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Introduction: In allergic diseases, group 2 innate lymphoid cells (ILC2s) play critical roles. Neuromedin U (NMU), a highly conserved multifunctional neuropeptide, is secreted by cholinergic neurons and involved in asthma pathogenesis by amplifying lung inflammation driven by ILC2s. However, the precise effects of NMU on ILC2s in allergic rhinitis (AR) and related diseases remain unclear. Methods: A total of 15 patients with persistent AR and 8 healthy controls (HCs) were enrolled in the study. Visual analog scale (VAS) scores are used to assess the severity of clinical symptoms in AR patients. The percentages of ILC2s in peripheral blood mononuclear cells (PBMCs) were enumerated using flow cytometry. The soluble or intracellular cytokines (IL-5 and IL-13) in PBMCs or sorted ILC2s were assessed in response to various stimuli with IL-33, NMU, IL-33 combined with extracellular signal-related kinase (ERK) inhibitor or NMU combined with ERK inhibitor in the presence of IL-2. Results: We confirmed the proportion of circulating ILC2s was significantly higher in AR patients than in HCs. ILC2s levels were found to be positively related to VAS scores. We also discovered that the release of IL-5 and IL-13 in AR patients’ PBMCs stimulated by NMU (p < 0.0001 and p < 0.0001, respectively) or IL-33 (p = 0.002; p = 0.044, respectively) was significantly higher than in HCs. In AR patients, NMU stimulated PBMCs or ILC2s to generate greater inflammatory factors IL-5 and IL-13 compared to IL-33. Furthermore, we observed that NMU-promoted ILC2s activation and proliferation functions were restricted when the ERK pathway was inhibited. Conclusion: NMU effectively activated ILC2s in AR patients to produce Th2-type cytokines, and this activation can be prevented by ERK pathway inhibitors. Our findings shed new light on the neuro-immune mechanism of AR and offer new insights into its prevention and treatment.

Polyadenylation of pre-messenger RNA (pre-mRNA) specific sites and termination of their downstream transcriptions are signaled by unique sequence motif structures such as AAUAAA and its auxiliary elements. Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism that processes RNA products depending on its 3′-untranslated region (3′-UTR) specific sequence signal. APA processing can generate several mRNA isoforms from a single gene, which may have different biological functions on their target gene. As a result, cellular genomic stability, proliferation capability, and transformation feasibility could all be affected. Furthermore, APA modulation regulates disease initiation and progression. APA status could potentially act as a biomarker for disease diagnosis, severity stratification, and prognosis forecast. While the advance of modern throughout technologies, such as next generation-sequencing (NGS) and single-cell sequencing techniques, have enriched our knowledge about APA, much of APA biological process is unknown and pending for further investigation. Herein, we review the current knowledge on APA and how its regulatory complex factors (CFI/IIm, CPSF, CSTF, and RBPs) work together to determine RNA splicing location, cell cycle velocity, microRNA processing, and oncogenesis regulation. We also discuss various APA experiment strategies and the future direction of APA research.

Jak2 is a nonreceptor tyrosine kinase that plays a critical role in signal transduction through an abundance of receptors, such as erythropoietin receptor. In this paper, we report two previously unknown transcripts of Jak2 gene. One transcript deletes the 77nt of 3' end exon 10 of the Jak2 gene, resulting in a frameshift that introduces a stop codon in the downstream exon and produces a truncated protein of 421 amino acids if translated. The other transcript skips the entire exon 10, leading to a premature stop codon in the adjacent exon 11, producing a truncated protein of 414 amino acids if translated. Therefore, the physiological significance of the expression of two novel transcripts in healthy volunteers and patients with myeloproliferative neoplasms, acute leukemia, and chronic myeloid leukemia needs to be investigated further.

Background Bone marrow mesenchymal stromal cells (BM-MSCs) are essential structural and functional components of the BM microenvironment and play an important role in acute myeloid leukemia (AML) pathogenesis. BM-MSCs isolated from AML patients (AML-MSCs) show distinct signatures from normal BM-MSCs. However, the exact abnormalities of AML-MSCs and the origin of these abnormalities are still unknown.Methods In this study, we evaluated the proliferative activity of AML-MSCs, and the influence of leukemia cells (LCs) on BM-MSCs. These two cell types were co-cultured using an in vitro co-culture system, and the biological functions of AML-MSCs, healthy donor derived MSCs (HD-MSCs), and LC-treated HD-MSCs (LCtrHD-MSCs) were compared by flow cytometry, and CCK-8 and chemotaxis assays. Student t-test (between two groups) and one-way ANOVA (more than 2 groups) were used to compare differences. Pearson correlation coefficients were used to assess correlations between two factors.Results AML-MSCs display a significant proliferative deficiency, which correlates with primary leukemic blast cell counts but not with patients’ age. Inhibition of BM-MSC proliferation could be induced by leukemia cells through direct contact. Co-cultured leukemia cells also increase expression of several inflammatory cytokines, and chemokines in BM-MSCs. Furthermore, LCtrHD-MSCs reduced apoptosis, and increased migration and chemoresistance in co-cultured AML cells, comparable with AML-MSCs.Conclusions Our results showed that leukemia cells can induce healthy donor derived BM-MSCs to exhibit AML-MSC-like characteristics and indicated that AML-MSC abnormalities may be partly induced by leukemia cells.

