A Deep Mesencephalic Nucleus Circuit Regulates Licking Behavior / 神经科学通报·英文版

Licking behavior is important for water intake. The deep mesencephalic nucleus (DpMe) has been implicated in instinctive behaviors. However, whether the DpMe is involved in licking behavior and the precise neural circuit behind this behavior remains unknown. Here, we found that the activity of the DpMe decreased during water intake. Inhibition of vesicular glutamate transporter 2-positive (VGLUT2+) neurons in the DpMe resulted in increased water intake. Somatostatin-expressing (SST+), but not protein kinase C-δ-expressing (PKC-δ+), GABAergic neurons in the central amygdala (CeA) preferentially innervated DpMe VGLUT2+ neurons. The SST+ neurons in the CeA projecting to the DpMe were activated at the onset of licking behavior. Activation of these CeA SST+ GABAergic neurons, but not PKC-δ+ GABAergic neurons, projecting to the DpMe was sufficient to induce licking behavior and promote water intake. These findings redefine the roles of the DpMe and reveal a novel CeASST-DpMeVGLUT2 circuit that regulates licking behavior and promotes water intake.

Abnormal expression of PDCD5 in the bone marrow cells of adult acute myeloid leukemia / 中国实验血液学杂志

The objective of this study was to estimate a novel apoptosis-promoting molecule PDCD5 expression in the bone marrow cells from adult acute myeloid leukemia (AML) for investigation of its significance in the pathogenesis of AML. Flow cytometry assay was used for detection of PDCD5 expression in the different groups of cells from bone marrow of AML patients and normal controls by using 21 monoclonal antibodies with different fluorescent markers. The PDCD5 expressions in bone marrow cells from some AML patients and normal controls were also detected by Western blot. The results showed that the mean PDCD5 fluorescence intensity in bone marrow nucleated cells (MNC) from the bone marrow of 36 untreated AML patients was significantly lower than that from the bone marrow of 30 normal controls (3059 +/- 1392) vs (7432 +/- 1261) (P < 0.01). The mean PDCD5 fluorescence intensity was lower in the marrow granulocytes, monocytes, blast cells, and lymphocytes from untreated AML patients than that from normal (3939 +/- 2121) vs (8367 +/- 1045); (3156 +/- 1635) vs (5917 +/- 2329); (2824 +/- 1592) vs (3998 +/- 2106); (1474 +/- 816) vs (3355 +/- 2042) respectively, (all P < 0.01). Western blot analysis demonstrated that PDCD5 expression was significantly decreased in the AML cells, as compared with normal cells. It is concluded that PDCD5 expression in MNC in untreated AML patients is lower than that in the normal. PDCD5 expression in the marrow granulocytes, monocytes, blast cells, and lymphocytes of untreated AML patients is significantly lower than that in the normal. It suggests that the abnormally low expression of PDCD5 may be involved in the pathogenesis of AML.

Adenovirus-mediated PDCD5 gene transfer sensitizes apoptosis of K562 cells induced by etoposide / 中国实验血液学杂志

This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.

