Ultra-rapid cardiotoxicity of the hepatitis C virus protease inhibitor BILN 2061 in the urokinase-type plasminogen activator mouse

BACKGROUND & AIMS: Because current therapies for chronic hepatitis C virus (HCV) infections are suboptimal and associated with severe side effects, novel treatment options are needed. A small animal model has recently been developed to study HCV infections. To examine the usefulness of this human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mouse for the development of HCV-targeted drugs, we evaluated the antiviral efficacy and safety of an HCV NS3-protease inhibitor, BILN 2061. METHODS: BILN 2061 was orally administered at clinical range doses for 4 days to SCID mice that differed in the presence of HCV infection, human hepatocyte grafts, and uPA zygosity. Treatment outcome was evaluated clinically, virologically, and morphologically. Using standard high-performance liquid chromatography-ultraviolet (HPLC-UV) methods and mass spectrometry, single-dose pharmacokinetics and multiple-dose drug exposures were analyzed. The (13)C-aminopyrine breath test was applied to compare in vivo liver function. RESULTS: A 4-day treatment with BILN 2061 of HCV genotype-1b infected chimeric animals reduced the viral load by >100-fold, but concomitant clinical and ultrastructural signs of cardiotoxicity appeared. BILN 2061 administration to uPA-transgenic mice induced mitochondrial swelling with aberrant cristae in cardiomyocytes, but not in skeletal muscle. Because both drug accumulation and liver function were identical in affected uPA-transgenic and nontransgenic SCID mice without cardiac involvement, the urokinase plasminogen activator transgene itself appears to be implicated. CONCLUSIONS: The human liver-uPA(+/+)SCID mouse is an interesting small animal model to evaluate the preclinical safety and efficacy of new antiviral compounds against HCV. The uPA-transgene increases the susceptibility of mice to BILN 2061-induced cardiotoxicity.     

Inhibitory effects of chitooligosaccharides on tumor growth and metastasis

Chitooligosaccharides (COS) are hydrolyzed products of chitosan and have been proven to exhibit various biological functions. The objectives of this study were to evaluate the anti-tumor growth, anti-metastatic potency and related pathways of COS extracted from fungi. In in vitro studies, we found that COS significantly inhibited human hepatocellular carcinoma (HepG2) cell proliferation, reduced the percentage of S-phase and decreased DNA synthesis rate in COS-treated HepG2 cells. Expressions of cell cycle-related genes were analyzed and the results indicated that p21 was up-regulated, while PCNA, cyclin A and cdk-2 were down-regulated. Moreover, we also found that the activity of metastatic related protein (MMP-9) could be inhibited by COS in Lewis lung carcinoma (LLC) cells. In in vivo studies, we found that COS inhibited the tumor growth of HepG2 xenografts in severe combined immune deficient (SCID) mice. In a LLC-bearing mouse tumor growth and lung metastasis model, COS inhibited tumor growth and the number of lung colonies in LLC-bearing mice as well as the lung metastasis, and it prolonged the survival time of the LLC-mice. These results suggest a potential anti-tumor growth and anti-metastatic potency of COS in cancer chemoprevention.     

Inhibition of a secondary human alloimmune response via the soluble active component of CD154 (CD40L) in severe combined immune-deficient mice engrafted with human lymphocytes

BACKGROUND: Alloimmunization requires a process known as co-stimulation. An important co-stimulatory pathway for most immune responses is mediated by the interaction of CD40 on antigen-presenting cells with CD154 (CD40L) on host T cells. Blockade of this co-stimulatory pathway simultaneous with exposure to challenge with HLA-incompatible cells is hypothesized to inhibit alloimmunization. STUDY DESIGN AND METHODS: Severe combined immune-deficient (SCID) mice were reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID mice) from a subject primed to HLA antigens and challenged with HLA-incompatible lymphocytes. Mice were challenged in the presence or absence of an 18-kDa soluble recombinant active form of human CD154 (18-kDa CD154). Human IgG production, alloimmunization, and in vitro T-cell responsiveness were assessed. RESULTS: There was no significant effect of 18-kDa CD154 on human IgG levels in these mice, but it inhibited the development of HLA-specific alloantibody in this model to five subsequent untreated white cell challenges. In vitro T-cell proliferation in a mixed lymphocyte culture was also prevented by 18-kDa CD154. CONCLUSION: The recombinant protein 18-kDa CD154 inhibited the ability of the Hu-PBL-SCID mice to mount a secondary immune response to allostimulation. This implies that transfusion-induced alloimmunization utilizes CD40-CD154 co-stimulation and that blockade of this pathway can inhibit T-cell function and interfere with the development of alloimmunization.     

A human anti-dsDNA monoclonal antibody caused hyaline thrombi formation in kidneys of 'leaky' SCID mice.

There are few studies assessing the pathogenicity of human monoclonal anti-DNA antibodies. The use of SCID mice avoids the problem of rejection of the human hybridoma cells thus allowing in vivo assessment of human immunoglobulins. Using electron microscopy we have shown that the human IgG anti-dsDNA monoclonal antibody, RH14, is nephritogenic in SCID mice, causing morphological changes in the kidney due to immunoglobulin deposition. The problem with using SCID mice is that they have an abnormal immune system; normally they are used at about 2 months of age, at which time they have virtually no functional T or B cells. It is known that older SCID mice become increasingly 'leaky', that is they develop some mature lymphocyte clones. Our aim was to assess if implanting anti-DNA antibodies into older 'leaky' SCID mice would result in pathology which was observable by light microscopy. Eight-month-old SCID mice were implanted with human hybridoma cells secreting either RH14 an anti-dsDNA IgG, CL24, an antiphospholipid antibody or an irrelevant human IgG control. As previously, RH14 deposited in the kidney and caused proteinuria but unexpectedly we also observed hyaline thrombi in the kidney glomeruli and peritubular capillaries. These thrombi occurred only in the case of RH14 implanted mice and were found to stain positively for human IgG and fibrin. However, apart from the interesting thrombi, we did not observe any greater pathological damage resulting from the anti-dsDNA antibody deposition than we had seen in the younger mice; indeed, the electron microscopic findings were more limited.     

