[Monitoring of plasma concentration of imatinib mesylate in patients with chronic myeloid leukemia].

OBJECTIVE: To analyze the clinical efficacy of imatinib mesylate (IM) for Ph-positive or BCR-ABL positive chronic myeloid leukemia (CML) to couple the trough plasma concentrations (C mins) of IM with clinical responses and adverse events (AEs). METHODS: One hundred and one CML patients received IM therapy, and Cmins of IM were determined in 30 patients. RESULTS: (1) Cumulative complete hematological response (CHR), major cytogenetic response (MCyR), complete cytogenetic response (CCyR) and negative BCR/ABL fusion gene rates were 96.6%, 86.5%, 77.5% and 47.2%, respectively, in CML-CP patients. In accelerated and blastic phases (AP and BC) patients, CHR, MCyR, CCyR and negative BCR-ABL fusion gene rates were 58.3%, 25.0%, 25.0%, 8.3%, respectively. (2) Mean Cmins of IM was significantly higher in the CCyR at 1 year [(1472 +/- 482) microg/L] group than in the non-CCyR at 1 years group [(1067 +/- 373) microg/L] (P < 0.05), and higher in the MMR at 1 year group than in the non-MMR at 1 years group [(1624 +/- 468) microg/L vs (1137 +/- 404) microg/L, P < 0.05]. CONCLUSION: IM significantly improves cytogenetic and molecular response, event-free survival, and overall survival for patients with Ph-positive CML. The Cmins of IM exerts a significant impact on clinical response (CCyR and MMR at 1 year).

[In vitro activation of bone marrow natural killer T cells of aplastic anemia patients].

OBJECTIVE: To investigate the quantitative and qualitative changes of TCRVα24(+)Vß11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer). METHODS: NKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry. RESULTS: In AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\]. CONCLUSION: Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.

Effects of garlic oil on n-hexane metabolized to 2, 5-hexanedione in mice serum / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of garlic oil (GO) on n-hexane metabolized to 2, 5-hexanedione (2, 5-HD) in mice.</p><p><b>METHODS</b>Adult healthy Kunming-mice were treated with n-hexane and GO. The serum was obtained and extracted with ethyl acetate, and the levels of the serum 2, 5-HD were determined by gas chromatography.</p><p><b>RESULTS</b>(1) The concentration of 2, 5-HD in serum increased firstly after a single exposure to n-hexane (4 000 mg/kg). The peak value occurred at 10 hours after n-hexane treatment, but could hardly be detected at 20 h. (2) There was no 2, 5-HD in serum of control mice. The content of 2, 5-HD in serum increased along with the exposure dose of n-hexane. The serum 2, 5-HD contents of the 2000, 4000 and 6000 mg/kg groups mice were 8.04, 16.68 and 22.38 microg/ml at 8 h in pretreated mice, respectively, and showed significant dose-effect relationship. (3) When the different age mice were exposed to the same dose of n-hexane, the contents of 2, 5-HD in serum were significantly different after 8 hours (P<0.05). The serum 2, 5-HD level of the 5 weeks old mice (22.83 microg/ml) was much higher than the 4 (19.59 microg/ml) and 6 (16.42 microg/ml) weeks old mice. (4) When the different gender mice were exposed to the same dose of n-hexane, the concentration of 2, 5-HD in serum of female mice (13.22 microg/ml) was higher than that of the female mice (10.34 microg/ml, P<0.05). (5) GO significantly inhibited the increase of the serum 2, 5-HD levels of both the pretreatment and post-treatment groups treated with 80 mg/kg n-hexane respectively, but the pretreatment with GO exhibited the more suppressive effects than the post-treatment (P>0.05). Compared with the n-hexane group, the concentrations of serum 2, 5-HD in GO-pretreated groups mice decreased by 16.2%, 20.8%, 22.8% (P<0.05) and 32.1% (P<0.01), respectively, and showed significant dose-effect relationship.</p><p><b>CONCLUSION</b>The serum content of 2, 5-HD, the metabolite of n-hexane, is different in different genders and age mice after exposed to the same dose of n-hexane. GO can effectively inhibit the production of n-hexane metabolized to 2, 5-HD in mice serum.</p>

Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

Effects of garlic oil on 2, 5-HD induced lipid peroxidation damage in rat nerve tissues / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of garlic oil (GO) against the peroxidation damage of rat nerve tissue and the peripheral motor neuropathy induced by 2, 5-HD.</p><p><b>METHODS</b>Male Wistar rats were divided into four groups, with 10 in each group. The model group, and low and high doses of GO groups were administrated with 2, 5-HD (ip, 300 mg/kg), respectively; The control group was treated with sodium chloride, five times per week for six weeks. Pretreatment with GO gavaged (40 mg/kg or 80 mg/kg) started one week be-fore 2, 5-HD treatment, and lasted to the end of the experiment. Neurobehavioral indexes were examined at the zero, second and fourth week. At the end of the experiment, the scores of the gait, and the concentration of MDA and GSH, the level of TAOC and the ability of inhibition of.OH in cerebrum, spinal cord and sciatic nerve were examined.</p><p><b>RESULTS</b>Compared with the zero week, except of the control group rats, the hind limb landing foot splay of three groups rats decreased by 44%, 50% and 49% at the fourth week, respectively without significant difference. The threshold value of balance in model, GO low and high doses groups rats decreased by 30%, 45% and 68% at the fourth week, respectively, and lower than the control group rats (P < 0.01). GO low and high doses groups rats showed the serious abnormality at the fourth week, before one week of the model group rats. The scores of gait of model, and GO low and high doses groups rats increased significantly compared with control group rats, and the GO high dose group rats were higher than model group rats (P < 0.05). Increase of the concentration of MDA, and decrease of the level of the ability of inhibition of.OH were induced by 2, 5-HD in cerebrum, spinal cord and sciatic nerve. The concentration of MDA increased, and the level of the ability of inhibition of.OH decreased (P < 0.05 or P < 0.01), respectively. The results showed that the concentration of MDA decreased, and the level of the ability of inhibition of.OH induced by GO in cerebrum, spinal cord and sciatic nerve increased, the concentration of MDA of GO low doses group rats decreased, the level of the ability of inhibition of.OH increased, the concentration of MDA of GO high doses group rats decreased (P < 0.01) respectively, and the level of the ability of inhibition of.OH increased (P < 0.01) in nerve tissue.</p><p><b>CONCLUSION</b>GO has antagonist effect on the 2, 5-HD induced peroxidation damage, but can not improve the function of the peripheral motor nerve, indicating that the lipid peroxidation does not play an important role in 2, 5-HD neurotoxicity.</p>

[Clinical implications of combined measurement bone marrow T cells intracellular IFNgamma and HLA-DRB1*1501 measurement in predicting the response to immunosuppressive therapy for aplastic anemia].

OBJECTIVE: To evaluate the clinical implication of combined measurement of bone marrow (BM) T lymphocyte intracellular IFNgamma with HLA-DRB1*1501 in predicting the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA). METHODS: Enrolled into the present study were 51 idiopathic AA patients treated with cyclosporine A (CsA) based IST. BM CD(8)(+) T lymphocyte intracellular IFNgamma was determined with flow cytometry and HLA-DRB1*1501 detected with PCR-sequence specific primer before treatment. The relationship between laboratory indices and clinical response were investigated and the potential usefulness of parameters in predicting the response to IST for AA was evaluated. RESULTS: These HLA-DRB1*1501 shows sensitivity of 45.7% (16/35) and specificity of 87.5% (14/16) respectively. Intracellular IFNgamma has sensitivity of 94.3% (33/35) and specificity of 62.5% (10/16), respectively. With combination of intracellular IFNgamma with HLA-DRB1*1501, the parallel test increases the sensitivity of 97.1% (34/35) and the negative predictive value of 90.0% (9/10). On the other hand, the serial test improves the specificity and positive predictive value which both achieve 93.7% (15/16). It could be calculated through a logistic regression equation that the probabilities of prediction of four subgroups of patients whose results are both positive reaction, a positive intracellular IFNgamma plus negative HLA-DRB1*1501, a negative intracellular IFNgamma plus positive HLA-DRB1*1501 and both negative reaction are 89.0%, 77.4%, 34.5% and 18.2%, respectively. CONCLUSIONS: Combination of BM T cells intracellular IFNgamma stain and HLA-DRB1*1501 phenotype can be a useful predictor for AA patients in immunosuppressive therapy. The patients with both positive results of the two tests may have more possibilities to response to IST. It may have an important implication for the majority of AA patients whose intracellular IFNgamma stain has a positive reaction.