Analysis of immunophenotype and leukemia associated immunophenotype in 610 patients with acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the immunophenotype and leukemia associated immunophenotype (LAIP) of leukemia cells from patients with acute myeloid leukemia (AML) in minimal residual disease (MRD) detection.</p><p><b>METHODS</b>Four-color multiparametric flow cytometry (FCM) with CD45/SSC gating was used to determine the expression of the following antibodies of CD7, CD117, CD33, CD34, CD10, CD19, CD56, CD38, CD13, CD14, CD64, CD9, CD16, CD2, CD5, CD11b, CD123, HLA-DR in 610 AML patients and 20 normal bone marrow (NBM) samples.</p><p><b>RESULTS</b>The mean percentages of CD34+ and CD117+ side scatter low (SSC(low)) cells in NBM mononuclear cells (BMMNCs) were (0.35 +/- 0.15)% and (0.76 +/- 0.31)%, respectively. The majority (84% -95%) of CD34+ SSC(low) cells co-expressed CD13, CD33, CD38, CD117 and HLA-DR. 33% and 20% of CD34+ SSC(low) cells were CD15+ and CD9, respectively. Only a small proportion (< 10%) of CD34 SSC(low) cell co-expressed CD11b, CD56, CD19 and CD64, while co-expressed CD7 was 12%. In CD117 SSC(low) cells, the relative proportions of CD19+, CD11b+, CD56+ and CD7+ were less than 10%, while CD9+ was 14%. CD38+ and HLA-DR+ were 87% and 91%, respectively. The expressions of CD15, CD34, CD13 and CD33 on CD117 SSC(low) cells were between 30% and 50%. In AML patients, most cases were CD117+ (95.08%), CD38+ (94.74%), CD9+ (84.93%), CD33+ (84.60%), HLA-DR+ (77.23%) and CD13 (75.25%). The proportions of CD64+ and CD34+ were 64.41% and 59.51%, and that of CD15 and CD11b+ were 43.06% and 22.07%, respectively. 86.39% of AML patients were found to have at least one LAIP, the highest incidence being in AML-M1 and M3 subtypes and the lowest in AML-M4Eo subtype. The cross-lineage antigen and asynchronous antigen expression were the most frequent aberrant phenotypes. CD7, CD19 and CD56 expressing on CD34+ cells were major cross-lineage antigen. For asynchronous antigen expression, CD34+ CD64+, CD117+ CD11b+ , CD34+ CD38(-/dim) and CD34+ HLA-DR(-/dim) were seldom expressed on normal BMMNCs (about 0.01%), and the logarithm difference between AML and NBM was larger than 3.0, being the more sensitive LAIP.</p><p><b>CONCLUSION</b>MRD detection by multiparameter flow cytometry can be applied to more than 80% of AML patients.</p>

Application of CD123 in detection of minimal residual disease in acute promyelocytic leukemia / 中国实验血液学杂志

The study was aimed to investigate the role and significance of CD123 with other immunological markers in detecting minimal residual disease (MRD) of APL patients. The immunophenotypes of 186 newly diagnosed APL patients and the percentages of cells identical with APL cell immunophenotypes in 20 normal bone marrow samples were analyzed using four-color flow cytometry. MRD in 172 specimens were monitored by mainly using CD34/CD117/CD123/HLA-DR four-color antibody panels, meanwhile 18 specimens were analyzed with the second antibody combination: CD9/CD117/CD34/CD33, simultaneously and the results were compared with real-time PCR. One hundred and sixteen of 172 bone marrow (BM) or peripheral blood (PB) specimens were from follow-up 19 newly diagnosed APL patients and the rest 56 samples were from 47 patients treated 3 to 24 months later. Among them, 117 samples and 55 samples were collected after achieving morphologic complete remission (mCR) and before achieving mCR respectively. The results of immunophenotyping demonstrated that except CD9, CD33 and CD117 were high-expressed and CD34 and HLA-DR were rarely expressed, the CD123 was expressed in 30/30 (100%) APL patients. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) and CD117(+)CD34(-)CD9(+)CD33(+) cells in nucleated cells were 0.066% +/- 0.012% and 0.089% +/- 0.066% in 20 normal bone marrow samples. The median time of achieving morphology complete remission in 19 APL patients was 4 weeks (3 - 6 weeks). The median time of FCM and PCR results turned to be negative in 13 APL patients was 7.5 weeks (5 - 11) and the median time of PCR results turned to be negative in 11 APL patients was 8 weeks (5 - 12). 41/117 (35.04%) samples were MRD positive by FCM after achieving mCR. The ratio of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells was < 5% in 33 specimens, but > 5% in another 8 specimens, their median percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells were 0.48% (range 0.02% - 4.70%) and 9.02% (range 5.26% - 18.14%) respectively. The median relative percentages of CD123(+)HLA-DR(-) cells in CD117(+)CD34(-) population were 63.59% (range 15.11% - 98.36%) and 86.77% (range 63.29% - 92.62%) respectively. In FCM MRD positive samples, 95.9% (93/97) were PCR positive, the false positive rate of FCM and the false negative rate of PCR were 4.1% (4/97) and 8.75% (7/93) respectively. In FCM negative samples, 92% (69/75) were PCR negative and 8% (6/75) were PCR positive. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells in 116 consecutive specimens and 117 specimens of mCR were related to PML/RARalpha quantified by real-time PCR (r = 0.824, P < 0.001 and r = 0.754, P < 0.001 respectively). It is concluded that the detection of APL patients by means of two sets of antibody panels is simple and suitable, which is complementary to PCR in monitoring MRD of APL patients.

