Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR⁺γδ T cells were expanded on CD19⁺ artificial antigen-presenting cells (aAPC), which resulted in >10⁹ CAR⁺γδ T cells from <10⁶ total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR⁺γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19⁺ tumor cell lines compared with CAR^negγδ T cells. CD19⁺ leukemia xenografts in mice were reduced with CAR⁺γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.     

Tinidazole in treatment of acute amoebic dysentery in children

The excellent results obtained in this trial indicate that tinidazole is a drug worthy of extensive evaluation in the treatment of amoebiasis, as three single daily doses is a simple form of treatment. The drug was well tolerated and free from any toxic effects.     

Treatment of respiratory failure in an infant with bronchopulmonary dysplasia infected with respiratory syncytial virus using inhaled nitric oxide and high frequency ventilation

A 2-month-old, former 28-week premature infant with brochopulmonary dysplasia infected with respiratory syncytial virus was treated with nitric oxide and high frequency oscillatory ventilation after conventional therapy failed. Nitric oxide and high frequency oscillatory ventilation rapidly improved oxygenation allowing recovery without the need for extracorporeal membrane oxygenation. This treatment regimen should be considered as an option in high-risk infants with respiratory syncytial virus infection who meet extracorporeal membrane oxygenation criteria.     

Haplotype and interspersion analysis of the FMR1 CGG repeat identifies two different mutational pathways for the origin of the fragile X syndrome

To understand the origins of the fragile X syndrome and factors predisposing alleles to instability and hyperexpansion, we have compared the haplotype (using markers FRAXAC1, FRAXAC2, and DXS548) and AGG interspersion patterns of the FMR1 CGG repeat for 214 normal and 16 premutation chromosomes. Association testing between interspersion pattern and haplotype reveals a highly significant (P < 0.002) non- random distribution, indicating that all three markers are useful in phylogenetic reconstruction of mutational change. Parsimony analysis of the FMR1 CGG repeat substructure predicts that loss of AGG interruptions has occurred independently on many haplotypes associated with the fragile X syndrome, partially explaining the haplotype diversity of this disease. Among haplotypes found in linkage disequilibrium with the fragile X mutation, two different modes of mutation and predisposition to instability have been identified. One pathway has involved the frequent and recurrent loss of AGG interruptions from rare asymmetrical ancestral array structures. Intergenerational transmission studies suggest that these predisposed chromosomes progress relatively rapidly to the disease state. In contrast, the second mutational pathway involves a single haplotype which has maintained two AGG interruptions. Parsimony analysis of CGG repeat substructure within this haplotype suggests that larger alleles have been generated by gradual increments of CGG repeats distal to the most 3' interruption. Pedigree analysis of the intergenerational stability of alleles of this haplotype confirms a gradual progression toward instability thresholds. As a result, a large reservoir of chromosomes carrying large repeats on this haplotype exists. These chromosomes are predisposed to disease. The present data support a model in which there are at least two different mutational pathways predisposing alleles to instability and hyperexpansion associated with the fragile X syndrome.      

Apoptosis in the early human placental bed and its discrimination from necrosis using the in-situ DNA ligation technique

Extensive areas of necrosis are present in the early human placental bed. Our aim was to determine whether apoptosis is also a feature. A method was therefore required to differentiate unequivocally necrosis and apoptosis. Initially, terminal deoxynucleotide transferase-mediated dUTP nick-end labelling was used to visualize apoptotic cells. However, non-specific labelling, probably due to free DNA released by necrotic cells, was excessive; thus, in-situ DNA ligation was employed. In this technique, two DNA fragments with single-base 3' overhangs and blunt- ends were labelled with a fluorochrome and then ligated to the DNA breaks on the sections. Immunolabelling for cytokeratin or leukocyte common antigen was performed to determine the phenotype of apoptotic cells identified by the in-situ DNA ligation technique. A proportion of the dying cells was confirmed to be trophoblasts. No co-localization with leukocyte common antigen was found in this region, suggesting that maternal macrophages and natural killer cells (CD56+) were not dying by apoptosis in significant numbers. In conclusion, in-situ DNA ligation in association with immunocytochemistry can readily distinguish apoptosis from necrosis in the placental bed. The results suggest that a proportion of invading trophoblast cells are eliminated by apoptosis in early pregnancy.      

人心脏型脂肪酸结合蛋白的纯化与鉴定

目的:以往的心脏型脂肪酸结合蛋白多来自牛、鼠等动物心肌组织,本实验构建了人左心室心肌组织纯化心脏型脂肪酸结合蛋白的新方法。方法:实验于2005-01/03在南京医科大学第一附属医院心血管病研究所完成。①实验材料:人心室肌由南京医科大学第一附属医院心血管病研究所提供。②实验过程:取20g人左心室肌在缓冲液中匀浆,多步骤离心后以(NH4)2SO4进行盐析→再次离心→沉淀缓冲液重悬透析得蛋白粗提液→Sephadex G-75凝胶过滤→电泳确定主要蛋白条带的收集管号合并收集→QSepharose Fast Flow阴离子交换层析→聚乙二醇浓缩→缓冲液透析得人心脏型脂肪酸结合蛋白。③实验评估:以SIGMA公司人心脏型脂肪酸结合蛋白为对照标准品;以Tricine-SDS-PAGE电泳鉴定相对分子质量;以鼠抗人脂肪酸结合蛋白单克隆抗体及羊抗鼠IgG对其进行Western印迹检测鉴定其特异性。结果:①Sephadex G-75凝胶过滤柱层析结果:各组分经Tricine-SDS-PAGE电泳检测证实,部分收集管中存在相对分子质量约14400的人心脏型脂肪酸结合蛋白。②阴离子交换柱层析结果:在洗脱液NaCl浓度达6mmol/L时,出现蛋白峰,人心脏型脂肪酸结合蛋白被洗脱;NaCl浓度达20mmol/L时,蛋白峰降至基线,蛋白洗脱结束。③纯化的人心脏型脂肪酸结合蛋白鉴定结果:经电泳显示其相对分子质量约为14000,纯度约为90%;Western印迹证实其可被抗人脂肪酸结合蛋白单克隆抗体特异识别,与对照标准品SIGMA公司人心脏型脂肪酸结合蛋白结果一致。实验从20g湿重心室肌中共纯化得到15.4mg心脏型脂肪酸结合蛋白,得率为0.77mg/g。结论:Sephadex G-75凝胶过滤柱层析及阴离子交换柱层析相结合,可用于人心脏型脂肪酸结合蛋白的纯化。