Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom / 中国天然药物

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.

Discrimination of Coptidis Rhizoma and its processed products by odor objectify / 中国中药杂志

Different processed products of Coptidis Rhizoma have its unique odor, which is an important assessment index for pro- cessed products identification of Coptidis Rhizoma. Objectify odor as an entry point in this study, an electronic nose technology was used, and a suitable method for Coptidis Rhizoma measurement was built firstly. Then different processed products of Coptidis Rhizoma were detected by the method built. Finally, different processed products were identified by combining with chemometrics based on the objective odor information obtained. Electronic nose detection indicated that a significant difference in odor between different processed products was performed. Coptidis Rhizoma processed or not can be distinguished based on statistical quality control (SQC) and soft independent modeling of class analogy (SIMCA). Principle component analysis (PCA) model showed that Coptidis Rhizoma and its various processed products discriminated obviously. In addition, in order to identify the processed products of Coptidis Rhizoma, a correct recognition rate of 100% was acquired by discriminant factor analysis (DFA) , and the initial identification rate and cross-validation recognition rate of linear discriminant analysis (LDA) is 100%, 94.4% respectively. In conclusion, differentiationin odor of different processed Coptidis Rhizoma was performed by the electronic nose technology used, and different products Coptidis Rhizoma were dis- criminated by combining with chemometrics. This research can be a reference for objective identification in odor of traditional Chinese medicine, and is good for the inheritance and development of traditional experience in odor identification.

Study on change rule of 6 ester-type alkaloids in process of Heishunpian / 中国中药杂志

To study the variation of six ester-type alkaloids and characteristic fingerprints in the process from Radix Aconite Lateralis to Heishunpian and lay a foundation for the study of the processing principle of Heishunpian, HPLC. analysis was performed on a Phenomenex Gemini C18 (4.6 mm x 250 mm, 5 microm) with acetonitrile and 40 mmol x L(-1) ammonium acetate (adjusted to pH 10 with concentrated ammonia water) as mobile phase. The detection wavelength was set at 235 nm. The flow rate was set at 0.8 mL x min(-1) and the injection volume was 10-20 microL. Six ester-type alkaloids were determined and characteristic fingerprints of the process were established. As the process continues, the contents of diester diterpene alkaloids were decreased step by step, while the contents varia tion of monoester diterpene alkaloids were not obvious. Each sample showed significant difference in characteristic fingerprints. With the exception of 6 known monoester diterpene alkaloids and diester diterpene alkaloids, 13 peaks were marked in the characteristic fingerprints, of which the total change rule of the other 7 unknown peaks were similar with 3 diester diterpene alkaloids. The established method is accurate, reliable and repeatable, and can provide reference for revealing change rule of index components and illuminating processing principle in the process of Heishunpian.

Clinical and laboratory characteristics of chronic active Epstein-Barr virus infection in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical and laboratory characteristics of chronic active Epstein-Barr virus (EBV) infection (CAEBV) in children and to provide a basis for the diagnosis and treatment of CAEBV.</p><p><b>METHODS</b>The clinical data of 13 children with CAEBV, as well as 15 cases of acute EBV infection (AEBV) as controls, were analyzed, including clinical manifestations, EBV antibodies, EBV DNA, and peripheral blood lymphocyte subsets.</p><p><b>RESULTS</b>Both groups of patients had infectious mononucleosis-like symptoms such as fever, hepatomegaly, splenomegaly, and lymphadenectasis, but CAEBV patients had a longer course of disease and continuous and recurrent symptoms. Compared with the AEBV group, the CAEBV group had a significantly higher EBV DNA load in peripheral blood (P<0.05), a significantly higher VCA-IgG titer (P<0.05), and significantly lower numbers of white blood cells, lymphocytes, B cells, total T cells, CD4+ T cells, and CD8+ T cells in peripheral blood (P<0.05). Among 13 CAEBV patients followed up, 8 cases died, 2 cases showed an improvement, 2 cases had a recurrence, and 1 case was lost to follow-up after being transferred to another hospital. All the AEBV patients were cured and had no recurrence during the one-year follow-up.</p><p><b>CONCLUSIONS</b>The clinical manifestations of CAEBV vary in children. It is difficult to distinguish CAEBV from AEBV early. More attention should be paid to CAEBV because of its severe complications, poor prognosis, and high mortality. Measurement of EBV DNA load, VCA-IgG titer, and lymphocyte subsets in peripheral blood may be helpful in the diagnosis and differential diagnosis of CAEBV.</p>

