Colonic delivery of dexamethasone from a prodrug accelerates healing of colitis in rats without adrenal suppression

Dexamethasone-β-d-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohn's colitis. The aim of this study was to evaluate colonic delivery and efficacy of this prodrug in the rat. Distribution of dexamethasone in luminal contents and tissues of the gastrointestinal tract and in plasma was measured after oral administration of dexamethasone-β-d-glucuronide or free dexamethasone. Efficacy of the prodrug and free drug was tested in an acetic acid-induced rat colitis model. Healing of induced colitis was assessed by measuring net intestinal fluid absorption, colonic surface area of ulceration, histology, and myeloperoxidase activity. Glucocorticosteroid toxicity was evaluated with serum corticosterone and plasma adrenocorticotropic hormone levels. The drug delivery index (a measure of relative targeting efficiency) was 6.7 and 8.6 in the cecal and colonic mucosa, respectively. The prodrug was significantly more potent than free drug in improving net colonic fluid absorption while significantly reducing surface area of ulceration and histological grade in colitic rats. Treatment with free dexamethasone significantly reduced serum corticosterone levels to subnormal levels, and treatment with the prodrug maintained serum corticosterone and plasma adrenocorticotropic hormone levels near control levels. The prodrug dexamethasone-β-d-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.     

Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation

In the current study, we tested whether higher numbers of hematopoietic stem cells correlate with the speed of immune reconstitution in a congenic transplantation model (C57BL/Ka, CD45.1, Thy1.1-->C57BL/6, CD45.2, Thy1.2) using purified hematopoietic stem cells (c-Kit(+)Thy1.1(low)Lin(-/low)Sca-1(+)). There were 3 different doses of stem cells used (400, 1000, and 5000). Phenotypic analyses in peripheral blood and spleen demonstrated that higher numbers of infused stem cells are associated with more rapid regeneration of T cells (CD4(+), CD8(+), naive CD4(+), naive CD8(+)) and B cells at early time points. The numbers of T and B cells eventually became equivalent between different dose groups at late time points. Production of interleukin-2 and inter-feron-gamma per T cell was similar regardless of stem cell dose even when tested at the time when there were significant differences in peripheral T-cell counts. The improved immune recovery was attributed to a more rapid regeneration of donor-type immune cells. Higher numbers of total thymocytes and signal joint T-cell receptor excision circles were observed in the higher dose stem cell recipients, suggesting that accelerated regeneration of T cells was due to enhanced thymopoiesis.     

拉米夫定治疗慢性乙型肝炎中变异病毒株的动态变化

目的:建立定量检测及监测乙肝病毒DNA的rtM204I/V(YMDD)及rtL180M变异的方法.方法:以克隆、测序鉴定及构建乙肝病毒DNA YMDD及变异(YIDD和YVDD)质粒作为标准,4例接受拉米夫定治疗(100 mg/d,疗程48 wk以上)的慢性乙型肝炎患者血清标本为对象,用焦磷酸测序的方法检测变异位点核苷酸的频率,并计算变异株的含量比例,通过检测不同变异株类型的标准质粒,确定测序峰图的背景信号.结果:通过检测标准质粒后,确定背景信号为5%,变异位点调整后的核苷酸频率转化为变异病毒株的比例后,4例患者治疗前均检测出变异的病毒株(4.5%-33%),并且随治疗时间延长呈增多的趋势.病毒载量及ALT分析提示基因型耐药发生后,将发生病毒学反跳及临床耐药.结论:焦磷酸测序可以定量检测及动态监测变异病毒株的含量.拉米夫定耐药突变株在拉米夫定治疗前已存在,并随治疗时间的增加而增长,出现病毒学反弹及临床耐药.     

抗巨噬细胞表面分子Mac-1单克隆抗体对利什曼原虫入侵巨噬细胞的抑制作用

应用抗巨噬细胞表面分子Mac-1单克隆抗体（M1／70和M18／2）处理巨噬细胞，观察M1／70和M18／2对杜氏利曼原虫前鞭毛体入侵巨噬细胞的抑制作用。结果，经上述单抗处理后巨噬细胞，对杜氏利什曼原虫的易感性明显降低，其原虫感染率和受染巨噬细胞内入侵的原虫数量减低，原虫对巨噬细胞的入侵过程及速度也减慢。M1／70和M18／2两种单抗同时应用，则对原虫侵入巨噬细胞的抑制作用更为显著，巨噬细胞受染率为13．8％，且受染巨噬细胞内入侵的原虫数量大多仅有1～2个。提示，M1／70和M18／2单克隆抗体可以通过与巨噬细胞表面Mac－1的结合，干扰巨噬细胞表面分子上与利什曼原虫相结合的连接位点，抑制利什曼原虫对巨噬细胞的入侵。     

