Urinary nuclear magnetic resonance spectroscopy of a Bangladeshi cohort with hepatitis-B hepatocellular carcinoma: A biomarker corroboration study

AIM: To establish if a distinct urinary metabolic profile could be identified in Bangladeshi hepatitis-B hepatocellular carcinoma(HCC) patients compared to cirrhosis patients and controls. METHODS: Urine samples from 42 Bangladeshi patients with HCC(39 patients with hepatitis-B HCC), 47 with cirrhosis on a background of hepatitis B, 46 with chronic hepatitis B, and seven ethnically-matched healthy controls were analyzed using nuclear magnetic resonance(NMR) spectroscopy. A full dietary and medication history was recorded for each subject. The urinary NMR data were analyzed using principal component analysis(PCA) and orthogonal partial leastsquared discriminant analysis(OPLS-DA) techniques. Differences in relative signal levels of the most discriminatory metabolites identified by PCA and OPLSDA were compared between subject groups using an independent samples Kruskal-Wallis one-way analysis of variance(ANOVA) test with all pairwise multiple comparisons. Within the patient subgroups, the MannWhitney U test was used to compare metabolite levels depending on hepatitis B e-antigen(HBe Ag) status and treatment with anti-viral therapy. A BenjaminiHochberg adjustment was applied to acquire the level of significance for multiple testing, with a declared level of statistical significance of P 0.05.RESULTS: There were significant differences in age(P 0.001), weight(P 0.001), and body mass index(P 0.001) across the four clinical subgroups. Serum alanine aminotransferase(ALT) was significantly higher in the HCC group compared to controls(P 0.001); serum α-fetoprotein was generally markedly elevated in HCC compared to controls; and serum creatinine levels were significantly reduced in the HCC group compared to the cirrhosis group(P = 0.004). A threefactor PCA scores plot showed clustering of the urinary NMR spectra from the four subgroups. Metabolites that contributed to the discrimination between the subgroups included acetate, creatine, creatinine, dimethyamine(DMA), formate, glycine, hippurate, and trimethylamine-N-oxide(TMAO). A comparison of relative metabolite levels confirmed that carnitine was significantly increased in HCC; and creatinine, hippurate, and TMAO were significantly reduced in HCC compared to the other subgroups. HBe Ag negative patients showed a significant increase in creatinine(P = 0.001) compared to HBe Ag positive patients in the chronic hepatitis B subgroup, whilst HBe Ag negative patients showed a significant decrease in DMA(P = 0.004) in the cirrhosis subgroup compared to HBe Ag positive patients. There were no differences in metabolite levels in HCC patients who did or did not receive antiviral treatment. CONCLUSION: Urinary NMR changes in Bangladeshi HCC were identified, corroborating previous findings from Egypt and West Africa. These findings could form the basis for the development of a cost-effective HCC dipstick screening test.     

MTHRF基因C677T多态性与ALL患儿MTX血药浓度和毒副反应的相关性

【目的】探讨亚甲基四氢叶酸还原酶（MTHFR）C677T基因多态性对急性淋巴细胞白血病（ALL）儿童甲氨蝶呤（MTX）血药浓度和毒副反应的影响。【方法】筛选50例采用大剂量MTX治疗的ALL患儿,采用多聚酶链反应一限制性内切酶片段多态性分析法（PCR-RFLP）检测MTHFRC677T多态性,应用荧光偏振免疫分析法（FPIA）24h、48h、72h监测患儿外周血中甲氨蝶呤动态血药浓度,记录相关毒副反应。【结果】24h时、48hMTX血药浓度均以突变型纯舍子（TT）最高（均P〈0．05）,突变型杂合子（CT）次元;MTHFRC677T基因型与肝损害、消化道反应和骨髓抑制相关,携带T突变基因患儿（CT＋TT组）发生肝损害的风险是野生型纯合子（CC）的5．12倍（95％CI：1．372～19．077）,发生3～4级消化道反应的风险是CC型的4．41倍（95％CI：1．066～18．266）,3～4级骨髓抑制发生率依次为TT型、CT型和CC型（均P〈0．05）。【结论】MTHFRC677T基因多态性影响ALL患儿大剂量MTX化疗期间MTX血药浓度,与肝损害、消化道反应和骨髓抑制等毒副反应相关。     