Leukemia-associated immunophenotypes in 415 childhood and adult patients with B lineage acute lymphoblastic leukemia by multiparametric flow cytometry analysis / 中国实验血液学杂志

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.

Clinical significance for minimal residual disease detection by 4 color flow cytometry in adult and childhood B lineage acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL).</p><p><b>METHODS</b>MRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available.</p><p><b>RESULTS</b>Of 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000).</p><p><b>CONCLUSION</b>Relapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.</p>

Comparison of the immunophenotype of patients with B lineage acute lymphoblastic leukemia at diagnosis and relapse / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the leukemia-associated immunophenotypes (LAIP) in patients with B lineage acute lymphoblastic leukemia (B-ALL) at diagnosis and relapse, and investigate its implications for minimal residual disease (MRD) detection.</p><p><b>METHODS</b>The immunophenotype of leukemia cells from 410 newly diagnosed and 6 relapsed patients with B-ALL were detected by four to six antibody combination, mainly CD34/CD10/CD45/CD19 of 4-color CD45/SSC gating flow cytometry (FCM).</p><p><b>RESULTS</b>The proportion of CD45 under-expressed or negative in relapsed patients was much higher than that in newly diagnosed patients, being 69.2% and 37.8% respectively. Immunophenotypic changes occurred in 9 relapsed patients (including 8 hematological relapse and 1 central nerves system relapse) when analyzed by paired samples analysis at diagnosis vs at relapse: 4 cases showed CD45 down-modulation and 2 up-modulation; 4 CD34 down-modulation and 2 CD10 up-modulation, while the expression of CD19 remained no change. MRD was observed in all 7 cases of hematological relapse 2 - 4 months before relapse, and the immunophenotype of MRD cells was the same as that in relapse.</p><p><b>CONCLUSION</b>A high frequency of immunophenotypic changes occurred at relapse and even in MRD before relapse, however the accuracy of MRD monitoring seemed not affected by the FCM strategy used in this investigation.</p>

Monitoring bcr/abl mRNA levels in imatinib mesylate treated chronic myeloid leukemia patients by real-time quantitative RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To quantify bone marrow bcr/abl mRNA levels in imatinib mesylate treated Ph chromosome positive chronic myeloid leukemia (CML) patients.</p><p><b>METHODS</b>Serial monitoring of bcr/abl mRNA levels by real-time quantitative RT-PCR technique (RQ-PCR) was performed in 34 cases (120 samples) of CML treated with imatinib mesylate. All the patients were IFNalpha based treatment failure before enrolled in this study and the percentage of Ph(+) bone marrow cells were over 95%.</p><p><b>RESULTS</b>The sensitivity of RQ-PCR was 10 pg RNA, with both coefficients of interassay and intraassay variation below 5% for standard samples. The median bcr/abl mRNA level of 10 patients' samples pre imatinib treatment was 5.79% with marked variation (0.24%-60.90%). In 72 samples post imatinib treatment, which the rates of Ph(+) cells [Ph(+)%] were between 0 and 94%, the mRNA level well correlated with Ph(+)% (r = 0.82, P < 0.001). The mRNA levels of 7 patients who achieved complete cytogenetic response (CCyR) within 12 months decreased markedly, the levels of 6 analysable patients decreased by 65.9% - 98.8% after 3 months'treatment accordingly. The level further decreased to 0 after achieving CCyR. For 4 patients who achieved major cytogenetic response (Ph(+) cells < 35%) later than 12 months, the mRNA levels decreased slowly. The levels of 3 analysable patients on 3 month therapy decreased by 2.5%, 18.5% and 61.6% compared with that before treatment. Out of 5 patients in chronic phase without cytogenetic response, 1 decreased, 2 increased gradually and 2 had no change. In 4 disease progression patients, the levels increased stepwise.</p><p><b>CONCLUSIONS</b>Serial quantifications of bcr/abl mRNA levels are necessary for imatinib treated patients, and are more informative than a single detection. A sharp decline of bcr/abl mRNA levels after the treatment implies a promise of CCyR.</p>

