Oncogenic potential of IDH1R132C mutant in cholangiocarcinoma development in mice

AIM: To investigate whether IDH1R132 C mutant in combination with loss of p53 and activated Notch signaling promotes intrahepatic cholangiocarcinoma(ICC) development.METHODS: We applied hydrodynamic injection and sleeping beauty mediated somatic integration to induce loss of p53(via sh P53), activation of Notch [via intracellular domain of Notch1(NICD)] and/or overexpression of IDH1R132 C mutant together with the sleeping beauty transposase into the mouse liver. Specifically, we co-expressed sh P53 and NICD(sh P53/NICD, n = 4), sh P53 and IDH1R132C(sh P53/IDH1R132 C, n = 3), NICD and IDH1R132C(NICD/IDH1R132 C, n = 4), as well as NICD, sh P53 and IDH1R132C(NICD/sh P53/IDH1R132 C, n = 9) in mice. Mice were monitored for liver tumor development and euthanized at various time points. Liver histology was analyzed by hematoxylin and eosin staining. Molecular features of NICD/sh P53/IDH1R132 C ICC tumor cells were characterized by Myc tag, Flag tag, Ki-67, p-Erk and p-AKT immunohistochemical staining. Desmoplastic reaction in tumor tissues was studied by Picro-Sirius red staining.RESULTS: We found that co-expression of sh P53/NICD, sh P53/IDH1R132 C or NICD/IDH1R132 C did not lead to liver tumor formation. In striking contrast, coexpression of NICD/sh P53/IDH1R132 C resulted in ICC development in mice(P < 0.01). The tumors could be identified as early as 12 wk post hydrodynamic injection. Tumors rapidly progressed, and by 18 wk post hydrodynamic injection, multiple cystic lesions could be identified on the liver surface. NICD/sh P53/IDH1R132 C liver tumors shared multiple histological features of human ICCs, including hyperplasia of irregular glands. Importantly, all tumor cells were positive for the biliary epithelial cell marker cytokeratin 19. Extensive collagen fibers could be visualized in tumor tissues using Sirus red staining, duplicating the desmoplastic reaction observed in human ICC. Tumors were highly proliferative and expressed ectopically injected genes. Together these studies supported that NICD/sh P53/IDH1R132 C liver tumors were indeed ICCs. Finally, no p-AKT or p-ERK positive staining was observed, suggesting that NICD/sh P53/IDH1R132 C driven ICC development was independent of AKT/m TOR and Ras/MAPK signaling cascades. CONCLUSION: We have generated a simple, nongermline murine ICC model with activated Notch, loss of p53 and IDH1R132 C mutant. The study supported the oncogenic potential of IDH1R132 C.     

输入性恶性疟原虫耐药相关基因Pfcrt和Pfmdr1单倍型及突变分析

目的了解目前山东省输入性恶性疟原虫抗药性基因Pfcrt和Pfmdr1的单倍型,分析突变基因型及其分布情况。方法根据恶性疟原虫Pfcrt和Pfmdr1基因序列设计套式PCR引物,对采自全省非洲务工返乡的输入性恶性疟感染者血样扩增,并对其产物进行基因测序和序列对比分析。结果 68例样本Pfcrt基因第72~76位点和Pfmdr1基因第86、1 042和1 246位点目的片段全部成功扩增和测序。Pfcrt基因中69.12%为野生单倍型CVMNK,30.88%为突变单倍型,突变型包括CVIET、CVIDT及混合型,其中CVIET数量最多。Pfmdr1基因中69.12%为野生单倍型NND,30.88%为突变单倍型,即YND及混合基因型。6个非洲输入来源国样本中,除几内亚Pfcrt基因全部为野生型外,其它国家Pfcrt和Pfmdr1基因均有突变型存在。5例样本Pfcrt和Pfmdr1基因共同表现为突变单倍型。结论山东省输入性恶性疟Pfcrt和Pfmdr1基因突变单倍型具有多样化特征。Pfcrt和Pfmdr1突变型比例均低于野生型,提示目前该省流行的输入性恶性疟未出现严重的氯喹耐药性。     

