[Significance of Morphological Examination, Cytochemical Staining Combined with Bone Marrow Biopsy in Differential Diagnosis of Myelodysplastic Syndrome with Low Blasts and Hemolytic Anemia].

OBJECTIVE: To explore the value of morphological examination, cytochemical staining combined with bone marrow biopsy in the differential diagnosis between myelodysplastic syndrome (MDS) with low blasts and hemolytic anemia (HA). METHODS: The clinical data of 85 cases of myelodysplastic syndrome with low blasts (< 5%) and 61 patients with hemolytic anemia in Chinese PLA's Gerneral hospital from September 2009 to March 2015 were retrospectively analysed. The clinical characteristics, cytogenetic and molecular features, bone marrow cell count and morphology features, cytochemical staining results and bone marrow biopsy features of above-methioned patients were compared. RESULTS: There was no significant difference (P > 0.05) in clinical data between MDS group and HA group. Megakaryocytic dysplasia-positive rate, and ring sideroblasts positive rate, and PAS positive rate were significantly higher in MDS group than those that in HA group (P < 0.05). Abnormal localization of immature precursors (ALIP) and megakaryocytic dysplasia positive rate in bone marrow biopsy were significantly higher in MDS group than those that in HA group (P < 0.05), 90.6% of MDS with low blasts patients were identifiable by combined detections. CONCLUSION: Combining detection of morphology, cytochemistry staining and bone marrow biopsy has been confirmed to be more useful for differential diagnosis between MDS with low blasts and HA.

Imbalance of Th17/Tregs in rats with smoke inhalation-induced acute lung injury.

T helper (Th) 17 cells and CD4(+) CD25(+) regulatory T (Treg) cells are supposed to be critically involved in regulating autoimmune and inflammatory diseases. The aim of this study was to investigate the Th17/Treg pattern in rats with gunpowder smog-induced acute lung injury. Wistar rats were equally randomized to three groups: normal control group, ALI 6 h group (smoke inhalation for 6 h) and ALI 24 h group (smoke inhalation for 24 h). We observed changes in cell counting in bronchoalveolar lavage fluid (BALF), alveolar-capillary membrane permeability and lung tissue pathology. Moreover, rats in ALI 6 h and ALI 24 h group showed increased expression of Th17 cell and related cytokines (IL-17 A, IL-6, TGF-ß and IL-23). Meanwhile, Treg prevalence and related cytokines (IL-10, IL-2 and IL-35) were decreased. Consequently, the ratio of Th17/Treg was higher after smoke inhalation. Additionally, Th1 cell decreased while Th2 cell increased at 6 h and 24 h after smoke inhalation. In conclusion, Th17/Treg imbalance exists in rats with smoke inhalation-induced acute lung injury, suggesting its potential role in the pathogenesis of this disease.

Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia.

BACKGROUND: The diagnosis of myelodysplastic syndrome (MDS), especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA). METHODS: The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA. RESULTS: The MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0-3.79]) than the AA group (P < 0.05). Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]). CONCLUSIONS: These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

[Clinical Implications on ZO-1 Gene Methylation in Myelodysplastic Syndrome Progression].

OBJECTIVE: To investigate the clinical significance of ZO-1 gene methylation level in MDS progression in order to provide a theoretical basis for evaluating progrosis of MDS patients. METHODS: The methylation specific PCR (MS-PCR) was performed to evaluate the ZO-1 gene methylation status in bone marrow samples of normal persons as control (NC). MDS and AML patients, the bisulfite sequencing PCR (BSP) was applied to detect the ZO-1 gene methylation status in serial bone marrow samples of MDS-RA, MDS-RAEB and AML stages of a MDS patients. RESULTS: The possitive rate of ZO-1 gene methylation in samples of NC, MDS and AML patients displayed significant difference; in sample of NC group the positive of ZO-1 gene methylation was not observed, but the positive rate of ZO-1 gene methylation in samples of AML patients was highest (65.0%), the proportion of ZO-1 gene methylation in myeloid blast count of MDS/AML patients was higher (P=0.000). The serial samples in one MDS patient showed that along with progress of disease, the positive rate of ZO-1 gene methylation in MDS-RA, MDS-RAEB and AML stages was found to be obvious different (P=0.000), the positive rate of ZO-1 gene methylation in AML stage was highest (64.65%). CONCLUSION: The high methylation in promoter region of ZO-1 gene has been found in MDS/AML patients, and along with clonal proliferation, the positive rate of ZO-1 methylation and positive froguency of methylation sites increase graduatly which suggests that the MDS progresses in a certain degree, and the ZO-1 gene methylation level may be used as an new indicator for monitoring desease progression from MDS to AML.

