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The JAK2 V617F mutation has been observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs), including polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. This mutation has also been observed in a small number of other myeloid malignancies, such as acute myeloid leukemia, chronic myeloid leukemia and myelodysplastic syndrome. The JAK2 V617F allele has rarely been evaluated in lymphoproliferative disorders. In total, 28 JAK2 V617F-positive B-cell lymphocytic leukemia (B-CLL) patients have previously been reported and all presented with Ph-MPN concomitantly. However, following investigation of the JAK2 V617F mutation in 63 B-CLL patients at the Shanghai First People's Hospital (Shanghai, China) between January 2008 and December 2012 via allele-specific polymerase chain reaction, two B-CLL patients without a history of Ph-MPN were identified to carry the JAK2 V617F allele.

To evaluate the role of a panel fluorescence in situ hybridization (Panel-FISH) for the detection of common cytogenetic abnormalities in patients with chronic lymphoblastic leukemia (CLL), multiplemyeloma (MM) and myelodysplastic syndrome (MDS).Three panels of probes were used to perform FISH assays in 46 patients with CLL, 53 with MM and 93 with MDS. Their results were compared with that obtain by conventional cytogenetic examination.The panel FISH detection in CLL and MM groups showed significantly higher sensitivity in revealing chromosomal abnormalities than that in conventional cytogenetics (73.8% vs 9.5%, 70.8% vs 22.9%, respectively). There were significant differences between these 2 technologies(P0.001, P0.001, respectively). However, there was no difference between Panel-FISH and conventional cytogenetics in MDS group (30.4 vs 27.2%, P=0.625).Panel-FISH can increase the detection rate in CLL and MM patients while it did not in MDS patients. However, it can increase the detection rate of aberration clones in MDS cases with normal karyotypes or without enough karyotypes to be analysis.

t(8;22)(p11;q11) is a rare but recurrent genetic alteration in various hematological disorders. Patients with t(8;22)(p11;q11) may be misdiagnosed with chronic myelogenous leukemia (CML), due to the similar clinical features. Thus, the current study presents a patient with t(8;22)(p11;q11) who was previously misdiagnosed with CML in the chronic phase. The current patient was a 26-year-old woman who was 4-weeks pregnant and in whom an increased white blood cell count (4.0×1010/l) was found upon physical examination. The patient had no history of hematological disease. Although cytogenetics showed a normal karyotype and no breakpoint cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR/ABL) fusion gene was detected by reverse transcription-polymerase chain reaction, a diagnosis of chronic myelogenous leukemia (CML) was initially made according to the clinical and morphological features. Another 6 weeks later, t(8;22)(p11;q11) rearrangement was present in 9 out of 10 analyzed metaphases. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction indicated a negative result for the BCR/ABL fusion, but gave a positive result for the BCR-fibroblast growth factor receptor 1 fusion. A hematological diagnosis of atypical CML was again formed.

Objective To investigate the effects of gene therapy with IL-10 on PTg-induced proliferation of splenocytes and Th1 cytokine production from PTg-stimulated splenocytes. Methods EAT rats were divided into four groups: group A (PBS+PLL), group B (pORF+PLL), group C (pORFmIL10+PLL), and group D (pORFmIL10+MEM). The substances mixed with lipofectamine were injected into the thyroid tissues of rats on the 18th dday after immunization. The rats were sacrificed at the 8th week. In vitro proliferative responses to ConA and different concentration of PTg were measured by culturing 4×105 splenocytes pulsed with 18.5KBq of [ 3 H] thymidine for the final 12h and then harvested for liquid scintillation counting. In vitro splenocytes were cultured with PTg (25 mg/L). Th1 cytokine IFN-γ, TNF-α and IL-2 were detected by ELISA. Results The proliferative response to PTg was suppressed in group C, compared with that of group A and B ( P P P P Conclusion IL-10 gene transmission in thyroid tissues could inhibit PTg specific proliferation of splenocytes from EAT rats and the secretion of Th1 cytokines from PTg-stimulated splenocytes.

Coexistence of chronic lymphocytic leukemia (CLL) and essential thrombocythemia (ET) in a patient is extremely rare, with only 10 cases reported thus far in literature. This paper describes a 94-year-old male having atypical B-CLL with CD5⁻ (CD5⁻) phenotype and ET. In this patient, we performed interphase fluorescence in situ hybridization (FISH) analysis which revealed 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei, but not in neutrophils and T-lymphocytes. Furthermore, we identified JAK2 V617F mutation in the peripheral blood nucleated cells and neutrophils, but not in the B- and T-lymphocyte populations. Therefore, it was concluded that the occurrence of CD5− B-CLL and ET in this patient was pathogenically independent.