Basal core-promoter mutant of hepatitis B virus and progression of liver disease in hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND/AIMS: The long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are distinct from those in HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in this special clinical setting remain largely unknown. We thus investigated the association of hepatitis B virus (HBV) genotypes as well as precore/basal core-promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative chronic hepatitis B in Taiwan. METHODS: HBV genotypes and sequences of precore and basal core-promoter regions of the HBV genome were determined in 174 HBeAg-negative chronic HBV infection patients including 62 inactive carriers and 112 with different stages of liver disease. RESULTS: HBV carriers with older age (> 50 years) (odds ratio, 9.09; 95% confidence interval (CI), 3.22-25, P < 0.001) and basal core-promoter mutant of HBV (odds ratio, 4.12; 95% CI, 1.41-12.03, P = 0.01) were associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). The gender-related risk factors associated with the development of liver cirrhosis and HCC were further analyzed, and basal core-promoter mutant was only associated with the development of liver cirrhosis and HCC in male carriers (odds ratio, 4.35; 95% CI, 1.30-14.52, P = 0.02). CONCLUSIONS: The risk of development of liver cirrhosis and HCC is significantly increased in patients with advanced age as well as with basal core-promoter mutant of HBV. In addition, basal core-promoter mutant might contribute to the gender difference of the progression of liver disease in HBeAg-negative chronic hepatitis B in Taiwan.     

Statistical diffusion tensor histology reveals regional dysmyelination effects in the shiverer mouse mutant

Shiverer is an important model of central nervous system dysmyelination characterized by a deletion in the gene encoding myelin basic protein with relevance to human dysmyelinating and demyelinating diseases. Perfusion fixed brains from shiverer mutant (C3Fe.SWV Mbp^shi/Mbp^shi n = 6) and background control (C3HeB.FeJ, n = 6) mice were compared using contrast enhanced volumetric diffusion tensor magnetic resonance microscopy with a nominal isotropic spatial resolution of 80 μm. Images were accurately coregistered using non-linear warping allowing voxel-wise statistical parametric mapping of tensor invariant differences between control and shiverer groups. Highly significant differences in the tensor trace and both the axial and radial diffusivity were observed within the major white matter tracts and in the thalamus, midbrain, brainstem and cerebellar white matter, consistent with a high density of myelinated axons within these regions. The fractional anisotropy was found to be much less sensitive than the trace and eigenvalues to dysmyelination and associated microanatomic changes.     

空肠弯曲菌galE变异株免疫原性及免疫保护性评价

目的评价galE变异株免疫原性及免疫保护效力。方法分别采用galE变异株低菌量及高菌量单次或重复免疫(口服)BALB/c小鼠,ELISA方法检测动物肠液及血清中特异性sIgA及IgG的滴度。免疫后26d,用大剂量CJ攻击被免疫动物,观察galE变异株的保护效应。结果①低菌量及高菌量galE变异株单次或重复免疫后,动物肠液及血液中sIgA及IgG滴度均明显增高,与阴性对照组比较差异非常显著(P<0.01);未显示低剂量与高剂量之间、单次免疫与重复免疫之间抗体水平有明显差异(P>0.05);与亲代株免疫组比较,抗体水平也无明显不同(P>0.05)。②galE变异株免疫可明显降低动物受CJ攻击后的疾病指数(P<0.01),变异株的临床保护效率为79.5%～83.1%;清除CJ肠定植的能力明显高于阴性对照组(P<0.05);低菌量与高菌量之间,单次及重复免疫清除肠道感染效力无明显差异(P>0.05)。结论①galE变异株保留与亲代株相似的免疫原性及免疫保护性,可刺激动物产生特异性抗体,有效保护细菌攻击,明显降低动物疾病指数,缩短细菌肠道定植时间。②galE变异株仅小剂量单次口服即可产生良好的免疫效果。     

突变型P53的存在状态与不同化疗药物敏感性相互性的研究

目的 研究胃癌细胞中突变型P53的存在状态与不同化疗药物敏感性的相互关系。方法 将突变型P53、sv40Tag（封闭P53）导入胃癌细胞株SGC．7901中，用MTI法比较各组细胞对化疗药的敏感性。结果 导入突变型P53细胞株对5-氟脲嘧啶（5-F）耐药性较导入竞变型P53＋sv40Tag细胞株及对照组差异均有统计学意义（P〈0．05）。导入突变型P53细胞株和导入突变型P53＋sv40Tag细胞株对阿霉素（ADM）耐药性较对照组差异均有统计学意史（P〈0．05）。导入突变型P53细胞株、导入突变型P53＋sv40Tag细胞株及对照组细胞株各组对顺铂（CDDP）耐药性差异均有统计学意义（P〉0．05）。结论 突变型P53与肿瘤细胞对化疗药敏感性有一定的相关性。     

