Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.     

Hemopoietic Recovery and Infectious Complications in Breast Cancer and Multiple Myeloma after Autologous CD34<Superscript>+</Superscript> Cell-Selected Peripheral Blood Progenitor Cell Transplantation

Autografting with CD34+ cell-selected peripheral blood progenitor cells (PBPC) is often associated with a prolonged recovery time and a higher incidence of infections. The aim of our study was to evaluate whether underlying disease influences hemopoietic recovery and the infectious complications occurring after transplantation. We studied 19 breast cancer (BC) patients and 17 multiple myeloma (MM) patients entered in a high-dose chemotherapy (HDC) program of tandem autografting with CD34+ cell-selected PBPC. PBPC were collected after mobilizing chemotherapy plus granulocyte colony-stimulating factor and were processed for selection of CD34+ cells. After selection, a median of 53% CD34+ cells was recovered with a median final purity of 92% with no significant differences between the MM (52% and 92%, respectively) and BC (53% and 89%, respectively) patients. Medians of 4.5 x 10(6)/kg CD34+ cells (BC, 4.4 x 10(6)/kg; MM, 5.4 x 10(6)/kg) and 18 x 10(4)/kg colony-forming units-granulocyte-macrophage (BC, 21 x 10(4)/kg: MM, 16 x 10(4)/kg) were reinfused after each HDC. Twenty-six patients (10 MM and 16 BC) underwent tandem autografting, and 10 patients received only 1 autograft because of inadequate collection (5 patients), clinical condition (3 patients), and refusal (2 patients). In the BC patients, the HDC regimen included a high-dose melphalan course followed by an ICE (ifosfamide, carboplatin, and etoposide) course. In the MM patients, the regimen consisted of a course of high-dose melphalan therapy and a course of ICBV (idarubicin, cyclophosphamide [Cytoxan], BCNU, and etoposide) or total body irradiation, etoposide, and Cytoxan. We found a significantly prolonged time for neutrophil recovery to > 500/microL in the MM patients (13 days versus 10 days; P < .002), whereas the times for platelet recovery to > 20,000/microL in the two groups were not different (13 days versus 12 days; not significant). No late engraftment failures and no toxic deaths were observed. The incidences of extrahematologic toxicity were similar for the two patient groups. All patients received similar anti-infection prophylaxis for 3 months after transplantation. After 12 months of observation, we found a statistically significant higher incidence of bacterial infections in MM patients in both the early (77.8% versus 48.6%; P < .034) and the late (41.1% versus 0%; P < .014) posttransplantation periods, whereas the incidences of fungal infections were similar in the two groups. Viral infections consisted of herpes zoster virus infection in 2 patients of each group, and cytomegalovirus infection was observed in 3 MM patients and no BC patients. Our experience demonstrates a prolonged neutrophil recovery time and higher incidences of bacterial and viral infections in MM patients compared with BC patients. These observations, although limited by the small sample size, suggest that the underlying disease may influence the incidence of infections after CD34- cell-selected transplantation and should be considered in the planning of appropriate antimicrobial prophylaxis in the autologous transplantation setting.     

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.     

Decreased treatment failure in recipients of HLA-identical bone marrow or peripheral blood stem cell transplants with high CD34 cell doses

We studied the association between CD34 cell dose and transplant outcomes in 359 bone marrow (BM) and 511 peripheral blood stem cell (PBSC) transplant recipients from human leucocyte antigen (HLA)-identical siblings, reported to the International Bone Marrow Transplant Registry (IBMTR). Transplants for leukaemia were performed between 1995 and 1998. Patients were divided into those receiving below or above the median CD34+ dose, for BM (3 x 106/kg) and PBSC (6 x 106/kg) grafts respectively. Cox proportional hazards regression was used to adjust for baseline patient-, disease- and transplant-related characteristics. Analysis of the BM recipients showed that high CD34 cell dose was associated with lower transplant-related mortality [relative risk (RR) = 0.60, P = 0.033] and treatment failure (inverse of leukaemia-free survival, RR = 0.69, P = 0.032). Among PBSC recipients, high CD34 dose was associated with faster recovery of neutrophils to > 0.5 x 109/l (RR = 1.38, P < 0.001) and platelets to > 20 x 109/l (RR = 1.34, P = 0.003), lower risk of relapse (RR = 0.62, P = 0.029) and treatment failure (RR = 0.74, P = 0.03). We conclude that higher CD34 cell doses decrease treatment failure in recipients of HLA-identical sibling BM and PBSC transplants.     

Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry

ABSTRACT— To clarify the relationship between angiogenesis and hepatocarcinogenesis on progression of hepatocellular carcinoma (HCC), we quantitatively evaluated angiogenesis by CD34 immunohistochemistry in liver cirrhosis (LC), adenomatous hyperplasia (AH), and HCC, and proliferative activity estimated by Ki-67 immunohistochemistry. Angiogenesis was evaluated by CD34 immunohistochemistry using monoclonal antibody HPCA-2, and tumor proliferative activity was evaluated using monoclonal antibody MIB-1. We used an image analysis system to assess the microvessel density as the area percentage of the endothelial area. Angiogenesis was generally observed in HCC and there was no significant difference among all clinical stages and histological grades of HCC. On the other hand, the staining of CD34 was partly observed in sinusoids of AH, although no positive staining was seen in any sinusoids of LC. The proliferative activity was significantly correlated with the clinical stage and histological grade of HCC. Our results indicate that the quantitation of angiogenesis does not provide significant prognostic information in HCC, but that it may have diagnostic value in distinguishing HCC from non-HCC. Meanwhile, AH, which is not morphologically diagnosed as cancer, shows positive staining for CD34, suggesting that some portion of AH contains cancerous characteristics.     