Preliminary study of odor change mechanism in Crataegi fructus stir-fried process based on correlation analysis / 中国中药杂志

In order to investigate the mechanism, the correlation between the odor change in Crataegi Fructus stir-fried process and 5-HMF were studied. Required samples were retrieved from Crataegi Fructus stir-fried process. Statistical quality control (SQC) was used to analyze the response values acquired by the electronic nose. At the same time, the content of 5-HMF was detected by high performance liquid chromatography (HPLC). Correlation analysis was used to analyze the relationship between the above two. Experimental results showed that SQC model established by response values of all samples could show the change law of odor in Crataegi Fructus stir-fried process and changes of 5-HMF content was dropped after the first increase. Correlation analysis showed that the odor change in Crataegi Fructus stir-fried process and 5-HMF were significantly correlated (P < 0.05). Sugar degradation reaction and the Maillard reaction may be one of the mechanisms of the odor change in Crataegi Fructus stir-fried process.

Effectiveness of immunosuppressive therapy for childhood aplastic anemia and its predictive factors / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the effectiveness of immunosuppressive therapy (IST) for childhood aplastic anemia (AA) and its predictive factors.</p><p><b>METHODS</b>The medical data of 110 children with AA who received IST between February 2003 and November 2009 were retrospectively studied. Of these patients, 83 were diagnosed as severe AA (SAA) and 27 were non-SAA. The former group was treated with antithymocyte globulin (ATG) + ciclosporin (CSA) +prednisone + androgens and the latter with CSA + prednisone + androgens.</p><p><b>RESULTS</b>Total response rates in the SAA and non-SAA groups were 69.9% and 70.4%, respectively. Univariate analysis showed that disease duration, CD34+ cell level in bone marrow (BM) and the expression of CD4+CD25+ regulatory T cell in BM were related to the severity of disease but not correlated with treatment response. Age, disease duration and proportions of CD3+ and CD8+ T cells in BM were predictive factors for response to IST. Multivariate analysis revealed that patients aged more than 10 years and patients with a CD8+ cell proportion in BM of over 25% had hazard ratios of 3.36 and 3.59 for treatment failure respectively.</p><p><b>CONCLUSIONS</b>IST is effective in the treatment of childhood AA. Age, disease duration and the proportion of CD8+ T cell in BM are predictive factors for response to IST.</p>

Establishment of a transgenic cell line with stable expression of human CD14 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a transgenic cell line with stable expression of CD14.</p><p><b>METHODS</b>Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay.</p><p><b>RESULTS</b>There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells.</p><p><b>CONCLUSION</b>The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.</p>

Diagnostic significance of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis / 中华儿科杂志