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.New therapeutic strategies are needed to treat complex human diseases. Because disease phenotypes are often regulated by interwoven genetic networks, exploiting combination therapy to target multiple pathways, as opposed to only single ones, can enhance treatment efficacy (1). However, discovering effective combination therapies for human diseases is challenging with existing methods, due to the cost, effort, and labor required to construct and analyze each combination (2). For example, the National Cancer Institute tested ∼5,000 pairwise combinations of 100 cancer drugs against the NCI-60 panel in a study that took 2 y and cost about USD $4 million (3). Thus, there is a need for technological advances to accelerate the identification of effective combinatorial therapies. Here, we used our combinatorial genetics en masse (CombiGEM)-CRISPR platform to perform rapid pooled screening of pairwise genetic knockouts against genes coding for epigenetic regulators and then translated our screen hits into drug combinations against human ovarian cancer cells.CRISPR-Cas9 technology has been used for large-scale genetic perturbation screens with single-guide RNA (sgRNA) libraries for gene knockouts (4–7), repression, and activation (8, 9). Despite its simplicity for multiplexed genetic perturbations (10–12), new methods are needed to enable high-throughput CRISPR-Cas9–based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13, 14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays. This strategy, termed CombiGEM-CRISPR, uses one-pot cloning steps to enable the assembly of combinatorial gRNA libraries, thus simplifying and accelerating the workflow toward systematic analysis of combinatorial gene functions.     

昆山市学龄期儿童学习困难影响因素研究

目的 分析儿童学习困难的影响因素,为提高儿童的学习状况提供有效的依据。方法 采用整群随机抽样的方法,对在江苏省昆山市抽取的5 132名6~12岁儿童(其父母、老师等)进行学习困难及其影响因素的问卷调查。结果 本研究调查发现昆山市儿童学习困难发生率为16.29%。多因素Logistic回归分析发现:年龄每增加1岁,学习困难的发生风险降低18%;女性与男性相比,学习困难发生风险相对较低,OR(95%CI)值为0.56(0.42~0.76);与主要照顾者为母亲的相比,主要照顾者为祖辈或其他可增加儿童学习困难发生风险,OR(95%CI)值为1.73(1.23~2.43);与民主的教养方式相比,保护式的教养方式可以增加儿童学习困难的发生风险,OR(95%CI)值为1.65(1.12~2.41);与家庭和睦的相比,不断争吵的家庭可以增加儿童学习困难的发生风险,其OR(95%CI)值为2.59(1.07~6.25)。结论 昆山市儿童学习困难的发生率仍较高,应从学校、家庭、主要照顾者等多个方面来提高儿童的学习状况。     

人工流产患者心理状态的护理干预及避孕宣教

目的：分析不同年龄层次人工流产孕妇的心理状态,进行有针对性的心理护理干预及避孕宣教,从而预防人工流产并发症的发生、降低人工流产率。方法：对本院2013年1月-2014年1月收治的1200例孕妇进行术前心理状态分析,并进行术前、术中、术后护理干预及术后中医艾灸应用和避孕宣教,统计护理干预结果。结果：干预后,1200例患者中,人工流产综合症72例（6.0%）,子宫穿孔6例（0.5%）,人流不全12例（1.0%）,感染4例（0.3%）。其中初产妇52例（4.3%）,未婚24例（2.0%）,经产妇18例（1.5%）。人工流产的并发症发生率为7.8%,明显低于国内报道13.5%（P〈0.05）。干预前后,患者的心理状态评分的比较差异有统计学意义（P〈0.05）。结论：有效的心理护理可以减少人工流产并发症的发生率,提高孕妇心理应激能力;另外,避孕宣教可降低人工流产率及未婚妊娠的发生率,这些措施均值得在临床中推广应用。     

两种敷料对层流手术间动态下空气质量的影响

目的研究无纺布手术衣及敷料与棉织布手术衣及敷料对层流手术室动态下空气质量的影响,为提高层流手术间动态空气质量措施提供科学依据。方法选取医院2013年2-10月骨科全髋关节置换手术患者100例,随机分成A、B两组,每组各50例,A组用无纺布手术衣及敷料,B组用棉织布手术衣及敷料;采用沉降法和浮游法对两组手术间动态空气质量进行监测,并将监测结果进行比较。结果沉降法监测中A、B两组手术间平均空气沉降菌菌落数整体比较差异有统计学意义(t=3.12,P<0.01),在不同时点比较中,麻醉前A、B两组手术间空气沉降菌菌落数比较差异无统计学意义,在切皮前、手术中、缝合前及手术结束后各时点两组比较差异均有统计学意义(P<0.05);浮游法监测中A、B两组手术间平均空气浮游菌菌落数整体比较差异有统计学意义(t=3.78,P<0.01),在不同时点比较中,麻醉前A、B两组手术间空气浮游菌菌落数比较差异无统计学意义,在切皮前、手术中、缝合前、手术结束后各时点两组比较差异均有统计学意义(P<0.05)。结论使用无纺布手术衣及敷料的手术其手术间动态空气质量明显优于使用传统棉织布手术衣及敷料。