Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals

BACKGROUND/AIMS: Clearance of hepatitis B virus (HBV) is characterised by a strong cytotoxic T cell response. Persistence of HBV in chronic hepatitis B carriers may be related to failure of this response. The aim of this study was to determine whether HLA class I restricted cytotoxic T lymphocyte (CTL) responses persist in anti-hepatitis B e (HBe) positive / HBV DNA negative individuals, and to correlate the presence of viral CTL epitope mutation with clinical outcome. METHODS: An HLA/HBV dual transfectant model was used to demonstrate these CTL responses in individuals chronically infected with HBV. Subsequently, a known hepatitis B core (HBc) CTL epitope was sequenced in a family of five chronically infected individuals all sharing a HLA allele (HLA-A68.1). RESULTS: Low level HLA class I restricted cytotoxic T cell responses were detected in the peripheral blood of five of eight anti-HBe positive individuals. In the family of HLA-A68.1 positive chronically infected individuals, mutation of the HLA-A68.1 restricted hepatitis B core antigen (HBcAg) CTL epitope STLPETTVVRR was found in all four anti-HBe positive individuals but not in the sole hepatitis B e antigen (HBeAg) positive patient. CONCLUSION: These data are consistent with a continued immune selection pressure on HBV in anti-HBe positive chronically infected individuals with low replicating HBV infection and suggest that mutation of a CTL epitope may be a consequence of the immune response, as opposed to the cause of viral persistence.     

Disruption of actin‐binding domain‐containing Dystonin protein causes dystonia musculorum in mice

The Dystonin gene (Dst) is responsible for dystonia musculorum (dt), an inherited mouse model of hereditary neuropathy accompanied by progressive motor symptoms such as dystonia and cerebellar ataxia. Dst‐a isoforms, which contain actin‐binding domains, are predominantly expressed in the nervous system. Although sensory neuron degeneration in the peripheral nervous system during the early postnatal stage is a well‐recognised phenotype in dt, the histological characteristics and neuronal circuits in the central nervous system responsible for motor symptoms remain unclear. To analyse the causative neuronal networks and roles of Dst isoforms, we generated novel multipurpose Dst gene trap mice, in which actin‐binding domain‐containing isoforms are disrupted. Homozygous mice showed typical dt phenotypes with sensory degeneration and progressive motor symptoms. The gene trap allele (Dst^Gt) encodes a mutant Dystonin‐LacZ fusion protein, which is detectable by X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactoside) staining. We observed wide expression of the actin‐binding domain‐containing Dystonin isoforms in the central nervous system (CNS) and peripheral nervous system. This raised the possibility that not only secondary neuronal defects in the CNS subsequent to peripheral sensory degeneration but also cell‐autonomous defects in the CNS contribute to the motor symptoms. Expression analysis of immediate early genes revealed decreased neuronal activity in the cerebellar‐thalamo‐striatal pathway in the homozygous brain, implying the involvement of this pathway in the dt phenotype. These novel Dst^Gt mice showed that a loss‐of‐function mutation in the actin‐binding domain‐containing Dystonin isoforms led to typical dt phenotypes. Furthermore, this novel multipurpose Dst^Gt allele offers a unique tool for analysing the causative neuronal networks involved in the dt phenotype.     

Identification of prognostic markers in diffuse midline gliomas H3K27M‐mutant

Pediatric diffuse midline gliomas are devastating diseases. Among them, diffuse midline gliomas H3K27M‐mutant are associated with worse prognosis. However, recent studies have highlighted significant differences in clinical behavior and biological alterations within this specific subgroup. In this context, simple markers are needed to refine the prognosis of diffuse midline gliomas H3K27M‐mutant and guide the clinical management of patients. The aims of this study were (i) to describe the molecular, immunohistochemical and, especially, chromosomal features of a cohort of diffuse midline gliomas and (ii) to focus on H3K27M‐mutant tumors to identify new prognostic markers. Patients were retrospectively selected from 2001 to 2017. Tumor samples were analyzed by immunohistochemistry (including H3K27me3, EGFR, c‐MET and p53), next‐generation sequencing and comparative genomic hybridization array. Forty‐nine patients were included in the study. The median age at diagnosis was 9 years, and the median overall survival (OS) was 9.4 months. H3F3A or HIST1H3B mutations were identified in 80% of the samples. Within the H3K27M‐mutant tumors, PDGFRA amplification, loss of 17p and a complex chromosomal profile were significantly associated with worse survival. Three prognostic markers were identified in diffuse midline gliomas H3K27M‐mutant: PDGFRA amplification, loss of 17p and a complex chromosomal profile. These markers are easy to detect in daily practice and should be considered to refine the prognosis of this entity.     

