Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

Abstract Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.     

An inducible caspase 9 safety switch can halt cell therapy-induced autoimmune disease

Transfer of either allogeneic or genetically modified T cells as a therapy for malignancies can be accompanied by T cell-mediated tissue destruction. The introduction of an efficient "safety switch" can potentially be used to control the survival of adoptively transferred cell populations and as such reduce the risk of severe graft-vs-host disease. In this study, we have tested the value of an inducible caspase 9-based safety switch to halt an ongoing immune attack in a murine model for cell therapy-induced type I diabetes. The data obtained in this model indicate that self-reactive T cells expressing this conditional safety switch show unimpaired lymphopenia- and vaccine-induced proliferation and effector function in vivo, but can be specifically and rapidly eliminated upon triggering. These data provide strong support for the evaluation of this conditional safety switch in clinical trials of adoptive cell therapy.     

地表火对红花尔基沙地樟子松种群空间分布格局的影响

利用全林木定位的方法, 对地表火干扰1年后的樟子松(Pinus sylvestris L. var. mongolica Litv.)林进行调查, 并通过假设检验和成对相关函数对其林火及林分结构特征和空间格局进行分析。结果表明, 林火强度相似的同一场地表火干扰下, 不同林分的密度均大大降低, 胸高断面积仅略有下降, 林分结构特征则有趋同的态势。不同林分的空间格局也有相似的变化趋势, 烧死木均表现为显著的双尺度聚集分布及显著的正相关, 活立木也表现出显著的正相关; 地表火干扰前后, 樟子松林的空间格局均为显著的聚集分布, 但地表火干扰后其聚集分布的尺度范围变小; 存活林木中, 大树和幼树则呈现出相互独立或略微排斥的关系。显然, 地表火驱动下, 不同樟子松林的空间格局呈现出相似的变化趋势, 并推动其向着成熟林方向演替, 这对天然樟子松林的资源保护和经营管理有着重要意义。     

Inheritance of inflorescence architecture in sorghum

The grass inflorescence is the primary food source for humanity, and has been repeatedly shaped by human selection during the domestication of different cereal crops. Of all major cultivated cereals, sorghum [Sorghum bicolor (L.) Moench] shows the most striking variation in inflorescence architecture traits such as branch number and branch length, but the genetic basis of this variation is little understood. To study the inheritance of inflorescence architecture in sorghum, 119 recombinant inbred lines from an elite by exotic cross were grown in three environments and measured for 15 traits, including primary, secondary, and tertiary inflorescence branching. Eight characterized genes that are known to control inflorescence architecture in maize (Zea mays L.) and other grasses were mapped in sorghum. Two of these candidate genes, Dw3 and the sorghum ortholog of ramosa2, co-localized precisely with QTL of large effect for relevant traits. These results demonstrate the feasibility of using genomic and mutant resources from maize and rice (Oryza sativa L.) to investigate the inheritance of complex traits in related cereals.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Prevalence and identity of Cryptosporidium spp. in pig slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America.

Analysis of Fusarium head blight (FHB) pathogen diversity revealed that 3ADON producing Fusarium graminearum are prevalent in North America and identified significant population structure associated with trichothecene chemotype differences (F(ST)>0.285; P<0.001). In addition, we identified a trichothecene chemotype cline in Canada and documented a recent and significant shift in FHB pathogen composition by demonstrating that the 3ADON chemotype frequency in western Canada increased more than 14-fold between 1998 and 2004. On average, isolates from 3ADON populations produced significantly (P<0.05) more trichothecene and had significantly (P<0.005) higher fecundity and growth rates than isolates from the 15ADON population. These results indicate that selection is driving the rapid spread of an introduced pathogen population that is more toxigenic and potentially more vigorous. The discovery of this previously unrecognized pathogen diversity has significant implications for food safety and cereal production in North America.     

人体动脉血气信号波浪式变化及连续动脉逐搏取血血气分析方法的建立*

目的: 建立动脉逐搏取血血气分析法,在人体实验中验证呼吸调控核心信号——PaO₂,PaCO₂和[H⁺]a是受呼吸影响的周期性、波浪式变化信号,而不是传统理念上误认的稳定水平信号。方法: 选择心功能正常、Allen试验阴性需要监测动脉血流动力学变化的患者6例。在左侧桡动脉穿刺,连接肝素化塑化管（3 mm×1 000 mm）,注满血液并计数血液注满所需心跳次数。用止血钳将塑化管钳闭成与心跳次数相对应的分段后,迅速置于冰水中,立即进行血气分析。选取每位患者的2个典型波浪式周期,用于分析2对最高-最低和最低-最高共4个测定值,取平均值。对相邻最高和最低值作统计学配对t检验。结果: 血液注满塑化管需要16±2次心跳,均覆盖超过2个呼吸周期。每个呼吸周期是5±0.6次心跳。PaO₂、PaCO₂、[H⁺]a和SaO₂都呈现出明显的波浪式变化（相邻高点与低点比较,P<0.05）,PaO₂、PaCO₂、[H⁺]a和SaO₂的波浪幅度分别是（11.28±1.13）mmHg,（1.77±0.89）mmHg,（1.14±0.35）nmol/L和（0.52±0.44）%;波浪幅度分别是其平均值的（7.7±1.1）%,（5.1±2.5）%,（3.1±1.0）%和（0.5±0.4）%。结论: 动脉延长管连续取血,按心跳次数分隔血样,血气分析法简单易行,为验证动脉血气受呼吸影响的周期性波浪式信号提供了可靠证据。本方法为原创,技术操作层面仍需提高熟练程度,增加志愿者和试验样本的数量进一步探索此类信号的临床检测可靠性及其与临床疾病的关系。