<p><b>OBJECTIVE</b>To illustrate the diagnostic value of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis (HLH) and its diagnostic accuracy.</p><p><b>METHOD</b>The BD(TM) CBA Human Th1/Th2 Cytokine Kit II was used to measure the serum Th1 and Th2 cytokines, including Interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-10, IL-6, IL-4 and IL-2 in 50 patients with de novo HLH admitted to our hospital from Oct. 2005 to Aug. 2009. The above cytokine levels were also determined in 250 healthy volunteers and 235 patients with sepsis as controls.</p><p><b>RESULT</b>The primary features of these patients were prolonged high-grade fever (50/50), hepatomegaly (44/50), splenomegaly (38/50), hemocytopenia (47/50), hyperferritinemia (49/50), coagulopathy (44/50), hemophagocytosis in bone marrow (42/50), liver dysfunction (42/50) and hypertriglyceridemia (42/50). The IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2 levels for healthy children were (4.6 ± 1.8) ng/L, (4.0 ± 1.2) ng/L, (6.5 ± 1.3) ng/L, (6.0 ± 1.5) ng/L, (2.9 ± 0.8) ng/L and (2.6 ± 0.7) ng/L, while the median levels of them in acute phase of HLH children were 1138.5 (49.2 - 5000.0) ng/L, 3.4 (1.0 - 25.1) ng/L, 740.5 (26.5 - 5000.0) ng/L, 66.1 (3.9 - 4472.6) ng/L, 3.9 (1.0-32.8) ng/L and 4.0 (1.0 - 51.1) ng/L, respectively. The cytokine levels decreased to 9.1 (1.9 - 180.1) ng/L, 2.9 (1.0 - 11.0) ng/L, 11.4 (2.9 - 184.2) ng/L, 6.5 (1.0 - 44.8) ng/L, 2.7 (1.0 - 6.5) ng/L and 4.1 (1.0 - 12.0) ng/L respectively after remission. The IFN-γ, IL-10 and IL-6 levels in acute phase were significantly higher than those after remission and those of the healthy control (P all < 0.001). IL-4, IL-2 and TNF slightly elevated or at normal range in acute phase of HLH. The patients with sepsis showed a different cytokine pattern, with an extremely high level of IL-6 (median: 251.3 ng/L, range: 8.4- > 5000.0 ng/L) and moderately elevated level of IL-10 (median: 46.5 ng/L, range: 3.1 - 5000.0 ng/L), whereas IFN-γ was only slightly elevated (median: 9.2 ng/L, range: 1.3 - 498.8 ng/L). When the criteria for HLH set as the following: IFN-γ > 100 ng/L, IL-10 > 60 ng/L and the concentration of IFN-γ higher than that of IL-6, the specificity reached as high as 98.7% and the sensitivity was 88.0% for the diagnosis of HLH among patients with HLH and sepsis. Meanwhile, the positive predictive value (PPV) and negative predictive value (NPV) could reach 93.6% and 97.5%, respectively.</p><p><b>CONCLUSION</b>The significant increase of IFN-γ and IL-10 with slightly increased level of IL-6 is a sensitive and specific cytokine pattern for childhood HLH, which is helpful for its diagnosis and differential diagnosis.</p>

Monitoring of minimal residual disease in children with acute lymphoblastic leukemia and its prognostic significance / 中华儿科杂志

