ALBERT

Description: Introduction and objectives Hemophogocytic syndrome (HPS) is an infrequent disease in children with a high mortality rate. Currently, the diagnosis of HPS largely depends on non-specific clinical manifestations, but the clinical features allowing to meet the criteria of diagnosis of HPS usually do not appear simultaneously which make the early diagnosis very difficult. According to the literature, hypercytokinemia has been noticed for this disease. However, the pattern of hypercytokinemia in HPS has not been proposed. In this study, the quick determination of Th1/Th2 cytokines in the sera from patients with HPS was performed and the patterns of the cytokines were analyzed to seek for the possibility of using the cytokine pattern for the early diagnosis of HPS and its prognostic significance. Patients and Methods A total of 16 children with HPS based on the 2004 HPS criteria from June 2005 ∼ December 2006 were enrolled into this study. Peripheral blood samples were collected at the time of diagnosis, during the induction therapy and at the time of relapse. Sera from 21 healthy children, 17 ALL at remission and 17 with sepsis were used as controls. A cytometric bead assay (CBA) was used to quantitatively determine the Th1/Th2 cytokines including IL-2, IL-4, IL-6, IL-10, TNFα and IFNγ. Results Levels of IL-6 (22.20 (10.60–139.25) pg/ml), IL-10 (195.60(63.85–918.70)pg/ml) and IFNγ (4740.20(1054.72– 〉5000.00)pg/ml) from patients with HPS were significantly higher than those of normal control (3.35 (2.50–5.40) pg/ml, P 〈 0.05; 2.90(2.20–4.80)pg/ml, P100pg/ml and IFNγ concentration 〉 4000pg/ml were both unfavorable prognostic factors for HPS (IFNγ, P =0.034; IL-10, P=0.034). Positive correlations between the levels of IFNγ and IL-10(r=0.893, P

Description: Introduction and Objectives As a potent ligand for an immunotoxin, many moieties have been tried. However, majority of those moieties were large molecules or proteins which may produce human anti-toxin antibodies or were radioisotopes which may generate the concerns of the radioactive contaminations. Thus a small, non-protein toxin is desired to avoid the above disadvantages. Our hypothesis was that there could be a water soluble toxin in the saline extract of a Chinese medicine Mylabris phalerata Pallas (SEM) and easy to be conjugated with antibody proteins to generate immunotoxin. In this study, the toxin (s) in SEM was used to conjugate with our anti-CD19 monoclonal antibody ZCH-4-2E8 (2E8) protein and targeting efficacy on leukemia cells was tested to elucidate its leukemia-specific targeting efficacy and its mechanisms. Materials and Methods The physical and biological activities of SEM were tested by heat treatment, dialysis, SDS-PAGE and cell culture analyses. SEM was conjugated with 2E8 antibody using direct incubation at 37°C for 24 hours. Targeting efficacy were evaluated by cell culture. Cell apoptosis was analyzed by using Annexin V-FITC/Propidium Iodide double staining and flow cytometry method. Results Different concentrations of SEM at 1:200, 1:400, 1:800 and 1:1600 had different cell growth inhibition on both Nalm-6 and K562 cells non-specifically. The concentration at 1:200 generated the most potent cell kill and all the cells were dead after 72 hr culture. Concentrations below 1:800 or lower generated a minimal efficacy of cell kill and the concentration at 1:1600 showed no significant impact as compared to that of control (P 〉 0.05). Time course investigations showed that the growth inhibition was increased with incubation time. The IC50 for Nalm-6 cells was 1:805 while that for K562 was 1:689. The prudent toxin was heat stable as treatment of 100°C for 5 min did not diminish its cytotoxic activity. The toxin could be dialyzed through the 10kDa pore-size membrane. Electrophoresis analyses with SDS-PAGE showed no protein bands on the gel after staining with Coomassie brilliant blue. The natural conjugation could occur when CD19 antibody supernatant was incubated with the same volume of 1:200 SEM at 37°C for 24 hr. After 144 hr incubation, the inhibition rate of 2E8-SEM (1:2 of 2E8 supernatant with ≤ 1:400 of SEM) on Nalm-6 cells was 55.11% If normal saline was considered as negative control (0%), which was significantly higher than that of non-SEM conjugated 2E8 antibody at 1:2 of supernatant (16.01% of inhibition rate, P 〈 0.05). The inhibitions were not observed when 2E8-SEM (12.38% of inhibition, P 〉 0.05) and non-SEM conjugated 2E8 antibody (8.98% of inhibition, P 〉 0.05) were co-cultured with CD19- K562 cells at the same conditions as compared to that (0%) of normal saline. The cell death was via apoptotic process based on the flow cytometry analysis using Annexin V-FITC/Propidium Iodide double staining. Conclusions The strong and non-specific cytotoxic water soluble toxin(s) existed in SEM which was able to bind to antibody protein using simple incubation at 37°C showing significant targeting efficacy on Nalm-6 leukemia cells. The further study on this new toxin and the optimal way of conjugation are currently under investigation to improve the binding and the targeting efficacies.