Detection of minimal residual disease in B lineage acute lymphoblastic leukemia by 4-color flow cytometry / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the method and value of detecting bone marrow minimal residual disease (MRD) in B lineage acute lymphoblastic leukemia (B-ALL) by multiparameter flow cytometry (FCM).</p><p><b>METHODS</b>The FCM immunophenotyping of B-lymphocyte precursors in regenerating stage and MRD was analyzed by using two sets of 4-color antibody panels, including mainly CD34, CD10, CD45, CD19, in 26 regenerating bone marrow samples after chemotherapy and 297 consecutive bone marrow specimens from 50 patients with B-ALL, respectively. The immunophenotype of leukemia cells of B-ALL was also detected by four to six antibodies combination of 4-color CD45/SSC gating FCM. The CD19, CD10, CD34 fluorescence intensity of B-cell precursors in normal and leukemic bone marrow was compared by relatively quantitative method.</p><p><b>RESULTS</b>Twelve patients were MRD positive (MRD(+)) among 50 patients during MRD monitoring, the percentages of residual leukemia cells were 0.06% to 7.73%. Eleven cases displayed CD45, CD34, CD19, CD10 antigens aberrant expression. Five patients were bone marrow relapsed and 4 of them were MRD (+) 7-17 weeks before relapse. The percentages of leukemia cells in all the 4 patients were over 0.1%. Only one patient relapsed with MRD negative. The regenerated B precursors could be divided into 3 stages according to the expression of CD45, CD34, CD19, CD10, CD22, and CD20 antigens. Abnormal expression of CD45, CD34, CD19, and CD10 were detected in 71.8% of 213 patients with B-ALL, the percentage of only aberrant expression of CD38 and myeloid antigen was 8.1%.</p><p><b>CONCLUSION</b>Detection of MRD by multiparameter flow cytometry is a rapid, quantitative and sensitive approach, and has a higher predictability for relapse.</p>

Significance of quantification of WT1 mRNA for monitoring minimal residual disease in acute myeloid leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the significance of quantification of WT1 mRNA for monitoring minimal residual disease (MRD) in patients with acute myeloid leukemia (AML).</p><p><b>METHODS</b>WT1 mRNA level was detected with real-time quantitative RT-PCR (RQ-PCR) technique in bone marrow samples from 15 normal subjects (NBM) and 123 AML patients. Sixty-two AML samples were also detected AML1-ETO mRNA expression by RQ-PCR. Simultaneously follow-up of WT1 and AML1-ETO levels were carried out in 50 samples from 8 AML patients. WT1 and AML1-ETO levels were normalized by internal control ABL gene.</p><p><b>RESULTS</b>All correlation co-efficiencies were over 0.99 for WT1, AML1-ETO and ABL standard curves. Co-efficiencies of both interassay and intraassay variation were below 4%. The WT1 expression levels in NBM were 0.001 to 0.019 with a median level of 0.008. Higher levels of WT1 expression were found in 61 of 67 (91%) newly diagnosed AML patients compared with NBMs and 37 of the 67 (55.2%) showed 100-fold higher WT1 levels than that in NBMs. WT1 mRNA levels were highest in M(4EO) and M(3) and lowest in M(1) and M(5) patients. There was an excellent correlation between WT1 and AML1-ETO gene expression levels (r = 0.88, P < 0.001). WT1 expression levels in three patients who were in continuous complete hematological remission (CHR) were within normal range. In three of four relapsing patients, WT1 expression levels increased 31.4, 11.4 and 4.0 fold respectively one month before hematological relapse.</p><p><b>CONCLUSIONS</b>Quantification of WT1 expression level by RQ-PCR may be used to monitor MRD for most AML patients, but it is less sensitive than fusion gene. Continuous or significant increase of WT1 expression in CHR patients predicts an impending relapse.</p>

