Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells

A number of targeted cytotoxic agents have been developed that selectively kill malignant or otherwise pathological cells. These engineered proteins consist of a potent cytotoxic element connected to a ligand domain that binds to specific molecules on the surface of the target cell. Several of these agents have shown promise in clinical trials and one is currently administered to patients. A significant technical obstacle that has impeded the development of some of these toxins is the difficulty of preparing certain recombinant proteins in properly folded forms. These fusion proteins have generally been produced in bacteria requiring them to be denatured and renatured in vitro. For some proteins this is an efficient process whereas for others it is not. We describe here a system to produce fusion toxins rapidly and efficiently by engineering mammalian cells to secrete them as properly folded molecules which can be purified in native form from cell culture medium. We have used this system to produce highly active preparations of DAB(389)-IL7, a molecule consisting of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7. This system is generalizable and can be used to produce and evaluate rapidly fusion toxins incorporating novel or uncharacterized ligands.     

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.     

铁死亡的机制及其在淋巴瘤中的研究进展

淋巴瘤是常见的血液系统恶性肿瘤，对人类健康造成极大的威胁。目前淋巴瘤的治疗方法包括化疗、分子靶向治疗、造血干细胞移植等，但耐药和复发难治仍是待解决的问题。铁死亡是新发现的一种细胞死亡模式，与铁依赖性的脂质过氧化损伤相关。靶向铁死亡为抑制淋巴瘤进展提供了新思路。本文从铁死亡的起始信号、中间事件、效应阶段、防御机制来阐述铁死亡的机制并回顾了铁死亡在淋巴瘤中的研究进展，为铁死亡在淋巴瘤治疗方法的应用方面提供新的思路。     

Targeting the DNA Damage Response to Increase Anthracycline-Based Chemotherapy Cytotoxicity in T-Cell Lymphoma

Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients’ outcomes.     

Histamine H4 Receptor Agonism Induces Antitumor Effects in Human T-Cell Lymphoma

The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 μM) in combination with histamine (10 μM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.     

Btk/JAK3双靶点共价抑制剂的设计、合成及其活性研究

布鲁顿式酪氨酸激酶(Btk)和两面神激酶3(JAK3)均是自身免疫性疾病和血液系统恶性肿瘤的潜力靶标.以4-氯吡咯并嘧啶为原料,经7步反应合成了一系列7H-吡咯并[2,3-d]嘧啶-4-胺衍生物,其结构经1HNMR、13CNMR和MS(ESI)分析证实.体外依次考察了所得化合物分别对Btk和JAK3激酶的活性、以及对部...     

Self‐Assembled Aptamer‐Nanomedicine for Targeted Chemotherapy and Gene Therapy

Chemotherapy is the mainstream treatment of anaplastic large cell lymphoma (ALCL). However, chemotherapy can cause severe adverse effects in patients because it is not ALCL‐specific. In this study, a multifunctional aptamer‐nanomedicine (Apt‐NMed) achieving targeted chemotherapy and gene therapy of ALCL is developed. Apt‐NMed is formulated by self‐assembly of synthetic oligonucleotides containing CD30‐specific aptamer and anaplastic lymphoma kinase (ALK)‐specific siRNA followed by self‐loading of the chemotherapeutic drug doxorubicin (DOX). Apt‐NMed exhibits a well‐defined nanostructure (diameter 59 mm) and stability in human serum. Under aptamer guidance, Apt‐NMed specifically binds and internalizes targeted ALCL cells. Intracellular delivery of Apt‐NMed triggers rapid DOX release for targeted ALCL chemotherapy and intracellular delivery of the ALK‐specific siRNA induced ALK oncogene silencing, resulting in combined therapeutic effects. Animal model studies reveal that upon systemic administration, Apt‐NMed specifically targets and selectively accumulates in ALCL tumor site, but does not react with off‐target tumors in the same xenograft mouse. Importantly, Apt‐NMed not only induces significantly higher inhibition in ALCL tumor growth, but also causes fewer or no side effects in treated mice compared to free DOX. Moreover, Apt‐NMed treatment markedly improves the survival rate of treated mice, opening a new avenue for precision treatment of ALCL.     

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X-ray microanalysis

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X-ray microanalysis. We use Burkitt's lymphoma cells in culture as the test sample and compare X-ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak-to-local continuum X-ray intensity ratio by electron probe X-ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X-ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm³) and cytology. We air dry cells on thin film supports and show by magnesium X-ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X-ray microanalysis can determine the variation in magnesium concentration among individual cells.