Mutation analysis should be performed to rule out gammac deficiency in children with functional severe combined immune deficiency despite apparently normal immunologic tests

To study the correlation between genotype and phenotype in x-linked SCID, we have characterized the presentation of 2 unrelated patients. Both had infections suggestive of immunodeficiency, but their immune function and lymphoid tissues were normal. They were found to have an identical R222C mutation in the gammac gene.     

人卵巢癌重症联合免疫缺陷小鼠腹腔移植瘤模型的建立及其生物学研究

目的建立人卵巢癌重症联合免疫缺陷小鼠(SCID鼠)移植瘤模型,为研究人腹水型卵巢癌的实验研究提供合适的动物模型。方法将2例卵巢癌患者手术切除标本移植于SCID鼠腹腔,原代移植瘤形成后进行鼠间传代,观察移植瘤的生长、转移及腹水形成情况,做腹水细胞学计数、涂片,检测荷瘤鼠血中肿瘤标记物CA125的浓度,肿瘤作病理学检查。结果成功建立3只原代人卵巢癌SCID鼠移植瘤模型,原代移植成功率为37.5%,自第四代后,移植瘤的传代成功率为100%,随着传代次数的增加,移植瘤的成瘤潜伏期及荷瘤鼠的生存期逐渐缩短,荷瘤鼠血中肿瘤标记物的浓度升高,病理组织学证实:移植瘤保持了原卵巢癌生物学特征。结论成功建立了人卵巢癌SCID鼠移植瘤模型,移植瘤保持了原卵巢癌生物学特征,是研究人卵巢癌较理想的动物模型。     

人正常子宫颈移行上皮重症联合免疫缺陷小鼠模型的建立

目的探讨人正常子宫颈移行上皮重症联合免疫缺陷小鼠(SCID鼠)模型建立的可行性及其生物学特性。方法获取正常子宫颈组织的移行带标本,接种于12只SCID鼠皮下,随机分成3组,分别在2、4、6周取出其皮下移植物行HE染色、免疫组化及人乳头瘤病毒(HPV)DNA的检测。结果所有小鼠的移植物均存活,病理证实存在宫颈鳞柱状上皮并具有基底膜结构,上皮细胞免疫组织化学染色ki-67阳性,HPV DNA检测为阴性。结论初步建立了人正常子宫颈移行上皮SCID鼠模型,为进一步研究子宫颈上皮HPV感染致癌变过程提供了理想的动物模型。     

多药耐药基因转染的脐血细胞对白血病小鼠化疗的骨髓保护作用

目的探讨转染多药耐药(mdr1)基因的脐血单个核细胞(MNC)对急性髓系白血病小鼠的骨髓保护作用及疗效。方法通过逆转录病毒介导的方法将含有人全长cDNAmdr1基因导入脐血MNC,即逆转录病毒上清液与脐血MNC体外共培养;将接种人髓系白血病细胞系(HL60细胞)的SCID小鼠分成3组A组(观察组)经鼠尾静脉注射转染mdr1的脐血MNC2×106/只,共2次;B、C两对照组小鼠以同样剂量、方法分别注射未转染mdr1的脐血MNC和等容积的生理盐水。3组白血病SCID小鼠在每周递增高三尖杉酯碱剂量化疗下,通过检测小鼠外周血白细胞数、瘤细胞阳性率、组织病理和HL60细胞表面抗原(CD33)等观察转基因小鼠和对照小鼠对抗癌药物的耐受性及抗肿瘤疗效。同时分别采用PCR技术、免疫组化方法和柔红霉素排出试验检测mdr1基因在小鼠体内的表达和功能。结果①体外成功地将mdr1基因导入脐血MNC,转染率达30%左右;②用HL60细胞2×106接种于经亚致死量照射的SCID小鼠可成功制成白血病动物模型;③采用程序性移植转基因细胞方法成功建立了mdr1转基因脐血细胞移植小鼠模型,并可作为白血病临床前期体内评价mdr1基因保护骨髓作用;④转基因脐血细胞移植小鼠对高三尖杉酯碱耐受性高于正常剂量的5~6倍,外周血白细胞维持在3.0×109/L左右,外周血涂片瘤细胞降至5%以     

131I-GM-CSF在人白血病SCID小鼠模型体内的生物学分布

目的 观察131I-GM-CSF在人急性髓系白血病SCID小鼠模型体内的生物学分布.方法 用SCID小鼠建立人白血病异种移植模型,用氯胺-T法制备131I-GM-CSF,观察131I-GM-CSF在白血病小鼠体内的生物学分布.结果①静脉接种的HL-60细胞在SCID小鼠体内植活,4周后发生白血病;②131I-GM-CSF主要滞留于模型小鼠的脾脏、骨髓及瘤体组织中,且前两者对131I-GM-CSF摄取于注入后30 min达峰值,组织摄取率分别为(442.9±86.4)%ID/g和(4283.8±252.8)%ID/g,滞留24h后高于其他脏器.而131I呈非特异性分布.结论 131I-GM-CSF集中分布于人白血病SCID小鼠模型脾脏、骨髓及白血病细胞浸润组织,具有组织器官相对特异性.