Two Ph chromosome positive chronic myelogenous leukemia patients with rare bcr/abl fusion gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the unusual bcr/abl fusion gene structures of two Ph chromosome positive chronic myelogenous leukemia (CML) patients in chronic phase (CP).</p><p><b>METHODS</b>By using general M- and micro -bcr/abl specific primers respectively, bcr/abl fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR products sequencing was performed, the DNA sequences were analyzed in Genebank and the bcr and abl sequences at the fusion site were identified. DNA was amplified by PCR using a set of primers designed according to the sequencing result of RT-PCR products.</p><p><b>RESULTS</b>Two patients showed typical manifestations of CML-CP. Their RT-PCR products were different from usual M- or micro -type; one was longer than M-bcr/abl but shorter than micro -bcr/abl, the other one was shorter than M-bcr/abl. The RT-PCR products sequencing showed that both products contained bcr and abl gene sequences. The first patient's bcr gene was broken within exon 18, and fused to abl gene exon 2(a2), and a 40 bp of partial abl intron 1b fragment was inserted between them, resulting in a novel in-frame bcr/abl fusion transcript-e18-int-a2 which has not been reported in the literature so far. In the second patient, deletion of abl exon2(a2) led to exon 13(b2) of bcr gene fusing with abl exon 3(a3).</p><p><b>CONCLUSION</b>Uncommon bcr/abl fusion gene may occur in typical Ph(+) CML patient.</p>

The significance of intracellular IL-1ra expression in the bone marrow cells from adult chronic myelogenous leukemia / 中国实验血液学杂志

To estimate intracellular interleukin-1 receptor antagonist (icIL-1ra) expression in the bone marrow cells from adult patients with chronic myelogenous leukemia (CML), flow cytometry (FCM) assay was used for detecting the mean icIL-1ra fluorescence intensity per cell (equivalent to it's expression level) in different groups of cells from normal and CML patient bone marrows by 15 monoclonal antibodies with different fluorescent marker. The results showed that all of marrow nucleated cells express IL-1ra, but its expression level in granulocytes was the highest and that in lymphocytes was the lowest. The icIL-1ra expression level was significantly lower in nucleated marrow cells, granulocytes and lymphocytes from the marrow of 17 untreated CML patients than that from the marrow of 8 normal. The mean icIL-1ra fluorescence intensity was significantly lower in marrow nucleated cells, granulocytes and lymphocytes in 13 CML patients with marrow blasts >or= 10% than that in 43 CML patients with marrow blasts < 5% or than that in 9 CML patients with marrow blasts 5% - 10%. It was concluded that the lower expression of icIL-1ra in CML marrow nucleated cells might be involved in the evolution of CML.

Analysis of immunophenotype of B-lineage acute lymphoblastic leukemia cells by 4-color flow cytometry / 中国实验血液学杂志

To investigate the characteristics of immunophenotype of B-lineage acute lymphoblastic leukemia (B-ALL) cells in Chinese cases, B-ALL cells from 181 patients were analyzed by 4-color flow cytometry with CD45/SSC gating. The results demonstrated that the antigen expression frequencies were as follows: CD19 (100%), HLA-DR (98.9%), CD38 (88.5%), CD10 (76.8%) and CD34 (76.8%). CD117 and T antigens (CD2 and CD7) were rarely expressed. Childhood group (<or= 14 years) had the highest frequency of CD10. Adolescent (15 - 18 years) and adult groups (>or= 19 years) had a higher expression rate of myeloid antigens (CD13 and/or CD33). Subtype CD10(+)/CD34(+) took the highest percentage in three age groups. Percentages of CD10(-)/CD34(+) group increased by age. Subtype CD10(-)/CD34(+) had the highest myeloid antigen co-expression. Comparing with CD45-positive cases, those with CD45-negative or CD45(+/-) always had higher CD10 expression. Bcr/abl mRNA was evaluated in 43 samples from patients with B-ALL. Myeloid antigen co-expression was not different in bcr/abl(+) and bcr/abl(-) groups, and m-bcr/abl(+) was mainly observed in subtype CD10(+)/CD34(+). In conclusion, typical B-ALL cells expressed CD19(+), HLA-DR(+), and CD117(-). CD34, CD10 and CD45 expression varied in different maturation stages. The immunophenotypic subtype of B-ALL in adolescents was similar to that in the adults. Blasts with CD45(+) were mostly seen in CD10(-) cases. CD13 and/or CD33 co-expression was fewer in children patients. m-bcr/abl(+) was mainly seen in subtype CD10(+)/CD34(+).