[Preliminary study of multivariable model in predicting response to immunosuppressive therapy in patients with aplastic anemia].

OBJECTIVE: To evaluate the potential usefulness of a multivariable model in predicting the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA), and its application to the clinical practice. METHODS: PB T cells subpopulation and BM T cells intracellular IFN-gamma and IL-4 were serially analyzed by flow cytometry (FCM) before and during treatment. HLA-DRB1 * 1501 phenotype was analyzed by PCR-SSP. The predictive potentials of different parameter combinations for clinical responsiveness were statistically assessed. RESULTS: In all evaluated parameters, CD8+ cell intracellular IFN-gamma had the relatively best diagnostic value with sensitivity and specificity of 94.3% and 62.5%, and positive and negative predictive value of 84.6% and 83.3% respectively. Positive CD8+ cell intracellular IFN-gamma plus Tc1/Tc2 < 50 could increase the positive predictive value to 92.3%. A multivariable model consisting of absolute neutrophil count (ANC), BM T cell intracellular IFN-gamma, Tc1/Tc2 ratio and HLA-DRB * 1501 phenotype of the patients was finally established. CONCLUSION: The multivariable model is superior to each of the single parameters in terms of predictive power of IST therapeutic outcome, and its higher accuracy and the clinical application make it potentially useful in practice.

[Influence of tumor necrosis factor-alpha and interferon-gamma on erythropoietin production and erythropoiesis in cancer patients with anemia].

OBJECTIVE: To explore impaired erythropoiesis and relative inadequacy of erythropoietin production in the anemic cancer patients and the correlation of tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) with inadequate erythropoietin (EPO) response and impaired erythropoiesis in cancer patients with anemia. METHODS: Fifty adult anemic and 15 non-anemic tumor patients were studied. Serum EPO levels were measured by radioimmunoassay (RIA) and serum soluble transferrin receptor (sTfR). TNF-alpha and IFN-gamma levels by enzyme-linked immunosorbent assay (ELISA). Log transformed EPO and sTfR values were used in statistical analysis. The R correlation analyses were performed. RESULTS: The mean serum immunoreactive erythropoietin level in anemic cancer patients [(23.11 +/- 10.00) IU/L] was not significantly higher than in healthy people (P = 0.053), but significantly lower than in IDA patients with similar degree of anemia [(43.00 +/- 22.00) IU/L, P < 0.01]. Both O/P EPO [0.88 (0.54-1.10)] and O/P sTfR [0.89 (0.57-1.22)] were significantly lower in anemic cancer patients than in controls and in non-anemic cancer patients. There was no significant difference between the latter two groups. Furthermore, the expected inverse linear relation between serum EPO and hemoglobin levels was absent in the anemic cancer patients, and so did the relation between serum sTfR and hemoglobin levels. There was no correlation between O/P EPO and O/P sTfR. The serum levels of both TNF-alpha and IFN-gamma in anemic cancer patients [(25.75 +/- 26.71) ng/L, (50.49 +/- 42.12) ng/L, respectively] were significantly higher than those in healthy controls (both P < 0.01) or in nonanemic cancer patients (both 0.01 < P < 0.05), and so did between non-anemic cancer patients and controls. The serum levels of TNF-alpha were inversely correlated with hemoglobin levels (r = - 0.40, P = 0.004), O/P EPO (r = -0.32, P = 0.025) or O/P sTfR (r = -0.36, P = 0.01); while serum levels of IFN-gamma were inversely correlated with hemoglobin levels (r = -0.36, P = 0.01) or O/P sTfR (r = 0.39, P = 0.006), but not with O/P EPO. Conclusions Anemia of cancer is due to impaired erythropoiesis and relative inadequacy of EPO production. TNF-alpha might inhibit EPO production and erythropoiesis, while IFN-gamma maybe directly inhibit erythropoiesis and be independent of EPO response inadequacy.

[Effectiveness and safety of rhIL-11 in the treatment of chemotherapy-induced thrombocytopenia].

