Renierosides, cerebrosides from a marine sponge Haliclona (Reniera) sp

Guided by the brine shrimp lethality assay, eight new cerebrosides (1-8) have been isolated from an extract of the marine sponge Haliclona (Reniera) sp. A novel feature of these cerebrosides was the presence of unprecedented amide-linked long-chain fatty acid moieties. The planar structures of the cerebrosides (1-8) were established by 1D and 2D NMR spectroscopic techniques, mass spectrometric analyses, and chemical degradation methods. The isolated compounds did not display cytotoxicity to a panel of five human solid tumor cell lines.     

Autopsy case of desminopathy involving skeletal and cardiac muscle

Desminopathy is a familial or sporadic skeletal and cardiac muscular dystrophy caused by mutation in the desmin gene. Desmin-reactive deposits in the affected muscles are the morphological hallmarks of this disease. Herein is reported an autopsy case of a 57-year-old Japanese man with adult-onset skeletal muscle weakness and atrioventricular (A-V) conducting block, with a missense A337P mutation in exon 5 of the desmin gene. Disease onset occurred when the patient was 45 years old. The initial presentation was lower limb weakness, and the weakness progressed to the upper limbs. When the patient was 51 years old, a cardiac pacemaker was implanted due to complete A-V block. When the patient was 53 years old, respiratory insufficiency occurred due to weakness of respiratory muscles, and the patient died at the age of 57 years. On autopsy, intrasarcoplasmic desmin-immunoreactive deposits were identified in the skeletal and cardiac muscle, and abnormal accumulations of granulofilamentous material were identified at the ultrastructural level. In the cardiac conducting system, calcification was observed at the bundle of His, and sporadic calcium deposits were observed at the left and right bundle branches.     

改性羟基磷灰石骨修复纳米复合材料的制备及生物学评价

目的:制备羟基磷灰石/聚乳酸聚乙醇酸骨修复材料,并对其进行生物学评价。方法:实验于2006-06/2007-02在中科院长春应用化学研究所完成材料制备,在吉林大学基础医学院实验动物中心完成动物实验。将低聚乳酸的羧基与羟基磷灰石表面的钙原子用化学键连接,得到表面接枝聚左旋乳酸的羟基磷灰石,将其与聚乳酸聚乙醇酸共混,得到复合材料PLLA-g-HA/PLGA。溶于氯仿后铺膜(厚0.2mm),用DMEM培养液浸泡材料膜制备浸提液。首先,进行材料生物安全性实验:①细胞毒性实验:将浸提液与培养液混合,接种兔成骨细胞,培养24h,MTT法检测细胞增殖,计算细胞增殖率和细胞毒性级(细胞毒性级0或1级为合格)。②全身毒性实验:小鼠以50mL/kg的剂量静脉注射浸提液,观察72h内小鼠中毒症状。③皮肤刺激实验:兔脊柱两侧皮内注射材料浸提液,观察72h内皮肤有无异常反应。④热原实验:自兔耳缘静脉注入浸提液(10mL/kg)。注射后每0.5h测肛温1次,共6次,以6次中最高的1次减去正常体温,计为升高度数。其次,对复合材料进行细胞黏附性检测:将复合材料制成1%氯仿溶液,涂于硅化的盖玻片上,置于6孔板,每孔接种1×105个成骨细胞,培养3d,在2,24,72h行FITC荧光染色,数码摄像系统拍摄细胞荧光照片。结果:制备了新型PLLA-g-HA/PLGA复合材料。①生物安全性实验结果:MTT实验检测复合材料细胞增殖率为94.8%,细胞毒性级为1级;全身毒性实验中动物无死亡、惊厥、瘫痪、呼吸抑制、腹泻和体质量下降等不良反应;热原实验中兔体温最大的变化值是0.25℃(国家标准为<0.6℃);皮肤刺激实验中未见任何刺激反应,无红斑、焦痂、水肿表现。②细胞黏附性实验结果:细胞接种后2h可见少量细胞开始贴壁;24h时可见贴壁细胞明显增多,并呈聚集生长;培养3d后可见细胞逐渐融合,细胞状态良好。结论:新型PLLA-g-HA/PLGA复合材料符合生物材料细胞毒性要求,按毒性剂量分级属无毒级,无致热原性、对皮肤无刺激作用,具有良好的生物相容性和细胞黏附性。     

The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active "phospholipid replacing" activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.     