Expression and prognostic significance of m6A‐related genes in TP53‐mutant non‐small‐cell lung cancer

BackgroundTP53 is an important tumor suppressor gene on human 17th chromosome with its mutations more than 60% in tumor cells. Lung cancer is the highest incidence malignancy in men around the world. N‐6 methylase (m6A) is an enzyme that plays an important role in mRNA splicing, translation, and stabilization. However, its role in TP53‐mutant non‐small‐cell lung cancer (NSCLC) remains unknown.MethodFirst, we investigated 17 common m6A regulators'' prognostic values in NSCLC. Then, after the establishment of risk signature, we explored the diagnostic value of m6A in TP53‐mutant NSCLC. Finally, gene set enrichment analysis (GSEA), gene ontology (GO) enrichment analysis, and differential expression analysis were used to reveal the possible mechanism of m6A regulators affecting TP53‐mutant NSCLC patients.ResultsStudy showed that nine m6A regulators (YTHDC2, METTL14, FTO, METTL16, YTHDF1, HNRNPA2B1, RBM15, KIAA1429, and WTAP) were expressed differently between TP53‐mutant and wild‐type NSCLC (p < 0.05); and ALKBH5 and HNRNPA2B1 were associated with the prognostic of TP53‐mutant patients. After construction of the risk signature combined ALKBH5 and HNRNPA2B1, we divided patients with TP53 mutations into high‐ and low‐risk groups, and there was a significant survival difference between two groups. Finally, 338 differentially expression genes (DEGs) were found between high‐ and low‐risk groups. GO enrichment analysis, PPI network, and GSEA enrichment analysis showed that m6A may affect the immune environment in extracellular and change the stability of mRNA.ConclusionIn conclusion, m6A regulators can be used as prognostic predictors in TP53‐mutant patients.     

耐利福平金黄色葡萄球菌突变株体内选择的临床研究

目的 对耐药突变选择窗（MSW）和金黄色葡萄球菌耐药突变株体内选择的关系进行初步研究。方法 选择前鼻腔携带金黄色葡萄球菌的结核患者为实验组,观察含利福平（RFP）联合化疗方案治疗前及治疗2、4、5周后金黄色葡萄球菌对RFP敏感性的变化,对获得性RFP耐药患者治疗前后分离的菌株进行分子分型。以前鼻腔携带金黄色葡萄球菌的糖尿病患者为对照组。结果 实验组58例患者治疗后有5例前鼻腔携带的金黄色葡萄球菌获得RFP耐药,脉冲场凝胶电泳和蛋白A分子分型结果表明这5例患者携带的菌株不同,但同一患者治疗前后分离的菌株同源。对照组39例患者中无耐药菌株出现。结论 耐利福平的金黄色葡萄球菌突变株的选择性富集是在MSW内完成的。     

Dissociation of ras oncogene-induced gene expression and anchorage-independent growth in a series of somatic cell mutants