This study was purposed to compare the significance of multiplex short tandem repeat polymerase chain reaction (STR-PCR) and fluorescent in situ hybridization (FISH) for monitoring chimerism after sex-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The chimerism of bone marrow or peripheral blood cells from 38 patients was analyzed by STR-PCR and FISH on days 14, 28 and at 3 months after allo-HSCT. The results indicated that on day 14, the complete chimerism (CC) was detected in 14 of 30 cases by STR-PCR and in 8 of 30 cases by FISH (p0.05). On day 28, the CC was detected in 26 of 31 cases by STR-PCR and in 15 of 31 cases by FISH (p0.01). At 3 months, the CC was observed in 22 of 24 cases by STR-PCR and 17 of 24 cases by FISH (p0.05). 14 cases were found to have a graft rejection or relapse among 28 cases which were continuously monitored more than 3 months post the transplants. Donor cell decrease in 9 of 14 cases was proved by FISH alone. It is concluded that FISH is more sensitive than STR-PCR in early monitoring chimerism status of post-transplant and in predicting graft rejection or disease relapse.

Hypothyroidism is often related with increased blood pressure, yet, gender-specific associations between subclinical hypothyroidism (SCH) and hypertension have not been previously assessed. We conducted a large-scale, cross-sectional study from six districts of Jiangsu Province to investigate the association of SCH and blood pressure in female and male adults. In the studied population, 4725 participants (3034 women and 1691 men) aged 20-60 years were included. The prevalence of hypertension was significantly higher in males as compared to females (37.34% vs. 27.39%, P 0.05), while the prevalence of SCH was much higher in women (9.36% vs. 5.32%, P 0.05). Furthermore, the hypertension rate was significantly higher in female SCH group compared to euthyroid (EUT) group(P 0.05), while no significant differences were observed between the two groups in male participants(P 0.05). After adjusting for age and body mass index (BMI), SCH was an independent predictor for increased SBP (OR = 1.47, 95%CI 1.08-1.99, P = 0.015) and elevated pulse pressure (OR = 1.45, 95%CI 1.05-1.99, P = 0.024) in females, and serum thyroid stimulating hormone (TSH) was significantly higher in female hypertensive group as compared to normotensive group (2.09 vs. 1.92 mIU/l, P = 0.0004). In male participants, SCH was not independently correlated with blood pressure, and no significant difference in TSH levels between hypertensive and normotensive groups was observed (1.74 vs. 1.66 mIU/l, P = 0.12). We concluded that SCH is an independent predictor of increased SBP and pulse pressure in females. Thus, thyroid function may influence blood pressure to a greater extent in females compared to males.

By inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle.The small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay.The transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively.The synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.

The relationship between subclinical thyroid dysfunction and blood pressure has been controversial and received unsufficient attention. Thus, we performed a cross-sectional study conducted among 6,992 inhabitants from six districts of Jiangsu Province to investigate the association of subclinical thyroid dysfunction with blood pressure in China. The data from 6,583 subjects (4,115 women and 2,468 men) were included and divided into three groups: euthyroidism (n = 5669, 86.11%), subclinical hyperthyroidism (n = 108, 1.65%), and subclinical hypothyroidism (n = 806, 12.24%). In the groups with subclinical hypothyroidism and hyperthyroidism, systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure were not significantly different from those in the groups with euthyroidism after being adjusted for age, sex, BMI, and smoking status (P0.05). More extensively, the SBP and DBP in the group of subclinical hypothyroidism with lower level of TSH (TSH 4.51-10.00 mIU/l, SCH(1)) were significantly higher than those of participants with euthyroidism (P0.05). Multivariable logistic analysis revealed that subclinical hypothyroidism with lower TSH (TSH 4.51-10.00 mIU/l) was an independent risk factor for increased SBP (OR = 1.28, 95% CI 1.03-1.59, P = 0.028). Similar results could not be found between groups of euthyroid and subclinical hypothyroid with higher level of TSH (TSH10 mIU/l, SCH(2)). Further subdivision of the euthyroid group on the basis of a TSH cut-off of 2.5 mIU/l, revealed still no significant difference in blood pressure after adjustment regardless of whether the TSH levels were in the lower reference (TSH 0.40-2.50 mIU/l, n = 4093) or in the upper reference ranges (TSH 2.51-4.50 mIU/l, n = 1576) (P0.05). We concluded that subclinical thyroid dysfunction was not associated with blood pressure. Neither subclinical hyperthyroidism nor subclinical hypothyroidism independently predicted increased blood pressure.