Peripheral blood harvest of unaffected CD34+ CD38- hematopoietic precursors in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) arises from somatic mutation of a bone marrow progenitor that disrupts glycosylinositol phospholipid (GPI) anchoring of cell surface proteins. We recently characterized the expression of GPI-anchored decay acclerating factor (DAF) and CD59 during hematopoietic development in PNH marrow. We found that, although a subset of early hematopoietic precursors identified by the CD34+CD38- phenotype exhibits normal DAF and CD59 expression, DAF and CD59 are absent on the majority of CD34+CD38- cells. Pluripotent CD34+CD38- hematopoietic stem cells normally circulate in the peripheral blood and can be collected by apheresis, cryopreserved, and later used for reconstitution of hematopoiesis. In this study, we examined the phenotypes of CD34+ cells that are released into the blood of PNH patients. Analyses of apheresis samples from three affected individuals showed discrete populations of circulating DAF+CD59+CD34+ and DAF-CD59- CD34+ cells. Variable proportions of CD34+CD38- cells were present within the peripheral blood CD34+ cells of each patient, but in all three cases the DAF+CD59+CD34+CD38- cell subset subset. Because CD34+ cells lacking CD38 antigen are highly enriched for self-renewing hematopoietic stem cells, these findings indicate that apheresis samples can serve as a source of unaffected stem cells for autologous marrow transplantation of PNH patients.     

肝型丙酮酸激酶启动子与人胰岛素基因逆转录病毒介导感染pT67、HepG2细胞的蛋白质表达

目的检测新构建肝型丙酮酸激酶启动子（LPKp）与人胰岛素基因（hInsg）逆转录病毒表达载体（pM54,LPKp-hInsg）在pT67、HepG2细胞的表达情况,为基因治疗或基因结合干细胞治疗糖尿病寻找新的优化生物载体。方法（1）限制酶切父、母本质粒p54、pMDN-SIN,取相关片段构建含人Ins基因一逆转录病毒载体pM54（pMDN-SIN＋p54;LPKp-hInsg）;pM54扩增、纯化、酶切鉴定;（2）脂质体FuGENE6转染pM54质粒进入pT67、PhoenixE和3T3细胞系,同时转染pCMVβGal和父本质粒p54做对照;（3）制备转染后细胞培养拟逆转录病毒上清液;（4）用转染后细胞培养上清液旋转感染pT67、HepG2细胞;（5）ELISA法检测转染、感染后细胞培养上清液Ins含量。结果酶切鉴定质粒与构建预期相符;ELISA法检测出转染、感染上清液中均含较高水平Ins。对照结果均为阴性。结论LPKp-hlnsg基因逆转录病毒表达载体pM54构建成功。人胰岛素基因逆转录病毒介导旋转感染pT67等细胞,目的蛋白Ins获得了预期的表达。实验为基因治疗或基因结合于细胞治疗糖尿病的进一步相关研究奠定了基础。     

肥厚型心肌病患者肌球蛋白结合蛋白C基因筛查及其相应的临床特征

目的 研究国人肥厚型心肌病(HCM)患者的致病基因一肌球蛋白结合蛋白C基因(myosin binding protein C gene,MYBPC3)的突变位点,分析基因突变类型与临床表型的相互关系.方法对66例HCM患者的MYBPC3基因进行扫描,聚合酶链反应扩增其外显子及剪接部位的基因组DNA片段,直接测序分析.分析各突变患者相应的临床表型特点.结果经测序分析,发现Lys30lfs移码突变、Asp463stop无义突变、Gly523Arg错义突变和Tyr847His错义突变.MYBPC3导致的HCM为3例,占病例总数的4.5%.其临床表型各异,患者H30(Lys301fs)47岁发病,活动后胸闷、气短,超声显示室间隔肥厚达18.7 mm,左心室后壁14.7 mm.患者H48(Asp463stop)为25岁男性,24岁发病,室问隔肥厚达15.4 mm.患者H53(Gly523Arg和Tyr847His)发病年龄36岁,活动后胸闷、憋气,伴心前区疼痛,室间隔肥厚达27 min.结论 MYBPC3突变为HCM主要致病原因之一.MYBPC3突变基因携带者临床表型差异大.国人MYBPC3突变患者多在青壮年期发病,不同于国外报道的多发病较晚.     