Immunophenotypic features of bcr/abl fusion transcript-positive B-lineage acute lymphoblast leukemia / 中国实验血液学杂志

To investigate the biological features of leukemic cells in bcr/abl fusion transcript-positive B-lineage acute lymphoblastic leukemia (B-ALL), 3- or 4-color flow cytometry with directly conjugated monoclonal antibodies was used to detect the immunophenotype of the cells in 26 patients with bcr/able-positive B-ALL and 32 patients with bcr/abl-negative B-ALL. bcr/abl fusion transcript was detected by RT-PCR. Immunoglobulin heavy chain (IgH) gene rearrangement was detected by PCR. The results showed that all of the B-ALL patients were positive for CD19. There was significant difference in expression of CD34 (96.2% vs 65.6%), CD10 (96.2% vs 71.8%) and CD38 (43.8% vs 95.4%) between bcr/abl-positive and -negative groups. In bcr/abl-positive B-ALL group, the co-expression rates of CD10(+)/CD19(+)/CD34(+), CD10(+)/CD34(+)/HLA-DR(+) and CD10(+)/CD34(+)/CD38(-) were 92.3% (24/26), 73.1% (19/26) and 56.2% (9/16), respectively. In bcr/abl-negative group, co-expression of CD10(+)/CD19(+)/CD34(+) and CD10(+)/CD34(+)/HLA-DR(+) were 43.8% (14/32) and 37.5% (12/32), respectively, there were significant differences (P < 0.05) between bcr/abl-positive and -negative groups, but none of the cases co-expressed CD10(+)/CD34(+)/CD38(-). The detection rate of monoclonal IgH gene rearrangement (58.8%, 10/17) was lower in bcr/abl-positive group than that (85.7%, 12/14) in bcr/able-negative group. It is concluded that the expression rates of CD34 and CD10 are higher, and CD38 and IgH gene rearrangement are lower in bcr/abl-positive B-ALL cases, CD10(+)/CD34(+)/CD38(-) is a unique feature of immunophenotype, and this phenotype of leukemia cells is closer to that of early B-lineage progenitor cells.

Restoring beta1 integrin activation function in K562 cells transfected with antisense VEGF121 cDNA / 中国实验血液学杂志

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.

Follow-up detection of M-bcr/abl and m-bcr/abl fusion transcripts in chronic myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation / 中国实验血液学杂志

In order to investigate the features of M-bcr/abl and m-bcr/abl fusion transcripts in patients with chronic myeloid leukemia (CML) after allogeneic stem cell transplantation (SCT), M-bcr/abl and m-bcr/abl fusion transcripts were sequentially detected by RT-PCR technique in 72 CML patients after SCT. The results showed that M-bcr/abl positive rate (79.2%, 42/53) within 6 months after SCT was remarkably higher than that in 6-12 months group (34.3%, 11/32) and >or= 12 months group (35.1%, 13/37) (P < 0.001), and the clinical relapse rates in corresponding periods were 1.9% (1/53), 0% (0/32) and 16.2% (6/37) respectively. M-bcr/abl and m-bcr/abl fusion transcripts occurred in 5 of 6 clinically relapsed patients. In period of more than 6 months after transplantation, none of 17 M-bcr/abl(+) samples from 14 patients in cytogenetic remission appeared positive reaction of m-bcr/abl. It is concluded that M-bcr/abl(+) fusion transcript still existed in most patients after SCT, and usually disappeared within 6 months. Existence of M-bcr/abl is not a clinical relapse marker in CML patients. Simultaneous detection of M-bcr/abl and m-bcr/abl fusion transcripts can be helpful for monitoring residual disease.