11p15 duplication and 13q34 deletion with Beckwith–Wiedemann syndrome and factor VII deficiency

Here we report a patient with 11p15.4p15.5 duplication and 13q34 deletion presenting with Beckwith–Wiedemann syndrome (BWS) and moderate deficiency of factor VII (FVII). The duplication was initially diagnosed on methylation‐sensitive multiplex ligation‐dependent probe amplification. Array comparative genome hybridization confirmed its presence and indicated a 13q34 distal deletion. The patient's clinical symptoms, including developmental delay and facial dysmorphism, were typical of BWS with paternal 11p15 trisomy. Partial 13q monosomy in this patient is associated with moderate deficiency of FVII and may also overlap with a few symptoms of paternal 11p15 trisomy such as developmental delay and some facial features. To our knowledge this is the first report of 11p15.4p15.5 duplication associated with deletion of 13q34 and FVII deficiency. Moreover, this report emphasizes the importance of detailed clinical as well as molecular examinations in patients with BWS features and developmental delay.     

Flow cytometry for CD34 determination in hematopoietic grafts

CD34 is a type I transmembrane protein that is expressed on lympho-hematopoietic progenitor and endothelial cells and has a potential adhesion function. Various monoclonal antibodies, whether characterized by glycosylated epitopes or not, are utilized to recognize different subsets of hematopoietic progenitors. Coexpression of membrane markers involving CD34 is an approach to the definition of those subpopulations of cells previously characterized by using in vitro cultures. Thus, in the autologous transplantation procedure, flow cytometry determination of CD34+ cells in the grafts themselves, especially as concerns cytapheresis products, was enhanced with CFU-GM enumeration. This methodology required a standardized protocol with regard to the choice of the monoclonal antibody, had to be to data acquisition and to computer analysis. Within the framework of a multicentric trial, various strategies had to be evaluated. Special attention was paid to obtaining a sensitivity level of 0.1 %. New, standardized approaches are currently in the planning stage, in particular with a view to determining absolute count on the basis of readings generated by the flow-cytometer.     

Electrophysiologic residua and sequelae of surgery for congenital heart defects

Postoperative arrhythmias may occur in any patient who undergoes intracardiac surgery for a congenital heart defect. The correction of certain intracardiac heart defects predisposes to a large incidence of cardiac arrhythmias. Ventricular arrhythmias and conduction disturbances are seen after correction of tetralogy of Fallot, ventricular septal defect and atrioventricular canal defect. Supraventricular arrhythmias and sinus nodal dysfunction may be seen after surgery for transposition of the great arteries or atrial septal defect. The identification, evaluation and treatment of these patients are discussed.     

Significance of increasing adhesion of cord blood hematopoietic cells and a new method: platelet microparticles

Hematopoietic recovery after transplantation with cord blood (CB) is slower than with bone marrow (BM) and mobilized peripheral blood. Adhesion molecules (AMs) on hematopoietic cells are involved in hematopoietic cells' homing. It may be possible to enhance CB CD34+ cells engraftment by increasing their expressions of AM. Twenty-three patients with childhood acute leukemia treated with unrelated CBT were studied. It was found that the time to neutrophil recovery correlated with CXCR4 and the time to platelet recovery correlated with both CD62L and CXCR4. Platelet microparticles (PMPs) carry some AMs such as aIIb b (CD41), P-selectin (CD62P), and CXCR4, CD34+ cells express platelet-binding antigens (CD162 and CD11b). It was found that AMs were increased dramatically on CD34+ cells surface in the presence of PMPs, and CD34+ cells covered with PMPs adhered better to human umbilical vein endothelial cells and fibronectin. These findings suggested that PMPs could increase adhesion of donor's cells to host BM in CBT.     

miR-34a通过靶向抗凋亡基因Survivin抑制皮肤鳞状细胞癌增殖的研究

[摘要]　目的　研究miR-34a在皮肤鳞状细胞癌(SCC)中的表达及对肿瘤细胞增殖、抗凋亡的调控作用及机制。　方法　采用实时定量PCR检测46例SCC病人肿瘤组织及15例正常皮肤组织中miR-34a及Survivin的表达;将miR-34a mimic转染进皮肤鳞状细胞癌A431细胞系,探讨miR-34a对SCC细胞增殖、Survivin表达的影响;评估miR-34a及Survivin表达对SCC预后的生物诊断价值。 　结果　　（1）低分化SCC患者miR-34a显著低于高分化组,而Survivin则显著高于高分化组（P均<0.05）。与正常皮肤比较,SCC病人的miR-34a显著降低,而Survivin表达则显著提高,P值分别为0.0013及<0.0001,差异均有极显著性意义。（2）与未转染组相比,miR-34a mimic可以显著抑制A431细胞增殖（P<0.05）;同时显著降低Survivin蛋白表达（P<0.01）。（3）高miR-34a表达的SCC患者生存率显著高于低表达者,P=0.020567;高Survivin表达的SCC患者生存率则显著低于高表达者,P=0.008198。　结论　　miR-34a可通过对Survivin表达调控影响SCC细胞增殖,同时它也可作为一个很好的生物学诊断指标为评估SCC病人预后提供参考。