Successful treatment with lamivudine may correlate with reduction of serum ferritin levels in the patients with chronic hepatitis and liver cirrhosis type B

Purpose  To study the changes in serum ferritin levels in lamivudine (LAM)-treated patients with chronic hepatitis and liver cirrhosis type B and determine whether successful treatment with LAM results in a reduction of serum ferritin levels. Methods  Thirty patients with chronic hepatitis B virus (HBV) infection were followed prospectively during their treatment with LAM for 12 months. Serum HBV DNA, ferritin levels, and emergence of YMDD mutants were monitored. A case of severe liver cirrhosis with hepatic hemosiderosis that was treated successfully with LAM also is shown as a representative case. Results  Serum alanine aminotransferase and ferritin levels decreased significantly more in the patients treated with LAM without YMDD mutants (n = 23) than those with mutants (n = 7). Hepatic hemosiderosis along with serum iron markers improved greatly in the representative patient. Conclusion  Successful treatment with LAM may reduce serum ferritin levels and improve hepatic siderosis in a subset of patients with chronic HBV infection. A study of Niigata-Zeffix investigation meeting.     

Prevalence of vaccine-induced escape mutants of hepatitis B virus in the adult population in China: a prospective study in 176 restaurant employees

BACKGROUND AND AIM: Hepatitis B virus (HBV) variants with mutations in the S gene would pose a substantial risk to the community as current HBV vaccines are not effective in preventing infection with them. The majority of such vaccine escape mutants so far reported have been found while studying vertical transmission of HBV; the vaccine failure rate in connection with vaccine escape mutants in adults is not clear at the moment. The purpose of this study was to evaluate the efficacy of immunization against HBV in the adult population by analysis using polymerase chain reaction (PCR) to detect HBV-DNA, and also to elucidate the type of mutation encountered in vaccine failure cases. METHOD: A total of 176 adult restaurant employees in China, who had been vaccinated according to the food epidemic law, were enrolled in a standard vaccination program. Their serum HBV-DNA was determined before and 1 year after the completion of the vaccination program. In those infected with HBV, despite having received the HBV vaccine, direct sequencing within the S gene of the amplified samples was conducted. RESULTS: Although only two cases were found to be hepatitis B surface antigen (HBsAg) positive 1 year after the completion of the vaccination program, six subjects (3.4%) were found to be HBV-DNA positive assessed by a nested PCR. Four out of these six cases had a point mutation within the 'a' determinant; they were Gly-145-Ala, and Ile/Thr-126-Asn/Ser. CONCLUSION: The HBV vaccine failure rate assessed by using PCR analysis was 3.4% (six of 176) in the Chinese adult population undergoing the HBV vaccination program. Hepatitis B virus variants with missense mutation within the 'a' determinant were responsible in most cases.     

Pharmacodynamics and pharmacokinetics of single doses of subcutaneous pegylated human G-CSF mutant (Ro 25-8315) in healthy volunteers: comparison with single and multiple daily doses of filgrastim

Ro 25-8315 is produced by conjugation of rhG-CSF mutant with polyethylene glycol (PEG). The purpose of this study was to examine the pharmacodynamics and pharmacokinetics of Ro 25-8315 in comparison with Filgrastim (rhG-CSF). Subjects received single subcutaneous doses of Ro 25-8315 ranging from 10 to 150 microg/kg using a double-blind, randomized, placebo-controlled design. Filgrastim was administered as a single dose (5 or 10 microg/kg) and, following a 14-day washout period, daily for 7 days. Ro 25-8315 increased absolute neutrophil count (ANC) by 6- to 8-fold and CD34+ cell count more than 30-fold at the highest doses tested. Single doses (60-150 microg/kg) of Ro 25-8315 and multiple doses of Filgrastim had similar effects on ANC and CD34+, although Ro 25-8315 had a greater effect on CFU-GM. The pharmacokinetics of Ro 25-8315 were dose-dependent, with peak concentrations and area under the serum concentration-time curve (AUC) increasing 100-fold over the range of doses studied. Time to reach peak concentration (T(max)) and half-life of Ro 25-8315 averaged 20-30 hr at all doses, approximately three times longer than with Filgrastim. Adverse events were not serious and occurred with similar frequency with both products. Pegylation of rhG-CSF mutant results in more desirable pharmacokinetic properties and a longer duration of action with effective increases in ANC and measures of peripheral blood progenitor cell mobilization for at least 1 week.