Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression

The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for α and β chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCR-transduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2′-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCR-based gene therapies targeting common epithelial malignancies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

VersusSNP:一种单碱基突变的筛选及分类工具

单碱基突变的筛选和分类是SNP分析的基础。为解决手工进行突变位点挖掘工作的困难,编写了VersusSNP软件。它可以解析并过滤序列比对结果,并根据突变类型将位点加以分类,以图形界面呈现给用户。使用VersusSNP,用户可以直观地了解基因组中单碱基突变的情况。其程序及源代码可以从http://sourceforge.net/projects/versussnp下载。     

A vertically stacked, polymer, microfluidic point mutation analyzer: rapid high accuracy detection of low-abundance K-ras mutations

Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1 h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-μl sample, equivalent to DNA from 42 mutant cells.     

成人弯刀综合症1例并相关文献复习

目的:探讨我院1例成人弯刀综合症患者的临床表现、诊断手段及治疗方法。方法:简要陈述我院1例成人弯刀综合症患者的入院基本情况及诊疗过程,调研并结合相关文献进行复习。结果:该患者结合临床表现,通过超声心动图、胸部后前位X片及右心导管造影等检查,结果证实该患者右下肺静脉畸形引流,诊断为弯刀综合症,并进行相关对症治疗及后续生活和治疗指导。结论:弯刀综合症为一种罕见而复杂的先天性心肺血管疾病,属于肺静脉畸形引流的一个类型(心下型),病因不明,此病在成人中更为少见,该病的诊断主要依赖于右心导管术和心血管造影以及一些影像学手段,成人型多以保守对症治疗为主,若出现严重症状者则选择外科手术纠正畸形引流治疗。     

Pluripotent stem cells: Maintenance of genetic and epigenetic stability and prospects of cell technologies

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources—inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia—as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.     

Epigenetic regulation of autophagy: A key modification in cancer cells and cancer stem cells

Aberrant epigenetic alterations play a decisive role in cancer initiation and propagation via the regulation of key tumor suppressor genes and oncogenes or by modulation of essential signaling pathways. Autophagy is a highly regulated mechanism required for the recycling and degradation of surplus and damaged cytoplasmic constituents in a lysosome dependent manner. In cancer, autophagy has a divergent role. For instance, autophagy elicits tumor promoting functions by facilitating metabolic adaption and plasticity in cancer stem cells (CSCs) and cancer cells. Moreover, autophagy exerts pro-survival mechanisms to these cancerous cells by influencing survival, dormancy, immunosurveillance, invasion, metastasis, and resistance to anti-cancer therapies. In addition, recent studies have demonstrated that various tumor suppressor genes and oncogenes involved in autophagy, are tightly regulated via different epigenetic modifications, such as DNA methylation, histone modifications and non-coding RNAs. The impact of epigenetic regulation of autophagy in cancer cells and CSCs is not well-understood. Therefore, uncovering the complex mechanism of epigenetic regulation of autophagy provides an opportunity to improve and discover novel cancer therapeutics. Subsequently, this would aid in improving clinical outcome for cancer patients. In this review, we provide a comprehensive overview of the existing knowledge available on epigenetic regulation of autophagy and its importance in the maintenance and homeostasis of CSCs and cancer cells.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors deve     

Structure-function analysis of the alpha5 and the alpha13 helices of human glucokinase: description of two novel activating mutations

It was recently described that the alpha5 and the alpha13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the alpha5 or the alpha13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.     

Differential epigenetic regulation of BDNF and NT-3 genes by trichostatin A and 5-aza-2′-deoxycytidine in Neuro-2a cells

To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I-IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB-II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells.     

A novel mechanism of phenotypic heterogeneity demonstrated by the effect of a polymorphism on a pathogenic mutation in the PRNP (prion protein gene)

Fatal familial insomnia (FFI) is a subacute dementing illness originally described in 1986. The phenotypic characteristics of this disease include progressive untreatable insomnia, dysautonomia, endocrine and motor disorders, preferential hypometabolism in the thalamus as determined by PET scanning, and selective thalamic atrophy. These characteristics readily distinguish FFI from other previously described neurodegenerative conditions. Recently, FFI was shown to be linked to a mutation in the prion protein gene (PRNP) at codon 178, which results in the substitution of asparagine for aspartic acid. As such, FFI represents the most recent addition to the growing family of prion protein-related diseases. The mutation that results in FFI had previously been linked to a subtype of familial Creutzfeld-Jakob disease (178^Asn CJD). The genotypic basis for the difference between FFI and 178^AsnCJD lies in a polymorphism at codon 129 of the mutant prion protein gene: 129^Met 178^Asn results in FFI, 129^Val 178^Asn in CJD. The finding that the combination of a polymorphism and a single pathogenic mutation result in two distinct conditions represents a singnificant advance in our understanding of phenotypic variability.