We evaluated the diagnostic utility of peripheral blood neutrophil distribution patterns obtained using the CELL-DYN SAPPHIRE hematology analyzer in patients with myelodysplastic syndrome (MDS). ...Peripheral blood was obtained from 467 individuals including 32 patients with MDS, and the respective neutrophil distribution patterns were observed using two light scatters 7-degree complexity (7D) and 90-degree lobularity (90D). These scattering intensities are shown as median (median neutrophil distribution: MND) and coefficient of variation (neutrophil distribution width: NDW). Generally, MDS patients showed lower 7D MND, higher 7D NDW, lower 90D MND and higher 90D NDW than other comparable groups. Whereas 90D parameters were more diagnostically efficient than 7D ones in patients with MDS. The sensitivity and specificity of 90D MND for MDS patients became 78.1 and 78.9%, respectively (cut-off value = 14,514). 90D NDW was most diagnostically effective with 87.5% sensitivity and 91.0% specificity (cut-off value = 21.2%). Both 90D parameters showed no evident correlation with the degree of either leukocytopenia or peripheral blood dysgranulopoiesis. In conclusion, neutrophil distribution parameters, especially 90D NDW, appear to provide convenient and objective markers for the screening of patients with MDS in routine laboratory hematology.
Osteoclasts are bone-resorbing cells which play an exclusive role in bone remodeling, but the molecular mechanisms of osteolysis, how osteoclasts are activated and how the lytic granules are finally ...released towards the bone matrix are poorly understood. Here we show that an energy molecule ATP induces osteolysis via P2X₇-nucleotide receptor and that deacetylation of α-tubulin is essential for the whole process of osteolysis under the control of a tyrosine kinase Syk. By developing a traceable and reproducible in vitro analyzing system for osteoclast function, we found that ATP-signaling gives rise to two events simultaneously (i) cytoskeletal reorganization for the formation of sealing zones, ring-like adhesion structures which delimit the contact surface, and (ii) the delivery and secretion of lytic granules towards the delimited site on the matrix. We further found that deacetylation of α-tubulin is a critical reaction for osteoclast function. Pharmacological inhibition of α-tubulin deacetylation resulted in (i) failure of the sealing-zone like structure formation and (ii) ceased secretion of lytic granules. Additionally, kinetics of deacetylation was found to be regulated by Syk. These data suggest a novel P2X₇ microtubular regulation pathway related to Syk for a therapeutic target in osteolytic diseases.
Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Survivin is a member of the inhibitor of apoptosis family ...and suppresses apoptosis. Survivin also functions as a subunit of the chromosomal passenger complex for regulating mitosis with Aurora-B. Survivin and Aurora-B play an important role in maintaining genome stability. The aim of this study was to determine the role of Survivin and Aurora-B kinase in disease progression and prognosis of myelodysplastic syndromes.
We evaluated the expression levels of these two genes in CD34(+) cells prepared from 64 patients with myelodysplastic syndrome or leukemic blasts from 50 patients with de novo acute myeloid leukemia using quantitative real-time PCR.
Survivin and Aurora-B expression levels were highly correlated with the type of myelodysplastic syndrome, were much higher in refractory anemia with excess blasts-1, refractory anemia with excess blasts-2, and secondary acute myeloid leukemia following myelodysplastic syndrome than in normal control, and increased during disease progression. There was a significant correlation between these expression levels and the International Prognostic Scoring System. Interestingly, these levels were remarkably higher in patients with secondary acute myeloid leukemia following myelodysplastic syndromes than in those with de novo acute myeloid leukemia.
This is the first report showing that high levels of Survivin and Aurora-B kinase expression in CD34(+) cells are distinctive molecular features of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia following myelodysplastic syndrome. Marked upregulation of Survivin and Aurora-B kinase may contribute to genetic instability and disease progression of myelodysplastic syndromes. Our data may explain why patients with high-risk myelodysplastic syndromes frequently show complex chromosomal abnormality.
When elderly patients present with persistent thrombocytosis, myeloproliferative disease, iron-deficiency anemia or post-splenectomy status are suspected along with autoimmune diseases. Reported here ...are the cases of two elderly patients with persistent thrombocytosis due to hyposplenia, which is very rarely diagnosed in old age. Case 1 was a 72-year-old man whose thrombocytosis was due to non-familial type isolated congenital asplenia. Case 2 was a 74-year-old man whose thrombocytosis was caused by an atrophied spleen resulting from perforated stomach ulcer-related panperitonitis that had been treated 20 years previously. Both patients had thrombocyte counts exceeding >500,000/μl in association with small vestigial spleen tissue on a computed tomography scan and positive Howell–Jolly bodies on the blood smear. A correct diagnosis is essential for the management of persistent thrombocytosis.
Complement-mediated phagocytosis plays an essential role in host defense against invading pathogens. Phagocytosis requires a dynamic and coordinated reconstruction of the membrane and the underlying ...cytoskeleton. Pathogens are entrapped into a phagosome and internalized into the cell. Subsequently, the phagosome fuses with lysosomes to form a phagolysosome with gradual acidification. Previous studies showed the pivotal roles of phosphoinositide-mediated signaling and the synchronous actin-remodeling in phagosome formation, but the mechanisms of phagolysosome generation, namely phagosome-lysosome fusion remain largely unexplored.
Here we show that phagosome-lysosome fusion requires the collapse of F-actin structure surrounding phagosomes and a tyrosine kinase, Syk plays a key role in this process. To reveal the mechanisms of phagosome-lysosome fusion particularly focusing on Syk, we performed complement-mediated phagocytosis assay using human leukemic HL60 and Syk-knockout (Syk-KO) HL60 cells established by the CRISPR/Cas9 system. Parental HL60 and Syk-KO HL60 were differentiated into macrophage-like cells and incubated with complement-opsonized Candida albicans(C. albicans) or fluorescence-labeled zymosan particles.