[Clinical significance of bone marrow morphological examination and tumor marker detection for lymphoma diagnosis and prognosis].

OBJECTIVE: This study was aimed to evaluate the significance of bone marrow(BM) morphological examination and many tumor marker(TM) detection, especially carcinoembryonic antigen (CEA), cancer antigen 125(CA125), cancer antigen 15-3 (CA15-3) and serum ferritin (SF) for lymphoma diagnosis and prognosis. METHODS: A total of 47 confirmed patients with lymphoma in our hospital from January 2012 to October 2013 and 20 health peoplels as normal controls were performed with bone marrow morphological examination, at the same time, the electrochemistry luminescent technique was applied for detecting levels of TM (especially CEA, CA125, CA15-3 and SF) in serum samples of lymphoma patient and normal controls, then the BM immature lymphocyte counts of these people and clinical parameters were analyzed for diagnosis and prognosis. RESULTS: There was significant differences in all the four TM levels between serum samples of lymphoma patients and normal control (P=0.029, P=0.000, P=0.005, P=0.000). These TM levels had no correlation with age, sex white blood cell, lymphocyte, platelet counts and anemia of lymphoma patients (P>0.05). It was also found that the patients with elevated TM levels had high BM immature lymphocytes (lymphoma cells) counts, B symptoms, advanced clinical stage and high IPI index (P<0.05). The CA15-3 and SF levels in serum samples of lymphoma patients with BM infiltration were higher than that in lymphoma patients without BM infiltration (P=0.002, P=0.000). CONCLUSION: Combination of BM morphological examination with serum TM level detection plays an important role in diagnosis, clinical stage and prognosis evaluation of lymphoma patients. It is also very important for assessing BM infiltration status of lymphoma patients.

[Clinical Significance of ID4 Gene Mehtylation in Demethylation-Treated MDS Cell Line and 2 MDS Patients].

OBJECTIVE: To evaluate significance of ID4 gene mehtylation in demethylating myelodysplastic syndrome(MDS) cell Line MUTZ1 and 2 patients with MDS. METHODS: The methylation-specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to identify the methylation status and gene expression of ID4 gene in MDS cell line MUTZ1, a patient with aplastic anemia(AA) and a donor with normal bone marrow (NBM). RT-PCR was applied to detect the ID4 gene expression status in MUTZ1 cell line treated with decitabine at 3 different concentrations. Then bisulfite sequencing PCR (BSP) was applied to detect ID4 gene methylation status in 2 MDS parients treated with decitabine. RESULTS: The MDS cell line MUTZ-1 displayed a complete methylation of ID4 gene promoter with little mRNA expression. Inversely, bone marrow of an AA patient and NBM showed complete unmethylation of this gene with intensity mRNA expression. With the increase of decitabine concentration, ID4 gene mRNA expression was more and more increased. After decitabine treatment, ID4 gene methylation-positive frequencies of both the 2 MDS patients were much more decreased than that of the first treatment. So, ID4 gene mRNA expression inhibited by promoter hypemethylation could be recovered by using demethylation medicine. CONCLUSION: ID4 as a new potential anti-oncogene suggests that its methylation may become a marker for selection and assessment of therapeutic schedules in patients with MDS.

