DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.     

Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.     

Helicobacter pylori-induced inflammation and epigenetic changes during gastric carcinogenesis

The sequence of events associated with the development of gastric cancer has been described as “the gastric precancerous cascade”. This cascade is a dynamic process that includes lesions, such as atrophic gastritis, intestinal metaplasia and dysplasia. According to this model, Helicobacter pylori (H. pylori) infection targets the normal gastric mucosa causing non-atrophic gastritis, an initiating lesion that can be cured by clearing H. pylori with antibiotics or that may then linger in the case of chronic infection and progress to atrophic gastritis. The presence of virulence factors in the infecting H. pylori drives the carcinogenesis process. Independent epidemiological and animal studies have confirmed the sequential progression of these precancerous lesions. Particularly long-term follow-up studies estimated a risk of 0.1% for atrophic gastritis/intestinal metaplasia and 6% in case of dysplasia for the long-term development of gastric cancer. With this in mind, a better understanding of the genetic and epigenetic changes associated with progression of the cascade is critical in determining the risk of gastric cancer associated with H. pylori infection. In this review, we will summarize some of the most relevant mechanisms and focus predominantly but not exclusively on the discussion of gene promoter methylation and miRNAs in this context.     

Effects of recombinant canine stem cell factor, a c-kit ligand, and recombinant granulocyte colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

The effects of recombinant canine stem cell factor (rcSCF) on hematopoiesis were studied in normal dogs and in dogs given otherwise lethal total body irradiation (TBI) without marrow transplant. Results were compared with previous and concurrent data with recombinant granulocyte colony-stimulating factor (rG-CSF). Four normal dogs received 200 micrograms rcSCF per kilogram body weight daily either by continuous intravenous infusion for 28 days (n = 2) or by subcutaneous (SC) injection in two divided doses for 20 days (n = 2). All dogs showed at least a twofold increase in peripheral blood neutrophil counts starting approximately 7 days after the initiation of treatment. Hematocrit level and monocyte, lymphocyte, eosinophil, reticulocyte, and platelet counts were not elevated. Marrow sections after rcSCF treatment showed panhyperplasia. The only toxicity was facial edema during the first few days of rcSCF administration, presumably caused by mast cell stimulation. Ten dogs were given 400 cGy TBI at 10 cGy/min from two opposing 60Co sources. They were given no marrow infusion and received 200 micrograms/kg/d rcSCF SC in two divided doses for 21 days starting within 2 hours of TBI. Five of the 10 dogs showed complete and sustained hematopoietic recovery and survived as compared with 1 of 28 control dogs not receiving growth factor (P < .005). RcSCF treatment allowed for hematopoietic recovery in two of seven dogs administered 500 cGy of TBI but in none of five dogs given 600 cGy of TBI. Results with rcSCF are similar to those obtained with rG-CSF. The rate of neutrophil recovery in rcSCF-treated dogs after 400 cGy TBI was not different from that of rG-CSF-treated dogs (P = .65), but the rate of platelet recovery was faster (P = .06) in the rcSCF-treated animals. Combined treatment with rcSCF and rcG-CSF after 500 cGy TBI did not result in strongly improved survival as compared with results obtained with either factor alone.     

Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)     

两种荧光显微成像系统亚细胞定位的对比

目的:应用高分辨率荧光显微成像系统采集细胞器探针图像,并与激光共聚焦显微成像系统进行对比。方法:实验于2003-05/2004-01在解放军总医院完成。①实验材料:鼠肺毛细血管内皮细胞株(1H11)由上海复旦张江生物公司提供;荧光探针Rhodamine-123,Lucifer Yellow,DiOC6[3],BODIPY(美国Sigma公司)。②细胞培养及荧光探针染色:细胞培养采用含体积分数为0.2胎牛血清的低糖DMEM培养基,密度5×107L-1。选择Rhodamine-123作为细胞线粒体特异性荧光探针,选择DiOC6[3]作为细胞内质网特异性荧光探针,选择BODIPY作为细胞高尔基体特异性荧光探针,选择Lucifer Yellow作为细胞溶酶体探针。前3个探针在完全避光条件下与培养的细胞共同孵育0.5h,后者则共同孵育15h。③高分辨率荧光成像系统的图像采集:线粒体荧光图像采集,选取经Rhodamine-123共孵育完成的细胞,选择激发滤色镜为BP460-490,吸收滤色镜为BA515,分光镜为DM500,另加一绿通道液晶滤光片,激发出Rhodamine-123的荧光。电荷耦合器件采集图像并送入计算机。重复上述步骤,采用DiOC6[3]标记内质网,BODIPY标记高尔基体,Lucifer Yellow标记细胞溶酶体,激发条件同Rhodamine-123。分别采集同一视野靶细胞DiOC6[3]、BODIPY或Lucifer Yellow的荧光图像,完成全部图像采集并储存在计算机中。④激光共聚焦显微成像系统的图像采集:选择经4种探针染色的靶细胞,使用氩离子激光器在488nm激发Rhodamine-123,Rhodamine-123荧光通过配置有530/60-G发射滤光片的通道1探测。重复上述步骤,在488nm激发DiOC6[3]和BODIPY,在457nm激发Lucifer yellow,3种荧光均由通道1探测,后2个探针的发射滤光片的配置为515/30-G,DiOC6[3]选择530/60-G。由光电倍增管接收信号并传输入计算机成像。结果:①高分辨率荧光成像系统所采集图像,靶细胞中由荧光探针Rhodamine-123染色的线粒体呈多个典型的小棒状或卵圆状,聚集在核周;Lucifer yellow染色的溶酶体呈多个非对称球型,在胞浆内随机分布,颗粒尺寸通常大于线粒体;荧光探针DiOC6[3]着色的内质网占据胞浆的很大空间,以囊状聚集为特征;BODIPY特异性地结合在高尔基体上,荧光图像显示围绕在细胞核周围呈条索状。②与高分辨率荧光成像系统比较,激光共聚焦显微成像系统所采集的图像其荧光强度基本相同,但分辨率低、细节显示模糊、胞浆中细胞器的准确分布信息和形态特征显示效果欠佳。结论:两种荧光显微成像系统均可采集到细胞器探针的荧光图像。但高分辨率荧光成像系统采集的荧光图像具有细节清晰、分辨度高、准确显示胞浆中细胞器的分布信息和形态特征等优点。     

Steuerliche Einordnung der wirtschaftlichen Vorteile aus einer Vertragsarztzulassung

Der wirtschaftliche Vorteil einer Vertragsarztzulassung stellt kein gesondert zu bewertendes Wirtschaftsgut dar, sondern einen wertbildenden Faktor des Wirtschaftsguts “Praxiswert” im Rahmen des Gesamtkaufpreises zum Erwerb der Vertragsarztpraxis.