This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

To identify the abnormal karyotypes by fluorescence in situ hybridization (FISH) and explore prognostic implications in patients with myelodysplastic syndromes (MDS).FISH was used to detect the frequently occurring chromosome abnormalities (-5/5q, +8, -7/7q-) in 37 MDS cases. SPSS 11.5 software and correlation analysis were used to analyze the relativity among the abnormal chromosomes, the prognosis and the disease conversion in 37 MDS patients.Karyotype abnormalities were found in 21 (56.8%) of 37 cases, among which 6 (16.2%) were complex karyotypes, 9 (24.3%) +8, 2(5.4%) -5/5q-, 2(5.4%) -7/7q-. In the median time of follow-up of 12 months, 12 cases transformed into acute leukemia. Complex karyotypes were significantly associated with the poor prognosis and leukemia transformation. + 8 and -7/7q- abnormalities were correlated with the death.FISH was more sensitive than conventional cytogenetics for detecting mini-clonal abnormality. There are some differences in abnormal karyotypes between patients in China and the western countries. Multi-probes used in cytogenetic detections may predict the patient' s prognosis more accurately. The higher proportion of abnormal karyotypes the poorer prognosis.

This study was purposed to explore the mechanisms of drug resistance of HL-60/ADR cells and to compare the reversal drug-resistance effects of antisense oligonucleotides (AS ODN) of XIAP (X-linked inhibitor of apoptosis protein) and AS ODNs of MRP (multidrug resistance-associated protein) by use alone or in combination. Reverse transcription-PCR and Western blot were applied to detect the expression of XIAP, BCL-2, MRP and MDR1 in mRNA and protein levels of HL-60 cells and HL-60/ADR cells, respectively. Fully phosphorothioated AS ODN of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine 2000 in the form of liposome-ODN complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODN of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to daunorubicin (DNR). Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP, MRP in mRNA and protein levels respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than those in HL-60 cells. AS ODN of XIAP and MRP down-regulated the expression of XIAP and MRP in HL-60/ADR cells and increased the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODN of XIAP + MRP did not enhance the inhibition expression of XIAP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly as compared with AS ODN of XIAP (P0.05). AS ODN of XIAP + MRP did not increase the concentration of DNR nor enhanced the inhibition expression of MRP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P0.05), as compared with AS ODN of MRP. It is concluded that both XIAP and MRP may be involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells can be reversed significantly when antisense oligonucleotides of XIAP and MRP were used in combination.

This study was purposed to investigate the clinical significance of the amount and activated status of T cell subsets, B cells, NK cells in peripheral blood from patients with myelodysplastic syndrome (MDS). The proportion of T cells, B cells, NK cells in peripheral blood from 30 patients with MDS and their surface activation markers of CD28, CD45RA, CD45RO, CD69, HLA-DR were analyzed by flow cytometry. Twenty-two patients were in the low risk group (RA + RAS) while eight patients were in the high risk group (RAEB + RAEBT). The result showed that the amounts of T cells (CD3+ cells) in peripheral blood from patients with MDS were lower than those in control group. The amounts of naive CD4+ cells (CD4+ CD45RA+ cells) in MDS patients were lower than those in control. The expression rates of early activation marker (CD69) and late activation marker (HLA-DR) on CD3+ cells in MDS patients were significantly higher than those in control. The abnormalities of the immunologically competent cells were mainly observed in the low risk group (RA + RAS), and were characterized by the high expression rates of CD69+ and HLA-DR+ on CD3+ cells, the decrease of B cell amounts. The amount abnormalities of T cell subsets were mainly observed in high risk group (RAEB + RAEBT), and were characterized by the decrease of CD3+ cells and CD3+ CD4+ CD8- cells (Th cells) amounts without high expression of the CD69 and HLA-DR, the decrease of NK cells amounts. It is concluded that there are the abnormalities of T cell subsets and function in the patients with MDS and may change with disease progression, so the measurement of amount and activated status of T cell subsets in peripheral blood from MDS patients can have predictive role for diagnosis of disease progression and guide of therapy.

To analyse the relationship of T lymphocyte and granulocyte chimerism following allogeneic peripheral blood cell transplantation and the occurrence of relapse, graft failure and graft versus host disease.21 patients underwent allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Fluorescence-activated cell sorter (FACS) sorted CD3+ T lymphocytes and CD15+ granulocyte from peripheral blood of all the patients were analyzed for short tandem repeats in 7 days interval for 1 month starting from the day of PBSCT, then 1 month interval for 6 months, and then 3 months interval to the end of one year.Chimerism of granulocyte was higher than T lymphocyte on day 7 posttransplant in 4 patients given myeloablative conditioning. The median donor chimerism of granulocyte and T lymphocyte was 95% and 55% respectively. The other 17 patients had higher chimerism of T lymphocyte than granulocyte on day 7, which was 60% (15%-76%) and 0% (0%-40%) respectively. 20 patients reached complete donor chimerism (CDC) on day 21 (14-102 days) for T lymphocyte and on day 14 for granulocyte, except one relapsed on day 28. Seven patients had decreasing mixed chimerism when disease relapsed or graft failure occurred. T cell donor chimerism decreased earlier than myeloid cells, however, bone marrow sample and granulocyte still remained in complete donor chimerism or stable mixed chimerism, bone marrow smear showed normal at the same time.Blood leukocyte subset chimerism analysis, especially T cell chimerism analysis may provide earlier information of engraftment, relapse and graft failure than blood and bone marrow samples, therefore immunomodulatory therapies may be given to recipients earlier and overall survival may be improved.