[Mechanism concerning antitumor effect of oridonin on multiple myeloma cell line U266].

This study was purposed to investigate the antitumor effect of oridonin on human multiple myeloma cell line U266 and its possible mechanism. The CCK-8 test was used to determine the inhibitory effect of oridonin on proliferation of U266 cells. The morphological changes of U266 cells were observed under optical microscope. The apoptosis rate of U266 cells was detected by flow cytometry. The mRNA levels of FGFR3, BCL2, CCND1 and MYC genes were quantified by using real-time quantitative PCR method, and the protein levels of BCL2, MYC, CCND1, FGFR3 and P53 were detected by Western blot. The results showed that the oridonin obviously inhibited the growth of U266 cell in dose-and time-dependent manners. As for morphological changes, characteristic apoptotic cells presented in U266 cells treated with 10 µmol/L oridonin for 24 hours. The apoptotic rate of U266 cells increased in dose and time dependent manners; after treatment of U266 cells with oridonin the mRNA levels of FGFR3, BCL2, CCND1 and MYC as well as the their protein levels decreased. Occasionally, the oridonin up-regulated the protein levels of P53 in the same manner. It is concluded that the oridonin can exert its anti-tumor effect by inhibiting proliferation and inducing apoptosis of U266 cell in dose dependent and time dependent manners, that maybe give the clues about new program of target therapy for multiple myeloma.

Detection and clinical significance of gene rearrangements in Chinese patients with adult acute lymphoblastic leukemia.

This study aimed to develop a novel multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) assay to accurately and effectively detect 10 common gene rearrangements in adult acute lymphoblastic leukemia (ALL) and to examine the clinicopathologic characteristics and other genetic aberrations of patients with ALL expressing different fusion genes. Our RT-nPCR assay had a positive detection rate of 35.15% (90/256) for the 10 fusion genes. BCR-ABL1, FUS-ERG, MLL-AF4, ETV6-RUNX1, E2A-PBX1, dupMLL, MLL-AF10, MLL-ENL, SET-NUP214 and SIL-TAL1 were detected in 36 (14.06%), 14 (5.47%), 14 (5.47%), four (1.56%), four (1.56%), five (1.95%), four (1.56%), two (0.78%), two (0.78%) and five patients (1.95%), respectively. The RT-nPCR results were further confirmed by split-out PCR, and cytogenetic and fluorescence in situ hybridization (FISH) analysis revealed corresponding translocations and fusions in 63 and 74 cases, respectively. JAK2 and IKZF1 mutations were commonly detected in patients with BCR-ABL1 ALL, and HOX overexpression was highly correlated with MLL fusions and SET-NUP214. This study demonstrates that RT-nPCR is an effective method for identifying 10 gene rearrangements in adult ALL, and it could potentially be developed for diagnostic use and prognostic studies of ALL.

Detection of ETV6 gene rearrangements in adult acute lymphoblastic leukemia.

ETV6 is an important hematopoietic regulatory factor and ETV6 gene rearrangement is involved in a wide variety of hematological malignancies. In this study, we sought to investigate the incidence of ETV6-associated fusion genes in B- and T-lineage acute lymphoblastic leukemia (ALL) by multiplex-nested reverse transcription-polymerase chain reaction (RT-PCR) in 176 adult ALL patients. Total RNA was extracted from bone marrow samples of ALL patients including 136 B- and 40 T-lineage ALL, and ETV6 fusion genes were detected by multiplex-nested RT-PCR. Changes of ETV6 fusion gene mRNA transcript levels were examined by real-time RT-PCR. We detected a total of 15 ETV6 gene rearrangements with a positive rate of 8.5%, involving seven ETV6-associated fusion genes in 13 B-ALL (13/136, 9.6%) and 2 T-ALL patients (2/40, 5.0%). ETV6-RUNX1 were observed in six cases (3.4%), ETV6-JAK2 in three cases (1.7%), ETV6-ABL1 in two cases (1.1%), and ETV6-ABL2, ETV6-NCOA2, ETV6-SYK, and PAX5-ETV6 each in one case (0.6%). ETV6-JAK2 was found in both B-ALL and T-ALL patients. Furthermore, real-time quantitative RT-PCR assays showed that the ETV6-RUNX1 mRNA transcript levels decreased during conventional chemotherapy or hematopoietic stem cell transplantation. This study shows that multiplex-nested RT-PCR is an effective and accurate tool to identify ETV6 rearrangements in adult ALL, which provides some clues into the diagnosis and prognosis of ALL but also molecular markers for the detection of minimal residual disease in adult ALL.