Description: Objective: Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated to antibody proteins reported were toxic proteins which presented immunogenicity to patients capable of producing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule of toxin derived from a Chinese medicine Mylabris phalerata Pallas. The chemical name of this agent is 7-Oxabicyclo(2,2,1) heptane-2,3-dicarboxylic anhydride with a molecular weight of 168.15 CAS. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro in order to provide the experimental fundamentals for the further development of this new targeting agent. Methods: 2E8 monoclonal antibody was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) to CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometery. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, Norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm6 cells or K562 cells were made. Results: The purity of the purified 2E8 antibody was more than 99% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm6 cells, while only 0.98% of K562 cells were reacted with this antibody. The new generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and Norcantharidin were shown to have significant inhibitory effects on the growth of CD19+Nalm-6 cells after 96 h of culture with inhibitory rates of 71.25% and 97.62%, respectively (P 〈 0.001), while the purified 2E8 antibody (inhibitory rate of 30.29, P 〉 0.05) didn’t show any significant influences on the growth of Nalm6 cells as compared to that of negative control. No significant inhibitory effects were identified among immunotoxin 2E8-NCTD and 2E8 antibody as compared to that of control group on CD19-K562 cells with inhibitory rates of 15.41% and 4.17%, respectively, P 〉 0.05, indicating that a significant targeting effect of the 2E8-NCTD against Nalm6 cells was noticed. Conclusions: The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing efficacy on CD19+ Nalm-6 leukemia cells in vitro, which lays the experimental fundamentals for the further development of this new targeting agent.

Description: Acute promyelocytic leukemia is a unique type of disease for which the all-trans retinoic acid is selected as the first line of induction treatment. Conventional AML regimen containing anthracycline + Ara-C with or without etoposide is dangerous to the patients with APL for its induction of disseminated intravascular coagulation (DIC) and intracranial hemorrhage while otherwise those complications would not happen if all-trans RA were used for induction. Furthermore, morphological diagnosis is usually not always informative at the time of early stage when cytogenetic and fusion gene results are usually not available at the first few weeks. Therefore, rapid and confirmative diagnosis of APL is crucial to the initial treatment of choice. Objectives The aims of this study were to investigate the new immunophenotypic marker(s) of APL and the quick diagnostic assays. Patients and Methods 83 cases of patients (45 males and 38 females, age range of 5 – 52 yrs with a median of 24 yrs) with APL were enrolled into this study. Leukemia cells were analyzed by multi-parameter flow cytometry with CD45/SSC gating strategy and 10% or more positive cells were considered positive. Samples from 21 patients with CML, 29 patients with MDS and 11 normal BMs were used as controls. Results Unsurprisingly, myeloperoxidase (MPO), CD13, CD33 were the most frequent expressing markers for APL cells accounting for 100%, 96% and 99% of the APL cases, respectively, while CD34 and HLA-DR were negative in almost all cases of this disease, with the positive rates of only 3.6% and 2.4%, respectively for these two antigens. The phenotype of high SSC, MPO+CD33+CD13+CD34−DR- strongly suggested the diagnosis. Interestingly, 12 APL cases (14.5%) showed positive for CD56, a neurogenic adhesion molecule, which could be used as leukemia associated phenotype for minimal residual disease (MRD) detection. The stem cell factor (SCF) receptor antigen CD117 was expressed in 78% of APL cases while it was not expressed on CML, MDS and normal bone marrows. Only 17% of APL cases expressed CD11b, while 100% of normal bone marrow neutrophils were positive for this antigen. A multi-color flow cytometry analysis revealed that a phenotype of CD117+CD11b− represented 72% of APL cases while no such phenotype existed in CML(29 cases, P 〈 0.01), MDS (21 cases, P 〈 0.01) and normal bone marrows (11 cases, P 〈 0.01) indicating the diagnostic effect of this particular phenotype. Immunophenotypic analysis of mononuclear cells from the APL patients consecutively detected at 1, 2, 3 and 4 months after RA induction showed that a phenotype switch from CD117+CD11b− to CD117−CD11b+ were clearly observed, indicating the gradual differentiation phenomenon during the induction treatment. Co-existence of CD117 expression and PML-RARα fusion gene presented in 14/16 (87.5%) of long(L)-type cases detected while the co-existence of these two markers presented only in 4/9 cases with short(S)-type fusion gene (P = 0.058), indicating the significant tendency of this two markers. Conclusions Immunophenotypic analysis with FCM is able to rapidly diagnose the APL patients. High SSC, MPO+CD33+CD13+CD117+CD11b−CD34−DR- can be considered as the useful phenotype suggesting APL, which may provide a quick diagnosis of APL.