Syk-KO cells permitted the survival of C. albicansand their escape from the phagosome.In general, after fusion with lysosomes, the phagosome acquires acidic milieu inside the organelle and enhanced bactericidal activity. To confirm the effects of Syk-KO on phagosome acidification, we used two types of fluorescence-labeled zymosan particles: one was labeled with fluorescein isothiocyanate (FITC) whose fluorescent intensity is reduced under decreased pH condition and the other was labeled with Texas Red whose intensity is conserved independent of pH change. Flow cytometric analysis indicated that Syk-KO cells showed insufficient phagosome acidification. These results suggest that Syk facilitates acidification of phagosomes and results in an enhanced bactericidal activity against pathogens following complement-mediated phagocytosis.
Since ourprevious study indicated the participation of Syk in dynamics of actin cytoskeleton, we examined the effect of Syk-KO on the distribution of F-actin around phagosomes. Two hours after phagocytosis, pathogen-containing phagosomes of both parental and Syk-KO HL60 cells were already intracellularly distributed. At that time, F-actin structure around phagosomes of parental HL60 cells was hardly invisible but the phagosomes of Syk-KO cells were circumscribed by dense F-actin structure. An F-actin-stabilizing agent, jasplakinolide induced a similar fusion defect in peripheral blood-derived monocytes as observed in Syk-KO cells. Presence of dense F-actin structure surrounding phagosomes seemed to be responsible for inhibiting phagosome-lysosome fusion. In other words, collapse of F-actin structure surrounding phagosomes may be a critical step of phagolysosome generation.
Our results demonstrate that actin-remodeling is essential not only for phagosome formation but also for phagosome-lysosome fusion in complement-mediated phagocytosis and that Syk promotes this process. The present study provides new insights into the mechanism controlling phagocytic progression that leads to host defense against pathogens.
No relevant conflicts of interest to declare.
Objectives
Effects of arsenic disulfide (As
2
S
2
) were investigated by focusing on growth inhibition, apoptosis induction, and erythroid differentiation in MDS-L, F-36p and HL-60 cells, derived ...from myelodysplastic syndrome (MDS), MDS/acute myeloid leukemia (AML), and de novo AML, respectively.
Methods
Cell viability was determined by MTT assay. Apoptosis induction was analyzed using Annexin V/propidium iodide staining. Erythroid differentiation was assessed by the expression level of CD235a, a marker for detection of the erythroid cell lineage. The activation of p38 MAPK and the expression profile of apoptosis-related proteins Bcl-2 and Bid were analyzed using western blot.
Results
As
2
S
2
inhibited cell growth of these cell lines. Of note, the IC
50
value of As
2
S
2
in MDS-L cells was comparable to that in F-36p cells, and was half of that in HL-60 cells. A dose-dependent decrease in cell viability and concomitant increase in the percentage of apoptotic cells were observed in F-36p cells treated with 8 and 16 µM As
2
S
2
for 72 hours. However, similar phenomena were only observed in HL-60 cells when treated with as high as 16 µM As
2
S
2
. Furthermore, As
2
S
2
exerted more potent erythroid differentiation-inducing activity on F-36p cells than HL-60 cells. Interestingly, negative correlation between p38 MAPK signaling pathway and As
2
S
2
-induced erythroid differentiation was observed in HL-60 cells. Treatment with relatively high concentration of As
2
S
2
resulted in the downregulation of Bcl-2 and Bid proteins in HL-60 cells.
Discussion
These results suggest that compared to AML cell line, MDS and MDS/AML cell lines are more sensitive to not only the erythroid differentiation-inducing activity of As
2
S
2
, but also its cytotoxicity associated with apoptosis induction. These findings further provide novel insight into As
2
S
2
action toward its use for clinical application in patients with hematological disorders.
We herein describe the case of a 60-year-old man with a history of Behçet's disease and myelodysplastic syndrome who received cord blood transplantation (CBT). The patient was given anti-thymocyte ...globulin conditioning and tacrolimus to prevent graft-versus-host disease. Two months after CBT, his blood Tac concentration measured by an antibody-conjugated magnetic immunoassay (ACMIA) was found to have increased >4-fold, even after the Tac treatment was stopped. This false response was caused by the interference of endogenous heterophilic antibodies with ACMIA. Therefore, physicians must be aware of possible false ACMIA results for patients with a history of autoimmune disease and/or treated by xenogeneic antibody therapy.
Calreticulin (
CALR
) and
JAK2
-V617F gene mutations, which are major genetic mutations in patients with primary myelofibrosis (PMF) and essential thrombocythemia (ET), exert different effects on the ...clinical features and outcomes of these diseases. We analyzed 88 and 9 patients with ET and PMF, respectively, and determined the differences in the clinical characteristics of ET patients with
JAK2
-V617F compared with
CALR
mutations. The frequency of the
JAK2
-V617F and
CALR
mutations were 64 and 22 %, respectively. Patients with
CALR
mutations were younger, had a lower white blood cell count, and had a lower rate of thrombotic events than patients with the
JAK2
mutation. The neutrophil alkaline phosphatase (NAP) score of 16 patients with
CALR
mutations was significantly lower than the normal controls, which was mainly due to the high proportion of NAP-negative neutrophils. This is the first report to show an association between
CALR
mutations in patients with myeloproliferative neoplasms (MPN) and the NAP score. Although the mechanism is unclear, the NAP score could be a useful and reliable biochemical marker to discriminate the mutational status of MPN patients. Further investigation is warranted to determine whether these characteristics contribute to the pathogenesis of MPN and the NAP score.
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