatoid arthritis and better predicts radiographic progression than does RF. DATA SOURCES: MEDLINE through September 2006 and reference lists of retrieved studies and review articles. STUDY SELECTION: Studies in any language that enrolled at least 10 participants and that examined the role of anti-CCP antibody and RF in the diagnosis or prognosis of known or suspected rheumatoid arthritis. DATA EXTRACTION: Two authors independently evaluated studies for inclusion, rated methodological quality, and abstracted relevant data. DATA SYNTHESIS: The DerSimonian-Laird random-effects method was used to summarize sensitivities, specificities, and positive and negative likelihood ratios from 37 studies of anti-CCP antibody and 50 studies of RF. The pooled sensitivity, specificity, and positive and negative likelihood ratios for anti-CCP antibody were 67% (95% CI, 62% to 72%), 95% (CI, 94% to 97%), 12.46 (CI, 9.72 to 15.98), and 0.36 (CI, 0.31 to 0.42), respectively. For IgM RF, the values were 69% (CI, 65% to 73%), 85% (CI, 82% to 88%), 4.86 (CI, 3.95 to 5.97), and 0.38 (CI, 0.33 to 0.44). Likelihood ratios among IgM RF, IgG RF, and IgA RF seemed to be similar. Results from studies of patients with early rheumatoid arthritis were similar to those from all studies. Three of 4 studies found that risk for radiographic progression was greater with anti-CCP antibody positivity than with IgM RF positivity. LIMITATIONS: Many studies had methodological limitations. Studies of RF were heterogeneous and had wide ranges of sensitivity and specificity. CONCLUSIONS: Anti-CCP antibodies are more specific than RF for diagnosing rheumatoid arthritis and may better predict erosivedisease.
Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/sangue , Peptídeos Cíclicos/imunologia , Fator Reumatoide/sangue , Biomarcadores/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Funções Verossimilhança , Prognóstico , Sensibilidade e EspecificidadeRESUMO
We herein introduce several clinically available methods to detect neutrophil function and oxidative stress. The flowcytometric detection of adhesive protein expression, such as CD11b(Mac-1), assessment of phagocytosis activity, and measurement of reactive oxygen species (ROS) production are relatively easy to apply as tools for laboratory medicine. A new device to simultaneously detect superoxide and calcium ion influx is also introduced. Oxidative stress induced by ROS produced not only from phagocytic cells but also from the mitochondria or endoplamic reticulum of all kinds of living cells is etiologically related to many disorders and also aging. A simple method using the FRAS4 instrument is demonstrated. These methods are expected to be clinically beneficial, especially in hematology, transfusion medicine, and the public health field.
Assuntos
Neutrófilos/imunologia , Estresse Oxidativo , Patologia Clínica , Animais , Antígeno CD11b/análise , Cálcio/análise , Moléculas de Adesão Celular/análise , Citometria de Fluxo , Humanos , Patologia Clínica/métodos , Fagocitose , Espécies Reativas de Oxigênio/análise , Superóxidos/análiseRESUMO
We discussed the usefulness of routine technologies of laboratory medicine in blood transfusion and transplantation medicine. New parameters that can be measured by automated hematology analyzers have been clinically evaluated and proven to be useful so far. Based on our experience, detection systems for fragmented red cells (FRC), immature platelets (immature platelet function, IPF), and hematopoietic progenitor cells (HPC) are useful for the diagnosis of thrombotic microangiopathy, differential diagnosis of thrombocytopenia, and decision regarding the optimal timing to collect peripheral stem cells, respectively. Moreover, IPF were suggested to be an indicator of the platelet transfusion requirement. The establishment of non invasive assaying technology has been eagerly anticipated. We evaluated a hemoglobin measurement tool, and revealed that it might be applicable in predeposited, autologous blood donation. Some adverse transfusion reactions are related to neutrophil activation. Thus, we investigated the effects of serum from patients and blood donors, in the context of adverse reactions, on adhesion molecule expressions of neutrophils from volunteers using flow-cytometry. This kind of simple technology is expected to be useful in future studies to clarify the mechanisms and prevent adverse reactions.
Assuntos
Transfusão de Sangue , Testes Hematológicos , Transplante de Células-Tronco Hematopoéticas , Transfusão de Sangue Autóloga , HumanosRESUMO
Activation of neutrophils by free heme is considered as one of the mechanisms for cellular dysfunction under the conditions of hemorrhage or tissue damage. We studied about the effects of hemin, ferriprotoporphyrin IX, on human neutrophil activation by measurements of adhesion molecule expression and reactive oxygen species (ROS) production. Human neutrophils purified from heparinized blood of healthy volunteers were stimulated with hemin. Surface expression of CD11b and L-selectin were evaluated by flow cytometry, and superoxide production was detected by chemiluminescence. Hemin increased the expression of CD11b and produced superoxide accompanying by increase in intracellular free calcium concentration. Thus, free heme-molecule is suggested to possess the activity to initiate or aggravate tissue injuries. Since neutrophils do not express CD163, scavenger receptor for hemoglobin-haptoglobin complex, the mechanisms by which hemin exerts these effects are still to be studied.
Assuntos
Hemina/farmacologia , Neutrófilos/fisiologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos CD11/análise , Cálcio/metabolismo , Células Cultivadas , Heme/fisiologia , Humanos , Selectina L/análise , Neutrófilos/imunologia , Receptores de Superfície Celular/análise , Superóxidos/análiseRESUMO
Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles. This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer. Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general-purpose conventional flow cytometer. In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained some vacuole-like structures of vacuoles or invagination of cell membrane. Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity.
Assuntos
Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Feminino , Humanos , Masculino , Microscopia Confocal/métodos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
FLT3 mutations and cell-surface antigen were investigated in 29 DR-negative (DR(-)) M1/M2 AML samples in comparison with 30 DR-positive (DR(+)) M1/M2 AML samples. FLT3-ITD was detected in 59.3% and D835 was detected in 7.4% of the samples. The incidence of FLT3-ITD was higher in the DR(-) group (59.3%) than in the DR(+) group (17.9%; P=0.002). The DR(-) status was associated with the CD34(-) (82.8%), CD7(-) (92.9%) and CD45RO(+) status (76%). Our results indicated that FLT3 mutation is the most common gene alteration found in the DR(-) M1/M2 AML. These results are important for further characterizing this phenotypic AML entity.