Drug-induced thrombocytopenia is associated with increased binding of IgG to platelets both in vivo and in vitro

Thrombocytopenia is a common serious adverse effect of drug treatment. A variety of in vitro diagnostic techniques to confirm the diagnosis are available, but the majority lack sufficient sensitivity to detect all cases of drug-induced thrombocytopenia. We studied 19 patients with suspected drug-induced thrombocytopenia and demonstrated that platelet- associated IgG (PAIgG) was elevated in all at the time of thrombocytopenia, and PAIgG returned to normal levels as the thrombocytopenia resolved. In the majority of patients, the platelet count rapidly returned to normal after the drug was discontinued; however, in six patients, the thrombocytopenia persisted well beyond the period of time that the offending drug would be expected to be cleared from the blood. In 13 patients, serum obtained after recovery was used to identify the drug responsible for the thrombocytopenia in an in vitro assay. In all cases, the addition of the drug historically associated with the thrombocytopenic episode was associated with an increased binding of IgG to control platelets. For uncertain reasons, the concentration of drug required to increase the in vitro binding of IgG to test platelets was often more than the concentration usually achieved in vivo. Wider application of these techniques may provide better understanding of the clinical characteristics and mechanisms responsible for drug-induce thrombocytopenia.     

Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins

The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non–small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1^G2032R mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.Receptor tyrosine kinases (RTKs) are critical mediators of extracellular signals that control key cell growth, survival, and motility pathways. Conversely, deregulated and constitutive RTK activation is responsible for the initiation and progression of many cancers. Multiple mechanisms contribute to aberrant RTK activation including chromosomal rearrangements, point mutations, and gene amplification. Oncogenic activation of the orphan RTK c-ros oncogene 1 (ROS1) is observed in a subset of patients with glioblastoma, non–small-cell lung cancer (NSCLC), and cholangiocarcinoma (1–5). In most cases, ROS1 signaling is activated by interchromosomal translocation or intrachromosomal deletion that results in N-terminal ROS1 fusion genes. Several ROS1 kinase fusion proteins have been identified, including the Fused in Glioblastoma–ROS1 (FIG–ROS) that was first discovered in a human glioblastoma cell line (2) and more recently in patients with NSCLC (4), cholangiocarcinoma (3), and serous ovarian carcinoma (6). The SLC34A2–ROS1 (SLC–ROS) fusion is present in a subset of patients with NSCLC (1, 7) and gastric cancer (8). Other ROS1 fusions include CD74–ROS1, EZR–ROS1, LRIG3–ROS1, SDC4–ROS1, and TPM3–ROS1 (5).Given the recent success of molecularly targeted therapies in treating cancers driven by oncogenic kinases, there is acute clinical momentum to identify inhibitors that selectively target ROS1 fusions. Because the ROS1 and Anaplastic Lymphoma Kinase (ALK) domains are partially homologous, the Food and Drug Administration (FDA)-approved ALK/MET kinase inhibitor crizotinib is being investigated via phase I/II clinical trials for its efficacy in ROS1-driven lung cancer patients (9). Although early results appear promising, consistent with the clinical experience of crizotinib in patients with ALK-positive lung cancer to date, as well as prior experience with kinase inhibitors in many other malignancies (10–13), recent evidence suggests that a subset of patients with crizotinib-treated ROS1 fusion-positive may acquire ROS1 kinase domain mutations that confer drug resistance, thus necessitating alternative therapeutic approaches.To identify additional and potentially more efficacious ROS1 inhibitors, we used an unbiased, high-throughput kinase inhibitor screening assay and discovered that foretinib (GSK1363089) and Gö6976 are potent inhibitors of ROS1. Foretinib selectively suppresses the growth of the SLC–ROS-driven human NSCLC cell line HCC78 and of FIG–ROS-driven murine cholangiocarcinoma, but not of EGFR-driven NSCLC or phosphatase and tensin homolog (PTEN)-suppressed murine cholangiocarcinoma cells. Further, treatment of tumor-bearing mice with foretinib resulted in specific and dramatic regression of FIG–ROS-driven tumors in contrast to non-FIG–ROS tumors that share similar histopathological features. Importantly, we also use a cell-based in vitro resistance screen to preemptively identify several ROS1 kinase domain point mutations that confer resistance to crizotinib and show that these crizotinib-resistant ROS1 mutants remain sensitive to foretinib. These data suggest that foretinib may provide an alternative front-line treatment for ROS1-positive tumors and an effective second-line approach for patients that develop crizotinib-resistant disease.     

Immune hemolytic anemia associated with tolmetin and suprofen

This article reports the first case of immune hemolytic anemia possibly associated with the ingestion of suprofen. The patient suffered from massive hemoglobinuria and acute renal failure. Serologic studies of the patient's serum revealed suprofen-dependent red cell antibodies. However, tolmetin-dependent antibodies were also found in the serum, showing the same properties as the suprofen antibodies and an even higher titer. The patient not only had drug-dependent antibodies in the serum, but also had developed autoantibodies, a phenomenon that has been described for several other drugs. The working mechanism by which suprofen and tolmetin caused immune hemolysis had properties of both the immune complex model and the induction of autoimmunity. Although it was unclear whether the immune hemolytic anemia was the result of suprofen, tolmetin, or cross-reacting antibodies, we feel that suprofen should be added to the list of nonsteroidal anti-inflammatory drugs associated with a positive direct antiglobulin test.