Study on relationship between different bcr/abl fusion transcripts and clinical manifestations in chronic myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between three types of bcr/abl fusion transcripts and clinical manifestation in chronic myeloid leukemia (CML).</p><p><b>METHOD</b>M-, m- and micro -bcr/abl fusion transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 537 fresh bone marrow samples of patients suspected CML clinically.</p><p><b>RESULTS</b>Of 573 patients, 479 expressed M-bcr/abl transcripts, among whom 370 were in chronic phase (CP), and 109 in accelerated (AP)/blastic phase (BP). The percentages of patients with b2a2 transcripts in CP and AP/BP were 32.4% (120/370) and 36.7% (40/109) (P > 0.05). The b2a2 transcript patients in blastic crisis were 52.6% (10/19) for lymphoblastic and 33.3% (30/90) for myeloblastic (P > 0.05). The platelet count of untreated patients with b3a2 isoform [(485.9 +/- 333.8) x 10(9)/L, n = 125] was distinctly higher than those with b2a2 isoform [(380.5 +/- 321.9) x 10(9)/L, n = 62] (P < 0.05). 66.0% (31/47) and 64.4% (29/45) of the patients in CP and AP/BP respectively co-expressed M- and m-bcr/abl transcripts (P > 0.05). One patient expressed only m-bcr/abl transcript was of typical acute myeloblastic leukemia (AML). Both two micro -bcr/abl(+) patients were of typical CML.</p><p><b>CONCLUSIONS</b>Almost all typical CML patients express M-bcr/abl transcripts, most of them coexpress M-bcr/abl and m-bcr/abl transcripts, a few possesses only micro -bcr/abl fusion gene. m-bcr/abl(+) are usually associated with AML or CML in myeloblastic crisis besides acute lymphoblastic leukemia (ALL). Patients with b3a2 isoform are prone to higher platelet count before treatment.</p>

Immunophenotypic characteristics of multiple myeloma cells / 中国实验血液学杂志

To investigate the immunophenotypic characteristics of multiple myeloma (MM) cells, 20 bone marrow samples from patients with multiple myeloma were analyzed by flow cytometry with three-color direct immunofluorescence staining. Results showed that all of myeloma cells expressed bright CD38, dim or negative CD45 and negative CD19. Most of the cells were CD56(+) and a small portions were ckappa(+) or clambda(+), or CD20(+). The phenotypes of normal plasmocyte, CD19(+) and CD56(-), except CD56(-) in one-third samples, were not appeared in all detected samples. It was concluded that the surface marker analysis of myeloma cells is a useful tool for diagnosis and further evaluating prognosis of multiple myeloma.

Follow up Detection of AML/ETO Fushion Transcripts after Chemotherapy or Bone Marrow Transplantation in Leukemia Patients / 中国实验血液学杂志

Expression of AML1/ETO mRNA was observed in bone marrow cells from 49 untreated leukemic patients, and continuously detected during different periods after chemotherapy (12 cases) or bone marrow transplantation (8 cases). The results showed that AML1/ETO mRNA could be expressed in cells from AML-M(2), AML-M(4) and MDS-RAEB-T patients. The positive expression changed into negative at different duration in patients who achieved complete remission either by chemotherapy (9 cases), allogeneic bone marrow transplantation (5 cases) and autologous peripheral blood stem cell transplantation (1 case), and they were sustained in complete remission status. In chemotherapeutic group, patients whose AML1/ETO expression turning from negative (2 cases) or faint positive (1 case) to positive relapsed later. Two patients treated with Allo-BMT showed continuously positive results and died of GVHD and relapse, respectively. These observations suggest that AML1/ETO chimeric mRNA could disappeared after chemotherapy or bone marrow transplantation. The patients have a great probability to relapse if the results of RT-PCR are continuously positive or change from negative to positive. Regular detection is necessary for leukemic patients.