OBJECTIVE: To investigate the effectiveness, safety and possible mechanism of recombinate human interleukin 11 (rhIL-11) in the treatment of chemotherapy-induced thrombocytopenia. METHODS: Thirty-four patients (totally 76 cycles) with chemotherapy-induced thrombocytopenia received subcutaneous injection of rhIL-11 at the dose of 25 microg.kg(-1).d(-1) for 4 to 16 days. Serum IL-11 level was measured by ELISA, and IL-11 R alpha expression was detected by RT-PCR. RESULTS: The mean baseline platelet count before chemotherapy was (135.0 +/- 54.3) x 10(9)/L for the 1st cycle and (259.4 +/- 64.5) x 10(9)/L for the 2nd cycle. The time to administer rhIL-11 was 7 to 16 days (median 12 days) in the 1st cycle and 4 to 10 days (median 6 days) in the 2nd, respectively (P < 0.05). The duration of post-chemotherapy platelet count below 50 x 10(9)/L was 7 to 13 days (median 10 days) for the 1st cycle and 3 to 8 days (median 5 days) for the 2nd, respectively (P < 0.05). Platelet count reached 300 x 10(9)/L or above in 30 chemotherapy cycles. The maximum platelet count was found to appear at D10 to D 17 (median D14), and negatively correlated with the pre-chemotherapy serum IL-11 level after administration of rhIL-11. Major adverse reactions included edema, headache, muscle and joint pain. CONCLUSION: rhIL-11 is effective and safe for the treatment of chemotherapy-induced thrombocytopenia, with a relatively slow but sustained effect on the recovery of platelet count. Pre-chemotherpy serum IL-11 level might predict the efficacy of rhIL-11.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel.

AIM: To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoli-posomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G(2)/M cells and induced apoptosis, but significantly decreased the percentage of cells in G(2)/G(1) and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

[Cellular ultrastructural changes of bone marrow of patients with hemorrhagic fever with renal syndrome].

BACKGROUND: To observe cytopathogenic effect of Hantaan virus (HV) on cultured human bone marrow cells. METHODS: Light and transmission electron microscopy and direct immunofluorescent technique were applied to study cellular structure especially ultrastructural changes of bone marrow cells from patients with Hantaan virus infection. Bone marrow cells of one healthy volunteer were also studied as control. RESULTS: The antigen of HV was found in bone marrow cells of 20 of 27 HFRS patients by the aid of direct immunofluorescent technique. It was found that the granulocytes had the highest percentage of HV antigen positive cells (76%), followed by monocytes (65%), lymphocytes (40%), megakaryocytes (20%) and the lowest was found in erythrocytes (3.7%). The injury of cell membrane after infection with HV was significantly more severe than that in the control group under the light and electron microscopy. CONCLUSION: This study demonstrated that HV could attack human bone marrow cells and cause cytopathogenic effect on them.

[Expression of three kinds of GPI-anchor proteins in paroxysmal nocturnal hemoglobinuria, aplastic anemia and myelodysplastic syndromes patients and their clinical implications].

OBJECTIVE: To investigate the expressions of three kinds of glycosyl-phosphatidylinositol anchor proteins (GPI-AP), the CD(55), CD(59) and CD(87) on the peripheral granulocytes and the soluble u-PAR (su-PAR) level in serum from patients with paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndromes (MDS), and to analyse their clinical implications. METHODS: Twenty-two PNH patients, including 4 complicated with thrombotic diseases and 5 with AA-PNH syndrome, 30 AA patients, including 9 being severe AA (SAA) and 11 chronic AA (CAA), 27 MDS-RA patients, and 20 healthy individuals were tested. The expressions of CD(55), CD(59) and CD(87) on peripheral granulocytes were analyzed with flow cytometry. Serum su-PAR was assayed by ELISA. RESULTS: The CD(55)(+), CD(59)(+) and CD(87)(+) granulocytes in peripheral blood of 20 normal controls were all more than 90%. The expressions of three kinds of GPI-APs in 22 PNH patients were significantly decreased as compared with that in normal controls, AA patients and MDS-RA patients. The serum level of su-PAR in PNH group was higher than that of the other three groups. The expression of CD(87) was significantly decreased in thrombotic PNH patients as compared with that in non-thrombotic PNH patients. The expression of CD(87) was significantly decreased in AA patients than in normal controls. The expressions of three kinds of GPI-APs in 5 AA-PNH syndrome patients were remarkably reduced as compared with AA patients, but no significant difference was found for these indexes between AA-PNH syndrome and PNH patients and between 27 MDS-RA patients and 20 normal controls. CONCLUSION: Measurement of CD(55), CD(59) and CD(87) expressions levels on the peripheral granulocytes and su-PAR in serum would be alternative approaches for diagnosis and differential diagnosis of PNH. Serum level of su-PAR may be helpful to monitor thrombosis in PNH patients.