<p><b>OBJECTIVE</b>Monitoring of minimal residual disease (MRD) is proven to be increasingly valuable for predicting relapse and outcome of childhood acute lymphoblastic leukemia (ALL) and is used to identify patients' risk groups in several current clinical trials. However, the limitation is that most studies focused on the cut-off value at 10(-4) and the time point after induction. The aim of this study was to investigate the predictive values of different MRD levels detected at different chemotherapy phases in childhood ALL.</p><p><b>METHODS</b>One hundred and two patients were enrolled in this study from January 2002 to December 2004 in our hospital. All the patients were treated with modified National Protocol of Childhood ALL in China 1997. MRD levels were detected on the 15th day, 29th day, at 3 months, 6 months and 12 months after initial chemotherapy. All samples were stained with a panel of four colour combinations of fluorochrome conjugated monoclonal antibodies according to the leukemia-associated immunophenotype (LAIP) defined at diagnosis and analyzed by multi-parametric flow cytometry. CD45CD19CD34CD10, CD45CD19CD34CD20 and CD45CD19CD10CD20 were the most common combinations in B lineage ALL, while CD45CD2CD3CD7 and CD45CD2CD3CD34 were the most frequently used immunophenotypes for T lineage ALL. The median follow-up time was 63.3 months ranged from 40.6 to 87.5 months.</p><p><b>RESULTS</b>Of the 102 patients, 64 were male and 38 were female, with a median age of 5.7 (0.2 - 14.8) years. Eighty-eight cases were diagnosed as B lineage ALL and the remaining 14 were T-ALL. The 5-year overall survival (OS) rate and event free survival (EFS) rate for this cohort were (86.9 +/- 3.4)% and (79.9 +/- 4.0)%, respectively. Twelve patients underwent relapse. Among the 102 patients, 14.3% had negative MRD (MRD < 10(-4)) on day 15, 43.9% on day 29, 39.1%, 39.7% and 45.6% had negative MRD at the third, sixth and twelfth month after chemotherapy. Patients who could achieve negative MRD within one year had superior outcome to the others [5-year EFS rates: (92.5 +/- 3.2)% vs. (58.3 +/- 8.6)%, P < 0.001]. The EFS for patients based on MRD levels measured at different stages of therapy were compared by Kaplan-Meier analyses. MRD was predictive of outcome at all 5 time points at a range of thresholds. The optimum threshold, selected for each time point on the basis of log rank analysis, progressively dropped from 10(-2) of day 15 [5-year EFS rates (79.8 +/- 10.3)% vs. (28.6 +/- 17.1)%, P < 0.001], to 10(-3) of day 29 [5-year EFS rates (88.3 +/- 4.9)% vs. (51.3 +/- 14.4)%, P < 0.003], to 10(-4) at 3 [5-year EFS rates (92.4 +/- 5.1)% vs. (65.5 +/- 7.5)%, P < 0.015], 6 [5-year EFS rates (96.3 +/- 3.6)% vs. (65.4 +/- 7.5)%, P < 0.003] and 12 [5-year EFS rates (100.0 +/- 0.0)% vs. (67.7 +/- 8.4)%, P < 0.002] months. And the hazard ratios for relapse and death at higher MRD level groups were 5.91 (95%CI: 1.9 - 18.9), 5.02 (95%CI: 1.5 - 16.5), 5.21 (95%CI: 1.2 - 22.9) and 11.10 (95%CI: 1.5 - 84.5) on day 15, day 29, at month 3 and month 6, respectively. And MRD >or= 10(-2) on day 15 was proven to be an independent predictor by multivariate Cox proportional-hazards regression model.</p><p><b>CONCLUSION</b>Dynamic MRD detection by multi-parametric flow cytometry is highly predictive of outcome for childhood ALL, and the cut-off values at different time points were different.</p>

Efficacy of chemotherapy using 10-hydroxycamptothecin on recurrent or refractory neuroblastoma in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The patients with recurrent or refractory neuroblastoma have a very poor prognosis and high mortality. 10-hydroxycamptothecin (HCPT) is a new agent extracted from comptotheca acuminate, a native plant. It has been shown to be very effective in some solid tumors such as gastric and colon cancers, lung cancers and ovary cancers. However, its efficacy in neuroblastoma has not been determined. This study aimed to investigate the therapeutic effects of HCPT in the treatment of recurrent or refractory neuroblastoma in children.</p><p><b>METHODS</b>Ten children with recurrent neuroblastoma and two children with refractory neuroblastoma were treated with HCPT. Of them, 5 children with recurrent neuroblastoma were treated with HCPT alone, and the other 7 patients received combination chemotherapy of HCPT plus other agents. The HCPT alone treatment group was injected with HCPT (7.5 mg/m2 daily) for 14 consecutive days. The combination chemotherapy group was alternately treated with the modified new protocol A1 (cyclophosomide 1 200 mg/m2 on day 1, etoposide 100 mg/m2 on days 1-5, HCPT 5 mg/m2 on days 1-3, cisplatin 90 mg/m2 on day 4) and the modified protocol B (ifosfomide 1.5 g/m2 on days 1-5, HCPT 5 mg/m2 on days 1-3, carboplatin 450 mg/m2 on day 2).</p><p><b>RESULTS</b>Four patients (33.3%) achieved partial remission and 8 patients (66.7%) had stable disease. The median remission duration was 3.5 months (2-5 months). HCPT treatment as a single agent resulted in mild side effects. Myelosuppression and digestive disorders were found as the main adverse events in the combined chemotherapy group. No chemotherapy related deaths were found.</p><p><b>CONCLUSIONS</b>HCPT is safe and effective in the treatment of recurrent or refractory neuroblastoma. The toxicities of HCPT are tolerable. The long-term efficacy of HCPT warrants further research.</p>

Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.</p><p><b>METHODS</b>2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.</p><p><b>RESULTS</b>After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.</p><p><b>CONCLUSION</b>2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.</p>

Long-term follow-up of treatment outcome and prognosis on 46 children with acute promyelocytic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Acute promyelocytic leukemia (APL) is a specific type of hematopoietic malignancy, accounting for 10% of the de novo acute myeloid leukemia (AML). The data on long-term outcome of APL in children are limited. The aim of this study was to investigate the clinical biological features, diagnosis, prognosis and long-term survival of childhood APL.</p><p><b>METHODS</b>A total of 46 children with newly diagnosed APL from April 1998 to October 2005 were enrolled into this study. Induction treatment containing all-trans retinoic acid (ATRA) plus daunorubicin (DNR) or pirarubicin (THP) was performed on these patients, followed by 6 courses of chemotherapy consolidation: DNR, homoharringtonine or etoposide plus Ara-C. A maintenance therapy was then administered once 3-6 months. The total period of treatment was 2.5 years.</p><p><b>RESULTS</b>Of the 39 patients who had completed the regular treatment, 36 (92.3%) achieved a complete remission. The 5-year cumulative incidence of relapse (CIR) was 28.6%. The estimated overall survival (OS) rates at 1, 3 and 5 years were (86.1 +/- 5.8)%, (76.1 +/- 7.5)% and (70.2 +/- 8.9)% respectively, while the event free survival (EFS) rates were (78.4 +/- 6.8)%, (63.6 +/- 8.7)% and (53.1 +/- 10.0)% respectively. The 5-year OS rate of patients with WBC less than or equal to 10.0 X 10(9)/L was (81.4 +/- 10.3)%, which was significantly higher than that with WBC greater than 10.0 X 10(9)/L[(51.6 +/- 14.7)%, P < 0.05]. Five patients with RT-PCR positive for PML/RARalpha S (short) subtype died eventually although all of them achieved CR, but none of the 13 patients with PML/RARalpha L (long) subtype died.</p><p><b>CONCLUSIONS</b>Remission induction therapy with ATRA + DNR or THP is effective and safe for newly diagnosed childhood APL. The remission induction therapy combined with chemotherapy containing high/intermediate dose Ara-C can improve the long-term survival rates of APL patients. High WBC count and S subtype of PML-RARa are two poor prognostic factors for children with APL.</p>

Reactivity of a novel monoclonal antibody ZCH-2B8a on normal hematopoietic cells and malignant cell lines and its significance / 中国实验血液学杂志

ZCH-2B8a (IgG2a) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to explore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells, natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34(+) cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells > or = 20% was considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC and Plt. The antibody reacted to all 3 marrow CD34(+) cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34(+) peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-60 (29.70%), K562 (28.19%), KG1a (16.23%) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and therapeutic applications among different types of hematopoietic malignancies.

Expressions of CD117 and CD11b in patients with APL at diagnosis and post-treatment / 中国实验血液学杂志