To explore the role of X-linked inhibitor of apoptosis protein (XIAP) in the fibronectin (Fn)-adhesion mediated drug resistance of HL-60 cells.Culture plates were coated with Fn and bovine serum albumin (BSA) (as control), respectively. Colorimetric CCK-8 assay was used to determine the effects of Fn on the cytotoxicity of DNR to HL-60 cells. Intracellular DNR accumulation was assayed with flow cytometry. Reverse transcription-PCR and Western blot were used to examine the mRNA expression and XIAP, bcl-2, MRP and mdr1 proteins, respectively. HL-60 cells were added to Fn coated Culture plates. The fully phosphorothioate antisense oligonucleotide (AS-ODNs) and the control ODNs of XIAP were delivered into HL-60 cells in the form of liposome-ODN complexes. IC(50) was calculated by linear regression of survival percent versus drug concentration.HL-60 cells adhered to Fn-coated plates had a significant survival advantage over those grown on BSA coated plates and in suspension when exposed to DNR, the IC(50) of Fn group being significantly higher than that of BSA group and suspension group (0.526 micromol/L vs 0.132 micromol/L, 0.123 micromol/L, respectively, P0.05). XIAP was up-regulated significantly in Fn group compared with BSA group and suspension group (P0.05), whereas there was no difference in the expressions of bcl-2, MRP and mdr1 among the three groups (P0.05). The intracellular concentration of DNR in Fn-adhered HL-60 cells was similar to that in BSA group and suspension group (P0.05). AS-ODNs of XIAP down-regulated the XIAP expression in Fn-adhered HL-60 cells. In addition, AS-ODNs sensitized HL-60 cells to the cytotoxic effects of DNR.The increased XIAP protein level contributes to the drug resistance induced by adhesion to Fn. AS-ODNs of XIAP might reverse the drug resistance.

To explore the specific locus deletion of the long arm of chromosome 13 and its relationship with the clinical behavior and prognosis of multiple myeloma (MM).FISH analysis was performed on bone marrow smears from 68 patients with MM to study the deletion of Rb-1 gene and locus 13q14.3 on chromosome 13. The statistic value of its effect on clinical features were determined.35 out of the 68 (51%) cases were found with deletion of chromosome 13; deletion of Rb-1 gene were found in 29 (43%) cases; deletion of locus 13q14.3 were found in 23 out of 44 (52%) cases; the analysis results were same in 66% of the cases (29/44) with the above two probes. Chi-square test showed that partial deletion of chromosome 13 was associated with clinical behavior, early chemotherapy response and 1 year survival.Deletion of Rb-1 gene and locus 13q14.3 were both common cytogenetic changes in MM patients with effect on the biological behavior of the disease.

To evaluate the effect of modified culture method used to cytogenetic analysis and the clinically significance of chromosomal abnormalities to multiple myeloma (MM).Mononuclear cells were isolated from bone marrow aspirate of 20 MM patients; and then cultured for 3 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL) and GM-CSF (30 ng/mL) before RHG banding analysis; the remained part of aspirates were treated directly. Eight cases of iron deficiency anemia were taken as control.The experiment was failure in 2 cases because of blood clot, and another 2 cases could be analyzed only by direct method due to inadequate cells. The karyotype abnormalities were found from 4 cases of 16 available patients. Of them, three cases had complex karyotypes. The abnormalities were detected after 6 days culture with addition of cytokines. No abnormalities were detected from those groups of directly analysis and 3 day culture. Meantime, the clinical data showed that the patients with cytogenetic abnormalities were in stage III, and had a high percentage of MM cells (25%-56%) in their bone marrow, and also poor responses to prior chemotherapy. No cytogenetic abnormalities were found from control individuals in all groups.Extended culture in the presence of cytokines could improve the efficiency of cytogenetic analysis to MM. Complex karyotype was common cytogenetic abnormalities in MM patients with poor response to chemotherapy.

To investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression.The small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay.The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively.At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.