[Study on the T lymphocytes early activation and soluble tumor necrosis factor receptor in patients with aplastic anemia].

OBJECTIVE: To investigate the expression of T cell early activation marker (CD(69)) on peripheral CD(4)(+) and CD(8)(+) lymphocytes and serum levels of soluble tumor necrosis factor receptor 1 and 2 (sTNF-R1 and sTNF-R2) in serum and bone marrow in patients with aplastic anemia (AA) and their pathophysiological significance. METHODS: In vitro activation of T lymphocytes was carried out by whole blood cell culture containing PHA (20 micro g/ml). The CD(69) expressions on CD(4)(+) and CD(8)(+) lymphocytes at 0 h and 4 h after PHA exposure were analyzed by two-color flow cytometry. The levels of sTNF-R1 and sTNF-R2 in serum and bone marrow were measured by ELISA. RESULTS: The CD(69) expression rates of CD(4)(+) and of CD(8)(+) cells in SAA patients were (8.96 +/- 7.23)% and (10.67 +/- 7.58)%, respectively, and that of CD(8)(+) cells in CAA patients was (7.36 +/- 5.49)% before PHA stimulation. The CD(69) expression rates of CD(4)(+) and of CD(8)(+) cells in SAA patients were (71.73 +/- 11.91)% and (61.74 +/- 13.44)% and in CAA (59.35 +/- 10.15)% and (48.78 +/- 8.25)% respectively, and were significantly elevated after PHA stimulation. CD(69) expression on CD(4)(+) cells was much higher than that on CD(8)(+) cells after stimulation. The levels of the two sTNF-R (sTNF-R1 and sTNF-R2) in peripheral blood and bone marrow of SAA patients were elevated and in the bone marrow of CAA patients were also increased. The serum levels of sTNF-R2 were positively related to the CD(69) expression rates of CD(8)(+) cells before PHA stimulation. CONCLUSION: Increased early activation and activated potentials of T lymphocytes, along with abnormally elevated immunologically active molecules might play a major role in the pathogenesis of AA.

[Immunophenotyping of peripheral blood leukemic cells by multi-parameter cytometry].

AIM: To explore the clinical application and significance of CD45/SCC gating strategy of multi-parameter cytometry and tricolor fluorescence-labeled monoclonal antibodies in immunophenotyping of peripheral blood leukocytes in leukemia. METHODS: Peripheral blood leukocytes were stained with CD45 combined with a panel of monoclonal antibodies to different lineage markers. CD45/SSC dot plot was formed and leukemic cell colony, once displayed, was gated for immunophenotyping. RESULTS: 144 patients with acute leukemia and 51 with chronic leukemia were enrolled. Distinguishable leukemic cell colony was found in 137 out of 144 specimens. 66 patients were immunologically classified as ALL and 67 as AML. 4 patients were unable to be classified immunophenotypically. 27 AL patients coexpressed markers of another cell lineage (20%). Immunophenotypic features of different types of leukemia were described. It is a common phenomenon that AL of one lineage coexpressed markers of another lineage. CONCLUSION: An accurate immunophenotyping of leukemia can be realised when leukemic cell colony is displayed and analyzed by CD45/SCC pattern and gating strategy, which is important for clinical treatment and prognosis judgment.

[Change of expression of cell adhesion molecules CD11a and CD49d in patients with chronic aplastic anemia before and after therapy].