The aim of this study was to evaluate the value of CD117/CD11b phenotypic analysis to diagnosis and prognosis of acute promyelocytic leukemia (APL). Three- or four-color flow cytometry with a series of 22 monoclonal antibodies and CD45/Side Scatter (SSC) gating strategy were used to identify immunophenotypic characteristics of APL as compared to CML in chronic phase (CML-CP). PML/RAR alpha fusion gene was detected by using reverse-transcription polymerase chain reaction (RT-PCR) technique. The results showed that MPO, CD13 and CD33 were almost expressed in all patients with APL and CML-CP whereas HLA-DR and CD34, the hematopoietic progenitor cell markers, were rarely expressed. The positive rate of CD15 in APL was significantly lower than those in CML-CP (P < 0.01). CD117 was positive in 78.3% of the APL cases and in none of the cases of CML-CP. On the other hand, CD11b was almost positive in all cases of CML-CP, but only 16.9% of the APL cases were found positive for this antigen. The CD117+ CD11b- phenotype was present in 72.3% of APL cases while none of cases with CML-CP with this phenotype. Almost all of the cases with CML-CP had the phenotype of CD117- CD11b+. CD117- CD11b+ phenotype was detected in all patients recovering from APL with CD117+ CD11b- phenotype at diagnosis and after treatment with all-trans-retinoic acid (ARTA) for 2 months. PML/RAR alpha fusion gene was positive in 80.6% (25/31) of the APL cases, of which, 64% of the cases belonged to the type L while only 36% of the cases were showed type S for this fusion gene. The positive rates of CD117 were 87.5%, 44.4% and 33.3% in type L group, S group and negative group respectively. It is concluded that analysis of both CD117 and CD11b phenotype may be helpful to the diagnosis, therapy and prognosis of APL in children and adults and to differentiation of APL from recovering benign myeloid proliferation.

Preparation and characterization of a directly labeled mouse anti-human CD14 monoclonal antibody ZCH-2F9-FITC / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare fluorescein isothiocyanate (FITC) directly conjugated to monoclonal antibody (McAb) anti-human CD14, ZCH-7-2F9 (2F9-FITC).</p><p><b>METHODS</b>After generation and purification, the purity and the murine immunoglobulin subtype of the antibody were evaluated with SDS-PAGE and multicolor flow cytometry (FCM). 2F9 McAb was directly labeled with FITC through modified Marsshall's method and the positive rate of the 2F9-FITC on different types of leukemic cells were compared with the standard CD14-FITC by FCM.</p><p><b>RESULT</b>A large quantity of purified 2F9 McAb was prepared. The subtype of 2F9 was murine IgG1kappa. 2F9-FITC was successfully manufactured with A295/A280 ratio of 0.44. The positive cell percentages of 2F9-FITC and CD14-FITC on the monocytes were 84.50% and 90.08%, respectively, while those on lymphocytes were only 0.52% and 1.01%. There was no significant difference between the CD14 expressions with 2F9-FITC and CD14-FITC on each type of leukemia (n=23, t=0.922, P=0.367).</p><p><b>CONCLUSION</b>2F9-FITC has been successfully prepared and it can be applied in diagnosis and differentiation of monoblastic leukemias.</p>

Study on elimination delay in high dose methotrexate therapy in childhood acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the incidence of elimination delay in high dose methotrexate (HDMTX) therapy, its side effects and influence to next course of chemotherapy and analyze the relationship between the dosage, the duration of MTX infusion and the morbidity of the elimination delay.</p><p><b>METHODS</b>A total of 121 childhood acute lymphoblastic leukemia (ALL) (497 infusions of HDMTX) were analysed in this study. The elimination delay rate and the adverse effects in different dose groups (3 g/m2 vs 5 g/m2) and different infusion duration groups (7 h vs 24 h) were compared. The adverse effect evaluation was based on the World Health Organization (WHO) Toxicity Grading Criteria. The rescue dosages of calcium folinate (CF) among these groups were compared through CF/MTX index.</p><p><b>RESULTS</b>The overall morbidity of elimination delay was 12.1% with a relative risk of 30.6% for the first time. The relative risk for the second time of occurrence was increased to 45.9% (P < 0.01) and it was not significantly increased for the third time (35.3%). Children with elimination delay had lower platelet count (P < 0.01) and higher CF rescue dosage (P < 0.01), while the damage of oral mucous membrane was more severe (P < 0.05) and the next course of chemotherapy would be postponed for a median of 4 days in 3 g group. There was no significant difference in elimination delay rates between 3 g and 5 g groups (12.1% vs 12.0%, P > 0.05), and between 7 h and 24 h MTX infusion groups (13.6% vs 11.9%, P > 0.05). The only side effect occurred in 5 g group was gastrointestinal morbidity. The CF/MTX index of 5 g group without elimination delay was less than that of 3 g group (P < 0.01).</p><p><b>CONCLUSION</b>Elimination delay in HDMTX therapy accompanies the suppression of bone marrow and damage of oral mucous membrane, which need more CF rescues and will postpone the following course of chemotherapy. Elimination delay is not associated with the duration of the infusion and the dosage of MTX within the range of 3 approximately 5 g/m2 but there are individual differences.</p>