[Detection of bone marrow involvement by lymphoma cells in patients with B-NHL by using flow cytometry analysis].

This study was purposed to investigate the significance of using (FCM) flow cytometry for detection bone marrow involvement by lymphoma cells in untreated patients with B cell non-Hodgkin's lymphoma (B-NHL). Bone marrow samples of 54 patients with B-NHL were analyzed by flow cytometry, morphological method and molecular biology technique. Bone marrow involvement was diagnosed based on the results of morphology, FCM and molecular biology. The results indicated that the positive ratios of bone marrow involvement were different detected by three methods, the most sensitive method was FCM (positive rate was 27.5%), the moderately sensitive method was molecular biology (positive rate was 22.2%), the inferior method was morphology method (positive rate was 5.6%). Combined with three methods, the positive rate increased to 38.9%. The monoclonal B lymphocytes were detected by FCM in fourteen patients, the ratios of kappa light chain/lambda light chain were far beyond the diagnostic threshold. Even in patients with early stage, lymphoma cells still could be detected by FCM in involved bone marrow. Monoclonal B lymphocytes were detected by FCM in three patients who were diagnosed bone marrow involvement by morphological method. The concordance between FCM and molecular biology was relatively high (70.4%, p=0.14). It is concluded that the detection of monoclonal B lymphocytes by using FCM has high sensitivity and accuracy in patients with B-NHL. Evaluation whether the bone marrow has been involved by lymphoma cells should be recommend to every patient with B-NHL before chemotherapy and every disease stages. Combined application of FCM and molecular biology method would enhance diagnostic efficacy.

[Changes of T-cell clonality after induction-cultivation of peripheral T lymphocytes in adoptive immunotherapy for leukemias].

This study was purposed to analyze the changes of T-cell clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor beta variable region (TCRBV) families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOXP3(+) cells to disclose the ratio change of cytotoxic T lymphocytes (CTL), helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 +/- 1.20 times. CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOX P3(+) cells were 71.1 +/- 11.8%, 26.7 +/- 11.4%, 35.7 +/- 12.9%, 3.1 +/- 1.6% and 0.12 +/- 0.1% respectively before the induction and cultivation, and changed to 95.4 +/- 3.2% (p < 0.01), 27.0 +/- 13.1% (p > 0.01), 55.5 +/- 13.8% (p < 0.01), 9.8 +/- 6.1% (p < 0.01) and 0.22 +/- 0.18% (p < 0.01) respectively after the induction and cultivation. It is concluded that the major action of this induction and cultivation method on T-lymphocytes in vitro is the promotion of CTL and NK T-cell proliferation. In leukemic patients at the remission stage, the TCRBV profile is characterized by the oligoclonal proliferation of T-lymphocytes. Several proliferated clones may have the same motif in CDR3, suggesting the recognition of the same antigen by these lymphocyte clones. Cytokine induction and co-culture with autogenous DCs can stimulate the T-lymphocytes to recover their immunocompetence as manifested by the polyclonal profile and the proliferation of CTL and NK-T cells.

Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line.

BACKGROUND: MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18 - 25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown. METHODS: miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A. RESULTS: Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A. CONCLUSION: HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.