The mechanism or mechanisms by which ras oncogenes induce morphological transformation and anchorage-independent growth are poorly understood but are thought to involve stable alterations in gene expression. We previously described a genetically dominant, mutant rat fibroblast cell line (ER-1-2) that is resistant to ras-induced anchorage-independent growth. We now describe a cell line derived from ER-1-2 cells, termed ER-1-2T, that has apparently sustained a second, dominant mutation that conferred on these cells the ability to form colonies in soft agar. Analysis of these and control cell lines demonstrated that deregulation of many of the genes commonly associated with the transformed phenotype could be dissociated from anchorage-independent growth. After infection with a ras-expressing retrovirus, both control and ER-1-2 cell lines constitutively expressed elevated levels of the c-jun, junB, fosB, c-myc, collagenase, ornithine decarboxylase, osteopontin, stromelysin, cathepsin L, and insulin-like growth factor 1 genes. These data indicate that signaling events downstream of ras were largely intact in ER-1-2 cells and that the defect in these cells lies either on a pathway separate from those that control stable, ras-mediated expression of these genes or at a point in the cell-division cycle distinct from those that control expression of the genes. In contrast, only c-jun, junB, c-myc, and ornithine decarboxylase were expressed at a significantly elevated level in ER-1-2T cells. Thus, deregulated expression of the genes analyzed was not sufficient for anchorage-independent growth. Furthermore, deregulation of most of them was also not necessary. © 1996 Wiley-Liss, Inc.     

A case of diffuse midline glioma,H3 K27M mutant mimicking a hemispheric malignant glioma in an elderly patient

Diffuse midline glioma, H3 K27M mutant arises from midline structures of the central nervous system and predominately affects pediatric patients. However, this disease entity was only recently established, and the clinical phenotypic spectrum remains largely unclear. We herein report a rare case of diffuse midline glioma, H3 K27M mutant with an unusual distribution in an elderly woman who presented with a diffuse glioma that invaded both sides of the thalami, and left hippocampus and frontoparietal lobes, thus mimicking a hemispheric malignant glioma. A biopsy of the lobular lesion led to a molecular diagnostic confirmation of diffuse midline glioma, H3 K27M mutant. The patient received concurrent bevacizumab and temozolomide therapy with radiation therapy and survived for 30 months. This case highlights the possibility that a glioma with cerebral hemispheric spread in an elderly patient may harbor the H3 K27M mutation.     

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM： To construct eukaryotic expression plasmids of full-length Hepatitis B Virus （HBV） genotype C genome, which contain lamivudine-resistant mutants （YIDD, YVDD） or wild-type strain （YMDD）, and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg）] of the recombinant plasmids in HepG2 cells. METHODS： Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 （＋）, between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.
RESULTS： Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.
CONCLUSION： Eukaryotic expression plasmids pcDNA3.1 （＋）-HBV/YIDD, pcDNA3.1 （＋）-HBV/YVDD or pcDNA3.1 （＋）-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.     

肿瘤坏死因子突变体对胃癌细胞肿瘤坏死因子受体的调节

本文采用放射配基结合分析法和蛋白合成抑制试验研究了肿瘤坏死因子突变体（ＴＮＦ－ｍ）对ＳＧＣ７９０１细胞ＴＮＦ受体的影响。结果表明，ＴＮＦ－ｍ可显著降低ＳＧＣ７９０１细胞表面ＴＮＦ受体数目并呈剂量、时间、温度依赖关系，对受体亲和力无影响，ＴＮＦ－ｍ可使胞浆ＴＮＦ受体数目增加，去除ＴＮＦ－ｍ３ｈ后，膜ＴＮＦ受体大约可恢复６０％，显著高于胰蛋白酶处理组ＴＮＦ受体的恢复率，放线菌素Ｄ对ＴＮＦ－ｍ处理的细胞ＴＮＦ受体的抑制作用显著低于对照组，且ＴＮＦ受体的半衰期约为９０ｍｉｎ．据此认为，ＴＮＦ－ｍ通过介导ＴＮＦ受体的内化从而使膜ＴＮＦ受体数降低。     

伤寒沙门菌CWDMs染色体的限制性内切酶谱分析

目的 了解伤寒沙门菌CWDMs染色体的特点，探讨限制性内切酶谱在伤寒沙门菌CWDMs检测的应用价值。方法 对伤寒秒门菌CWDMs染色体的特异性PCR扩增产物进行限制性内切酶谱分析。结果 限制性内切酶谱分析显示伤寒沙门菌CWDMs具有与其亲代细菌型相同的酶谱。结论 可用限制性内切酶谱分析对伤寒沙门菌CWDMs进行检测。