Clinical characteristics and drug sensitivity in children with invasive pneumococcal disease: a multicenter study / 中国当代儿科杂志

OBJECTIVE@#To study the clinical characteristics, drug sensitivity of isolated strains, and risk factors of drug resistance in children with invasive pneumococcal disease (IPD).@*METHODS@#The clinical characteristics and drug sensitivity of the isolated strains of 246 hospitalized children with IPD in nine grade A tertiary children's hospitals from January 2016 to June 2018 were analyzed.@*RESULTS@#Of the 246 children with IPD, there were 122 males and 124 females. Their ages ranged from 1 day to 14 years, and among them, 68 (27.6%) patients were less than 1 year old, 54 (22.0%) patients were 1 to 2 years old, 97 (39.4%) patients were 2 to 5 years old, and 27 (11.0%) patients were 5 to 14 years old. Pneumonia with sepsis was the most common infection type (58.5%, 144/246), followed by bloodstream infection without focus (19.9%, 49/246) and meningitis (15.0%, 37/246). Forty-nine (19.9%) patients had underlying diseases, and 160 (65.0%) had various risk factors for drug resistance. The isolated Streptococcus pneumoniae strains were 100% sensitive to vancomycin, linezolid, moxifloxacin, and levofloxacin, 90% sensitive to ertapenem, ofloxacin, and ceftriaxone, but had a low sensitivity to erythromycin (4.2%), clindamycin (7.9%), and tetracycline (6.3%).@*CONCLUSIONS@#IPD is more common in children under 5 years old, especially in those under 2 years old. Some children with IPD have underlying diseases, and most of the patients have various risk factors for drug resistance. Pneumonia with sepsis is the most common infection type. The isolated Streptococcus pneumoniae strains are highly sensitive to vancomycin, linezolid, moxifloxacin, levofloxacin, ertapenem, and ceftriaxone in children with IPD.

MiR-21 promotes the EMT and invasion of prostate cancer cell by inducing FOXO1 / 临床外科杂志

Objective To investigate the mechanism of EMT and invasion promoted by miR-21 in prostate cancer cells. Methods The sequence of miR-21 mimic/inhibitor was firstly designed and synthesized. Then miR-21 mimic/inhibitor and its control were transfected into prostate cancer cells C4-2 and DU145, respectively. And cells were collected for mRNA isolation and RT-qPCR analysis for miR-21 and FOXOl. FOXOl, E-cadherin and N-cadherin were detected by Western blot, and the invasion of prostate cancer cells were detected by transwell assays. Results The expression of miR-21 increased in both C4-2 and DU145 after transfection, and the expression of FOXOl mRNA increased at the same time (P＜0.01).The expression of miR-21 and FOXOl mRNA in C4-2 and DU145 was decreased by miR-21 inhibitor(P＜ 0.05). The protein expression of FOXOl and N-cadherin in C4-2 and DU145 increased after the treatment of miR-21, while that of E-cadherin decreased. The protein expression of FOXOl and N-cad-herin in C4-2 and DU145 decreased after the treatment of miR-21 inhibitor, while that of E-cadherin increased. The invasive level in C4-2 and DU145 increased after the treatment of miR-21, while that decreased after the treatment of miR-21 inhibitor. Conclusion MiR-21 promotes EMT and invasion by inducing FOXOl in prostate cancer cells.

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>

Evaluation of the P-gp pump function on leukemic cell membrane and proper application of its reversal agents with Calcein-AM and flow cytometry / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia (ALL) have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia (AML) remain low due to the multiple-drug resistance (MDR). P-glycoprotein (P-gp) is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A (CSA) and verapamil (VER) through the observation of Calcein-AM (C-AM) metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR.</p><p><b>METHODS</b>The mean fluorescence intensity (MFI) of C-AM inside the cytoplasm was analyzed with flow cytometry (FCM). The events of K562 and K562/VCR cells treated and untreated with CSA, VER and CSA + VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software.</p><p><b>RESULTS</b>The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA + VER groups co-cultured with K562/VCR cells were 4014 +/- 219, 3879 +/- 116 and 4158 +/- 302, respectively after 120 min of incubation, significantly higher as compared to that of control group (3251 +/- 107, P < 0.05). On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 +/- 155, 1932 +/- 233 and 2231 +/- 147, respectively in the three groups, which was significantly higher than that of control group (1622 +/- 191, P < 0.05). CSA, VER and CSA + VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed.</p><p><b>CONCLUSIONS</b>CSA, VER and CSA + VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA + VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.</p>

Comparison between CD19 and CD20 expression patterns on acute leukemic cells / 中国实验血液学杂志

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.

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>

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>

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>