To explore the cell adhesion molecules (CAMs) CD11a and CD49d in patients with chronic aplastic anemia (CAA) and its clinical implications, the expression of CD11a and CD49d in mononuclear cell (MNC) of bone marrow (BM) and peripheral blood (PB) were measured using APAAP techniques in 20 patients with CAA before and after SSL/C therapy. The results showed that the expression of CD11a and CD49d in MNC of BM and CD11a in MNC of PB increased significantly (P < 0.05) after SSL/C therapy, and there was no significant change of CD49d in MNC of PB in both groups. In conclusion, the decrease of CAMs of CD11a and CD49d participated in the pathogenesis of CAA. The expression of CAMs increases with effective treatment, so the restoration or improvement of altered CAMs of CAA might be beneficial to the proliferation and differentiation of hematopoietic stem cell, and improvement of hematopoiesis in CAA.

[A study on early activation of T lymphocytes and soluble tumor necrosis factor receptor in patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of T cell early activation marker (CD(69)) on CD(4)(+) and CD(8)(+) lymphocytes in peripheral blood and the levels of soluble tumor necrosis factor receptor 1 (sTNF-R1) and sTNF-R2 in serum and bone marrow in patients with myelodysplastic syndrome (MDS). METHODS: Whole blood cell culture procedure was applied to activate T lymphocytes with phytohemagglutinin (PHA) (20 mg/L) in vitro. The expression rates of CD(69) on CD(4)(+) and CD(8)(+) lymphocytes at 0 h and 4 h after culture were analyzed with two-color flow cytometry. The levels of sTNF-R1 and sTNF-R2 in serum and bone marrow were measured with ELISA. RESULTS: There was an increase in the expression rates of CD(69) on CD(4)(+) and CD(8)(+) cells in RA and RAS patients (8.32% and 9.88% respectively) and in the expression rate of CD(69) on CD(8)(+) cells in RAEB and RAEB-T patients (7.92%) before PHA stimulation. CD(69) expression on CD(4)(+) and CD(8)(+) cells were significantly elevated in MDS patients after PHA stimulation (53.46% and 51.63% in RA + RAS; 42.93% and 41.96% in RAEB and RAEB-T) and the expression rate on CD(4)(+) cells was similar to that on CD(8)(+) cells. The levels of the two sTNF-R in MDS patients were elevated. sTNF-R1 in RA and RAS (1.58 +/- 0.68) micro g/L (PB), (2.10 +/- 0.26) micro g/L (BM); sTNF-R2 in RA and RAS (1.41 +/- 0.50) micro g/L (PB), (1.95 +/- 0.64) micro g/L (BM); sTNF-R1 in RAEB and RAEB-T (2.62 +/- 2.55) micro g/L (PB), (3.12 +/- 0.67) micro g/L (BM); sTNF-R2 in RAEB and RAEB-T (1.96 +/- 0.56) micro g/L (PB), (3.09 +/- 0.62) micro g/L (BM). The levels of sTNF-R2 in serum positively correlated to the expression rate of CD(69) on CD(8)(+) cells before PHA stimulation. CONCLUSION: It is indicate that the increased early activation and activated potentials of T lymphocytes, along with abnormally elevated immunologically active molecules play an important role in immune pathogenesis of patients with MDS.

[Synergistic effect of Apo2L and chemotherapeutic agents on leukemia cells].

OBJECTIVE: To explore if the antileukemic drugs Vp16 or Ara-C are able to upregulate DR5 gene expression and enhance Apo2L-induced apoptosis of HL-60 cells. METHODS: Cell apoptosis was determined by flow cytometry after annexin V/PI staining, the effect of Apo2L on fresh leukemia cells by MTT reduction assay, the expression of DR5 gene in HL-60 cells by semi-quantitative RT-PCR. RESULTS: 1. Apo2L induced apoptosis of HL-60 cells in a dose-dependent manner. 2. Apo2L inhibited the proliferation of fresh leukemia cells, but there was difference among different subtypes. 3. Vp16 or Ara-C upregulated DR5 gene expression and augmented Apo2L-induced apoptosis in HL-60 cells. CONCLUSION: Apo2L could induce apoptosis of HL-60 cells and inhibit the proliferation of fresh leukemia cells. Ara-C or Vp16 upregulated DR5 gene expression and increased the sensitivity of HL-60 to Apo2L-induced cytotoxicity. Apo2L might be a promising antileukemic agent for the treatment of leukemia.