Molecular analysis of VH and VL regions expressed in IgG-bearing chronic lymphocytic leukemia (CLL): further evidence that CLL is a heterogeneous group of tumors

We report the heavy (H) and light (L) chain variable (V) region sequences of cDNAs encoding the Ig receptor of two cases of CD5+ IgG- bearing CLL P87 and P103. In both CLL cases the H chain was encoded by members of the VH3 gene family. The L chain expressed by P87 belonged to the V lambda IV subgroup, whereas P103 used a member of the V kappa III subgroup. The VH3.P87 gene differed by only three nucleotides from 38P1, a VH3 gene previously cloned from a fetal liver cDNA library. Nucleotide sequence analysis demonstrated that the V kappa III.P103 gene differed by seven nucleotides from its most homologous germline counterpart, the Humkv325 gene, a highly conserved gene frequently expressed in IgM-bearing CLL. The nucleotide sequences of VH3.P103 and V lambda IV.P87 could not be reliably matched with reported germline V genes. The analysis of multiple independently obtained VH and VL cDNA clones from each tumor showed a lack of intraclonal diversification. The data show that V regions expressed in isotype-switched CD5+ CLL may be either in/near germline configuration or somatically mutated. Furthermore, these tumors, like their IgM-bearing counterparts, do not seem to undergo intraclonal diversification.     

Artificial urinary sphincters: plain radiography of malfunction and complications

Inflatable artificial urinary sphincters provide excellent voluntary continence. Eighty-four consecutive patients underwent implantation of artificial urinary sphincters for intractable urinary incontinence; 33 patients had 58 episodes of sphincter malfunction, and eight patients had eight complications involving a functional prosthetic sphincter. Retrospective analysis was performed to determine the value of plain radiography of the pelvis in patients with sphincter malfunction or complication. The cause of malfunction in the majority of patients was a system leak and subsequent loss of hydraulic fluid (31 occurrences; 53%). Plain radiography permitted correct identification of all instances of fluid leakage in patients with opacified prostheses. Plain radiographs were of no value in examining patients with nonopacified prostheses or the complications of cuff erosion or wound infection. Due to the low cost and noninvasive nature of plain radiography of the pelvis, we conclude that it should be used as the initial diagnostic modality in patients with previously opacified but currently dysfunctional artificial urinary sphincters.     

p18INK4C基因敲除加重顺铂诱导的小鼠急性肾损伤

目的 探讨周期素依赖性激酶抑制基因p18INK4C敲除对顺铂诱导的小鼠急性肾损伤(AKI)的影响,并分析其可能机制.方法 利用已引种的杂合p18INK4C基因敲除小鼠p18INK4C+/-繁育p18INK4C基因敲除小鼠P18INK4C-/-(KO组),以同窝野生型小鼠p18INK4C+/+作为对照(WT组).腹腔注射顺铂制备AKI模型,观察两组小鼠的存活情况,并检测给药后第2、3、5天血肌酐、尿素氮的变化及肾组织的病理变化.结果 WT组小鼠在第3天出现死亡,第7天存活率为46.7%(14/30).KO组小鼠自第2天出现死亡,至第7天全部死亡(100.0%,30/30).KO组的死亡率显著高于WT组(P<0.05).自给药后第2天起,KO组小鼠的肾功能恶化明显,此后继续加重.给药后第2、3、5天,KO组的尿素氮、肌酐均显著高于WT组(P值均<0.05).WT和KO组小鼠均以给药后第3天肾脏病变最为严重.结论 p18INK4C基因敲除加速了顺铂诱导的小鼠AKl的进展,可能与其增加小管上皮损伤及加剧炎性反应有关.