Expression of leucocyte cell-surface antigens on colon cancer cell line HR8348 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To define the immune phenotype of colon cancer cells.</p><p><b>METHODS</b>Using a panel of 40 anti-human monoclonal antibodies (MoAbs), the cells of colon cancer HR8348 were analyzed with three-color flow cytometry after direct immunofluorescent staining.</p><p><b>RESULTS</b>HR8348 cell line did not express CD2, CD3, CD4, CD5, CD7, CD8, TCR, CD10, CD11b, CD14, CD16, CD19, CD22, CD25, CD28, SmIg, CD33, CD35, CD36, CD41a, CD45, CD45RA, CD45RO, CD56, CD61, CD64, CD66b, CD69, CD71, CD117, CD122 and P-glycoprotein but expressed CD13, CD15, CD20, CD38, CD95 and HLA-DR.</p><p><b>CONCLUSION</b>The results demonstrate that colon cancer cell line HR8348 shares some antigenic determinants with leucocyte lineage.</p>

The expression of CD19 in 210 cases of childhood acute leukemia and its significance / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of CD19 on childhood acute leukemia (AL) and its significance, and to provide evidence for the diagnosis and differential diagnosis as well as monoclonal antibody-targeting treatment of leukemia.</p><p><b>METHODS</b>There were 210 cases of childhood AL, of which 130 cases were male and 80 were female with a mean age of 9 years old. Using a panel of 27 fluorochrome directly labeled monoclonal antibodies, 210 samples from the patients were analyzed with CD45/SSC double parameters and multi-color flow cytometry to determine the expression of CD19.</p><p><b>RESULTS</b>In the 93 cases of B lineage acute lymphoblastic leukemia (ALL), the positive rate (98.9%, 92/93) of CD19 was significantly higher than that of the other B cell related antigens, such as CD10 (88.2%, 82/93, P = 0.003), CD20 (24.7%, 23/93, P = 0.001) and CD22 (60.2%, 56/93, P = 0.001). CD19 was expressed on all 8 cases of B/myeloid (My) hybrid acute leukemia (HAL) and 1 case of B/T HAL, but was not expressed on all 24 cases of T lineage leukemia and 5 cases of T/My HAL. In the 79 cases of acute myeloid leukemia (AML), only 5 (6.3%) cases expressed CD19. The positive rate (6.3%) of CD19 on AML was significantly lower than that on B lineage ALL (98.9%, P = 0.001). The percentage of CD19 positive cells in B/My HAL (41.6% - 88.7% with a mean of 73.8%) was significant higher than that in CD19(+)-AML (21.4% - 50.4% with a mean of 24.2%; Run Sum test, P = 0.0084). Of the 210 cases, 102 were B lineage related AL including B lineage ALL, B/My HAL and B/T HAL. In B lineage related AL, the sensitivity and the specificity of CD19 was 99.0% (101/102) and 95.4% (13/108) while the positive predictive and the negative predictive values to B lineage were 95.3% (101/106) and 99.0% (103/104), respectively. Using CD19(+) as a single reagent to diagnose B lineage, the false positive rate was 4.6% (5/108) and the false negative rate was 1.0% (1/102) with a general diagnosis index (GDI) of 94.4% [GDI = 1-(false positive rate + false negative rate)].</p><p><b>CONCLUSION</b>CD19 is continuously and stably expressed on all stages of B lineage differentiation. It is a reliable cell membrane marker for diagnosing B lineage ALL and an ideal target for antibody-targeting treatment of leukemia as well; the expression degree of CD19 can be used to distinguish B/My HAL from CD19(+)-AML; CD19 didn't express on normal myeloid cells but did on some AML cells. Therefore it can be used to detect the minimal residual disease.</p>