Background Blast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T‐LBL/ALL) is rare, and the molecular mechanism is still unclear. Case report A 28‐year‐old woman who developed T‐ALL with coexpressing both p210 and p190 BCR‐ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t(9;22)(q34;q11) and t(10;11)(q25;p15). Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3. Conclusion It is the first T‐cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T‐lymphocyte transformation depend on potential molecular and pathological mechanism.

Objective To investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL) and explore the sensitivity and specificity of various antibody combinations for the timely and accurate diagnosis APL. Methods A retrospective analysis was performed using morphological, immunological, genetic, and molecular biological data from 92 patients diagnosed with APL and 190 controls diagnosed with non-APL acute myeloid leukemia. Results For APL diagnosis, the CD9/CD11b/human leukocyte antigen (HLA)-DR antibody combination had 85% sensitivity and 95% specificity, AUC = 0.85. However, the sensitivity and specificity were 39% and 92%, AUC = 0.65, respectively, for the HLA-DR/CD34/CD117 combination, and 80% and 80%, AUC = 0.80, respectively for the CD11b/HLA-DR combination. Significant differences were observed between the different antibody combinations. Conclusions The CD9/CD11b/HLA-DR antibody combination displays high sensitivity and specificity and can be used to diagnose APL.

OBJECTIVE This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2 ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P

More than 100 cases harboring both myeloproliferative neoplasms (MPN) and chronic lymphocytic leukaemia (CLL) have been reported, suggesting that the two diseases can coexist in one patient. However, the mechanism by which this phenomenon is caused remains unclear. In this study, one patient with polycythemia vera (PV) and CLL is examined. Identifications of the

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib-resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib-resistance of K562/G01 cells, and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Most patients with acute myeloid leukemia (AML) achieve complete remission (CR) after induction chemotherapy, however, in some patients, the disease subsequently relapses and may lead to death. Leukemia stem cells (LSC) have been identified as the main cause for recurrence. Increased aldehyde dehydrogenase (ALDHhigh) activity in a variety of cancer stem cells prevents effective action of chemotherapeutic drugs. In this study, we found that approximately 50.7% of AML patients had ALDHhigh, and the presence of ALDHhigh stem cells was associated with poor cytogenetic prognosis. Lentiviral vector transduced ALDHhigh leukemia cell lines are insensitive to the conventional chemotherapy drug cytarabine, and inhibition of ALDH activity by disulfiram (DSF) can increase the sensitivity of ALDHhigh leukemia cells to cytarabine. Unlike traditional chemotherapy drugs, DSF is not toxic to healthy umbilical cord blood stem cells. An ALDHhigh leukemia cell xenograft model was established using immunodeficient mice to mimic the disease environment, and DSF and cytarabine were found to eliminate the ALDHhigh leukemia cells in transplanted mice while not affecting the healthy blood cells of mice. These findings suggest that DSF may have therapeutic potential by inhibiting ALDH activity and thereby increasing chemosensitivity.

To study the association between TET2rs2454206, TET2rs12498609 and ASXL1rs3746609 and Myelodysplastic syndromes (MDS), a total of 90 MDS patients and 143 healthy volunteers were included. The clinical data, bone marrow samples of patients and peripheral blood samples of volunteers were obtained. We found TET2rs2454206 G/A genotype, TET2rs12498609 G/C genotype and ASXL1rs3746609 A/G genotype in 13.3%, 11.1%, 10.1% MDS patients and in 42.7%, 22.4%, 23.8% healthy volunteers (P 0.001; P = 0.029; P = 0.009, respectively). TET2 rs2454206 G/A genotype was associated with higher serum LDH level in MDS (P = 0.025). Patients with TET2rs12498609 G/C genotype were characterized with higher frequency of mutated SRSF2 gene (P = 0.042) and lower occurrence rate of anemia (P = 0.026) than those with C/C genotype. ASXL1rs3746609 A/G genotype linked with higher thrombocyte counts (P = 0.02) and percent of total T lymphocyte (P = 0.029), whereas with lower percent of NK cell (P = 0.032) and B lymphocyte (P = 0.007). None of these three SNPs had impact on the overall survival and disease progression to AML. We concluded that People with TET rs2454206 G/A genotype, TET2rs12498609 G/C genotype or ASXL1rs3746609 A/G genotype were related to lower prevalence of MDS. All of the three SNPs were associated with certain laboratory features in MDS patients.

To investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL) and explore the sensitivity and specificity of various antibody combinations for the timely and accurate diagnosis APL.A retrospective analysis was performed using morphological, immunological, genetic, and molecular biological data from 92 patients diagnosed with APL and 190 controls diagnosed with non-APL acute myeloid leukemia.For APL diagnosis, the CD9/CD11b/human leukocyte antigen (HLA)-DR antibody combination had 85% sensitivity and 95% specificity, AUC = 0.85. However, the sensitivity and specificity were 39% and 92%, AUC = 0.65, respectively, for the HLA-DR/CD34/CD117 combination, and 80% and 80%, AUC = 0.80, respectively for the CD11b/HLA-DR combination. Significant differences were observed between the different antibody combinations.The CD9/CD11b/HLA-DR antibody combination displays high sensitivity and specificity and can be used to diagnose APL.

C-KIT gene mutations result in the constitutive activation of tyrosine kinase activity, and greatly affect the pathogenesis and prognosis of core-binding factor acute myeloid leukemia (CBF-AML). C-KIT mutations are often found as single point mutations. However, the rate of double mutations has recently increased in AML patients. In this study, we detected six cases (18.8%) harboring double C-KIT exon17 mutations in 75 patients with CBF-AML. The clone composition and dynamic evolution were analyzed by sequencing and droplet digital PCR (ddPCR). Results revealed that these double mutations can be occurred in either the same or different clones. Different clones of double mutations may result in different sensitivity to the treatment of CBF-AML. The clones with N822 mutation responded better to treatment as compared to those with D816 mutation. Moreover, D816 clone was readily transformed into a predominant clone at relapse. Meanwhile, the predominant clones in the same patient may change during the progression of disease. The emerging mutation can originate from a small quantity of clones at diagnosis or newly acquired during the course of disease. Furthermore, patients with double mutations had better overall survival (OS) and event-free survival (EFS) than those with single mutation, but the differences did not reach statistical significance (P > 0.05). The ddPCR is an effective method for monitoring clonal evolution in patients with CBF-AML.

An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable.

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outwardfacing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription. [BMB Reports 2013; 46(3): 163-168]

BACKGROUND Current diagnostic methods of acute promyelocytic leukemia (APL) have some inevitable shortcomings; therefore, further studies are needed to develop more effective diagnostic methods. We used functionalized graphene oxide (GO) to detect the promyelocytic leukemia/retinoic acid receptor, α fusion gene (PML/RARα fusion gene) in bone marrow of APL patients. This method was more convenient and time-saving, and we can obtain the detection results in 1 hour. METHODS Our group consists of 36 cases, among them are 16 cases which are PML-RARα positive, 20 cases which are PML-RARα negative. Firstly, samples were fixed, drilled, and incubated with antibody CD45. Secondly, GO, fluorescent probes, and buffer liquid were added. One hour later, samples were washed with PBS (1 x) buffer, centrifuged, and fluorescent signals were detected with flow cytometry. RESULTS Using functional GO to carry the fluorescent probe we ascertained whether bone marrow samples contain the L type PML/RARα fusion gene. Using the probe, only cells which contain L type PML/RARα fusion gene will have fluorescent signals compared to no signals (p < 0.05). The GO detection method was accurate and has clinical diagnostic values (p < 0.05). CONCLUSIONS The GO detection method has the advantages of accurate, time-saving, energy-saving simple operation, and no pollution.

To identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN).MPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR).A novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people.MPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.

Alternative splicing is associated with human disease. In acute promyelocytic leukaemia (APL) patients with the long (L)-type promyelocytic leukaemia-retinoic acid receptor α fusion gene (PML-RARA), three alternative splicing isoforms can be detected: E5(+)E6(+), E5(-)E6(+), and E5(-)E6(-). This study is the first to demonstrate that alternative splicing of L-type PML-RARA is associated with time to achieve complete remission (CR) in APL. Higher expression of the E5(-)E6(-) isoform, the short isoform, was related to longer time to achieve CR. Each isoform was constructed into recombinant lentiviral vector and transfected into U937 cells. Compared with the E5(-)E6(+) and E5(+)E6(+) groups, the U937 cells with E5(-)E6(-) showed lower sensitivity to all-trans-retinoic acid treatment.

Tetraploidy is a rare chromosome number aberration in de novo acute myeloid leukemia (AML), and may be associated with erythrophagocytosis by leukemic blast cells. We report a 48-year-old female patient with minimally differentiated acute myeloblastic leukemia (AML-M0) exhibiting tetraploidy and erythrophagocytosis. The karyotype was 46,XX[2]/92,XXXX[18]. Bone marrow aspirate smears showed large and prominent nuclei, with erythrophagocytosis in leukemic cells. Fluorescence in situ hybridization using RUNX1 dual color break probes detected four fusion signals, accounting for 95 % (190/200), in one interphase nucleus. The mutations of TP53 and the fusion genes RUNX1/ETO, CBFβ/MYH11, and PML/RARα were all negative. This patient showed a poor response to chemotherapy, and died 66 days after the onset. To our knowledge, this is the first reported case of AML-M0 with tetraploidy and erythrophagocytosis and without additional chromosome aberrations. This case of tetraploid AML with poor prognosis suggests that further biological study of more cases of tetraploid AML will be of great importance in improving the understanding and prognosis of this tetraploid AML.