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Objective To investigate the clinical efficacy of different treatment in 35 chronic lymphocytic leukemia (CLL) patients. Methods Patients were treated with different regimen according to Binet stage. Patients at stage A were subcutaneously injected with interferon (3-6) MU/day, consecutive for 5 days every week. The dosage could be reduced to 2-3 times a week in long term maintenance phase after the 6 months loading treatment if there was no disease progression. Those at stage B or C were initially treated with chemotherapy regimen of FC/FC -R or CHOP/COP, and interferon were administered during chemotherapy interval, after complete remission (CR) or partially remission (PR) as maintenance therapy.Results Twenty patients were at stage A and treated with interferon, with 5 patients(25%) achieving partial remission (PR), 14 patients at stable status while no patients acquiring CR. Three of the 5 patients who achieved PR collapsed after 36.3 months at average. Eight of the 14 patients at stable status deteriorated to stage B and received chemotherapy after mean 74 months interferon maintenance treatment. In total, 27 patients in the current observation were finally included at stage B or C. Patients at stage B or C in FC/FC-R chemotherapy regimen achieved CR at 38.9% and total effective rate 77.8%, which were superior to that of CHOP/COP prescription (CR 11.1 %). The mean survival time for patients at stage A, B and C were 155.2,97.5 and 82.9 months, respectively and were statistically significant via Kaplan-Meier analysis method (P =0.032). Ten patients died in this observation, 2 at stage A, 4 at stage B and C, respectively, among whom 9died of infection and 1 for gastric cancer bleeding. The side effects of interferon were generally mild during the long term treatment. Conclusion Patients with CLL need to be individually treated with different regimen by considering disease stage and other prognosis criteria. Interferon could be applied at early phase of CLL and may reduce occurrence of infection after long term treatment.
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BACKGROUND: Cord blood stem cells are one of ideal target cells for gene therapy, but low gene transferring rate is the main difficulty at recent. Janus kinase tyrosine 2 (JAK2) plays an important role in self-renewing of cord blood stem/progenitor cell12s. Therefore, cord blood CD34+ cell line modified by target-amplified JAK2 genes has been developed yet by using gene regulating expression technique in order to overcome low transferring rate of cord blood genes.OBJECTIVE: To investigate the feasibility and reliability of a long-term amplified regulation for cord blood stem/progenitor cells mediated by transgene JAK2. SETTING: Department of Hematology, Beijing Hospital, Ministry of Health.MATERIALS: The experiment was carried out in the Laboratory of Hematological Department, Beijing Hospital, Ministry of Health from June 2003 to April 2006. Cord blood was derived from umbilical cord which was immediately cut from healthy, full-term and natural-parturition infants and was provided by Department of Obstetrics & Gynecology, Beijing Hospital. The experiment was approved by the local ethical committee, and informed consent was obtained from expectant mothers and their relatives for the use of cord blood cells. MiniMACS magnetic separation apparatus and immunomagnetic beads adsorbing CD34 single antibody were provided by Miltenyi Biotec Company, Germany; flow cytometer by FACScalibur, USA; recombinant human stem cell factor (rhSCF), Flt3 ligand (FL), human interleukin-6 (hIL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and thrombopoeitin (TPO) by PeproTec Company; nude mice of the SPF level by Animal Center of Beijing Medical University.METHODS: Retroviral vector MGI-F2JAK2, which was composed of functional catalytic domain of JAK2 genes and two site proteins (2xF36v, F2) combined with synthetic drug (AP20187) of target gene of small molecules, was constructed. AP20187 might specially combine with F36v to cause dimerization of JAK2 so as to activate signal conduction in cells. In addition, the vector included green fluorescence protein reporter gene, which was regarded as a label to detect proliferation. MiniMACS magnetic separation apparatus was used to purify and separate cord blood CD34+ cells. While, retrovirus supernatant including JAK2 was used to transfer cord blood CD34+ cells. After transduction, CD34+ cells were cultured with stem cell factor (SCF), Flt3 ligand, TPO and IL-6 and divided into control group (not adding AP20187) and experimental group (AP20187).MAIN OUTCOME MEASURES: ① Flow cytometer was used to detect percentage of green fluorescence protein reporter gene in the CD34+ cells and to determine gene transferring rate. ② Colony culture results of cord blood stem/progenitor cells after amplification. ③ Nude mice were given subcutaneous injection of ten-week cultured cord blood CD34+ cells at costa and neoplasia was observed after 30 days. RESULTS: ① Plentiful amplification of CD34+ cells was observed in both experimental group and control group. With the culture time passing by, positive rate of gel-filtered platelet of amplified CD34+ cells in the experimental group was gradually increased based on the basic level and more than 95% in the 11th week; however, positive rate of green fluorescence protein reporter gene in the control group was gradually decreased below the basic level and disappeared finally. ② Transgenic CD34+ cells in the experimental group still could generate brust forming unit-erythroid (BFU-E), colony-forming units granulocute/monocyte (CFU-GM) and multipotential hematopoietic progenitors (CFU-Mix); especially, CFU-GM was the main cell in hemopoietic progenitor cell (HPC). ③ Nude mice did not have neoplasia. CONCLUSION: Human cord blood CD34+ cells of transferring JAK2 genes may cooperate with other cytokines to amplify cord blood stem/progenitor cells in vitro for long. Therefore, this is potentially valuable for stem cells to treat some hereditary hematologic disease.
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Objective To explore the clinical characteristics,therapy reactions and prognosis of the elderly patients with idiopathic thrombocytopenic purpura(ITP). Methods A total of 43elderly ITP patients(age≥60 years old)including 16 men and 27 women were reviewed and further followed up for 1 month to 15 years. Results Until June 2007,35 elderly ITP patients survived,platelet counts were sustained(30-50)×109/L in 7 cases,but no significant bleeding was found.Thirty-six patients had adrenocorticosteroid therapy first, 25 patients were sensitive to adrenocorticosteroid therapy,4 patients underwent splenectomy,and 3 patients achieved a normal platelet count. Immunosuppressive agents(vinscristine,cyclophosphamide, azathioprine and Cyclosporin A)treatments were held in 5 6 case-times,Cyclosporin A and azathioprine were more effective than vinscristine and cyclophosphamide.The refractory rate was 13.9%.One patient progressed to monoclonal gammopathy of unknown significance and 1 to lymphoma.Eight patients died.1 patient died of brain bleeding after trauma,3 patients died of malignant neoplasm,4 patients died of heart failure induced by infection. Conclusions The clinical features of elderly ITP patients are atypical.the mortal bleeding in them was rare,treatment strategy should be individualized tO each elderly patient.
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AIM:To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34+ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer, dimerization (AP20187), was cloned (designated MGI-F2JAK2). CD34+cells were enriched from cord blood with a MiniMACS system. The purified CD34+cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2. Following transduction, cells were expanded into four groups: AP20187 alone, FL alone, TPO, alone, AP20187+FL+TPO, respectively. The expanded cells were monitored by GFP expression, immunophenotyping, progenitor colony assay, karyotype analysis as well as tumorigenesis in nude mice. RESULTS: The purity of selected CD34+ cells was over 91% and gene transfer rate was 49.32%?6.21%. Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34+ cord blood cells. The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture. Flow cytometry analysis showed that the phenotype of the expanded cells was CD33+, CD61+ and Gly-A+ partial positive; CD38+ and HLA-DR+ strong positive, while CD2, CD7 and CD19 were almost negative. Colony assays performed in methycelluos, which can give rise to BFU-E, CFU-GM and CFU-Mix, the CFU-GM was predominantly in all colonies. The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34+ cells in combination with FL and TPO. This system may have applications for studies in signaling transduction, hematopoiesis, and for gene and cell therapy.