Immunophenotyping of 222 children with acute leukemia by multi-color flow cytometry / 中华儿科杂志

<p><b>OBJECTIVE</b>Acute leukemia (AL) is one of the most common malignant diseases in children. AL immunophenotypes are known to be benefit to the predictable prognoses and specific therapy. Recently, the accuracy of AL immunophenotyping was dramatically improved with the application of the flow cytometry, the new monoclonal antibodies, the improvement of the gating strategies and the multi-parameter analytic techniques. The aim of this study was to evaluate the value of multi-color flow cytometry in the immunophenotyping of acute leukemia in children.</p><p><b>METHODS</b>Three- or four-color flow cytometry and CD(45)/Side Scatter (SSC) gating were used to analyze the surface and cytoplasmic (Cy) antigen expressions in 222 successive cases of childhood acute leukemia.</p><p><b>RESULTS</b>Cells from 222 cases of children with acute leukemia were analyzed. Based on the diagnostic criterion proposed by European Group for the Immunological Characterization of Leukemia (EGIL), four categories could be identified: the undifferentiated type accounted for 0.9%, acute myeloid leukemia (AML) 35.1%, acute lymphoblastic leukemia (ALL) 55.9%, and mixed lineage AL 8.1%. Of 124 patients with ALL, 94 patients (75.8%) were classified as B lineage and 30 patients (24.2%) T lineage ALL. Antigen aberrant expressions were found in AML (24.4%), B lineage ALL (36.2%) and T lineage ALL (30.0%). CD(7) was the most commonly expressed lymphoid antigen in AML (12.8%), followed by CD(19) (6.4%) and CD(2) (5.1%). CD(13) was the most commonly expressed myeloid antigen in ALL (18.5%), followed by CD(15) (11.3%), CD(11b) (6.5%) and CD(33) (4.3%). CD(117) and CD(56) presented in 73.3% and 38.0% cases of AML, respectively, but were generally absent on blast cells of ALL. CyCD(22), CyCD(3) and CyMPO were specifically expressed in B lineage, T lineage and myeloid lineage leukemia, respectively, and the first two could be more sensitively detected than they were on cell membrane surface.</p><p><b>CONCLUSIONS</b>Multi-color flow cytometry is a reliable technique in the diagnosis, differential diagnosis and classification of acute leukemia in children.</p>

Analysis of CD117 expression on leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the expression of CD(117) on different types of leukemia and its significance.</p><p><b>METHODS</b>CD(117) expression was analysed by three-color flow cytometry with CD(45)/SSC gating strategy.</p><p><b>RESULTS</b>CD(117) was expressed in 68% of acute myeloid leukemia (AML) and 80% of chronic myeloid leukemia in blast crisis (CML-BC) cases, but in only 2% of acute lymphoblastic leukemia cases. CD(117) was negative in CML in chronic phase and chronic lymphocytic leukemia. Cases in AML, CD(117) was expressed in 72% of M(0)/M(1), 88% of M(2), 50% of M(4), 75% of M(5a), 100% of M(6) and 100% of M(7) cases but was less expressed in M(3) (39%) and M(5b) (29%) cases. Expression of CD(117) was more frequent than CD(34) and HLA-DR in M(3) and some M(2) cases. There was a significant inverse relationship between CD(117) and CD(14) expression in AML.</p><p><b>CONCLUSIONS</b>Analysis of CD(117) expression may be of help in distinguishing myeloid from lymphoid leukemia and leukemia clone from normal cells.</p>