Infertility: Avoidance of triplet pregnancies by elective transfer of two good quality embryos

Attempts to increase the probability of a successful pregnancyin in-vitro fertilization (IVF) treatment by increasing thenumber of embryos transferred automatically also increase theprobability of multiple pregnancies and their attendant risks.Even where the number of transferred embryos is limited to amaximum of three as in this and other centres, there is a highincidence of twins and triplets. The question therefore ariseswhether the number of transferred embryos should be furtherlimited to a maximum of two in cases where the prognosis isotherwise good. The only objection to this idea is a possiblelowering of pregnancy rate. The present study set out to investigatethis question. No significant lowering of pregnancy rate wasfound, so that limiting the number of transferred embryos totwo where the prognosis is otherwise good has now become standardpractice in our centre. A good IVF prognosis was defined bythe following criteria: first attempt for IVF, less than 37years old, and good embryo development. From 183 patients fulfillingthese criteria, 80 agreed to the transfer of two embryos (group1) and 103 opted for a triple transfer (group 2). Patient characteristicsand embryology results were similar in the two groups. In group1, 34 patients (42.5%) became pregnant and in group 2, 50 (48.5%).This difference is not significant. Similarly, twin pregnancyrates in both groups were high; eight twin pregnancies (23.5%)in group 1 and 12 (24%) in group 2. For the triplet pregnancyrate of 18% (nine triplet pregnancies) in group 2, there wasobviously no parallel in group 1. After thawing about half ofthe cryopreserved embryos and subsequently replacing them, preliminarycumulative pregnancy rates of 52.5% in group 1 and 53.4% ingroup 2 were obtained. Future results from cryopreservationshould provide relatively better outcomes for group 1 sinceall the patients in this group had at least one embryo frozenand fewer embryos replaced in the fresh cycle.     

流行性出血热病毒特异性转移因子的研究

对流行性出血热病毒(EHFV)特异性转移因子(EHFV-TF)的制备、特性及在小鼠体内的活性作用进行了研究。其理化性状与普通 TF 非常相似,能激活淋巴细胞的 E 受体(激活率为65.54%),明显提高 Et-RFC 的形成率。E-HFV-TF 还具有特异性抗原依赖活性:①促进 BALB/C 小鼠和 NIH 裸鼠特异性抗体的产生;②促进 LACA 小鼠脾细胞对 EHFV 反应的特异性应答;③促进脾细胞在 EHVF 存在时对 PHA 诱导下的增殖反应(促时率为151.3%)。EHFV—TF可推迟动物感染 HEFV 后的发病期,延长病程和推迟死亡时间;减轻发病动物脑肺充血、出血、水肿现象和肝肾病理损害程度。结果表明 EHFV-TF 不仅具有提高细胞免疫的非特异活性,而且具有对 EHFV 抗原的依赖活性。本研究提示,E-HFV-TF 可用于 EHFV 感染病人的治疗。     

Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells.

Tumor cells infected with a retrovirus vector (VIK) containing the herpes simplex virus thymidine kinase (HSV-TK) gene can be selectively killed by treatment with nucleoside analogues, such as ganciclovir. To mediate delivery of the HSV-TK gene to "recipient" tumor cells, "donor" C6 rat glioma cells infected with the VIK vector (C6VIK) were superinfected with wild type Moloney murine leukemia virus (WT Mo-MLV). These modified donor cells (C6VIKWT) produced both wild type retrovirus and the VIK vector. In culture, C6VIKWT cells were 300-fold more sensitive to the toxicity of ganciclovir than were C6VIK cells, suggesting that the presence of wild type retrovirus contributed to the toxicity. Co-culture of C6VIKWT cells with the C6 subline, C6BAG, sensitized the latter to ganciclovir treatment. Nude mice inoculated subcutaneously with a mixture of C6VIKWT and C6BAG cells showed regression of subsequent tumors when treated with ganciclovir. The observations show that tumor cells modified in culture by infection with a retrovirus bearing the HSV-TK gene and wild type retrovirus are not only sensitive to ganciclovir, but can transfer this sensitivity to neighboring "naive" tumor cells in culture and in vivo.     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Single top quality embryo transfer as a model for prediction of early pregnancy outcome

BACKGROUND: Single embryo transfer (particularly of a top quality embryo) is an excellent model to correlate embryo quality in terms of morphological criteria to early pregnancy. We investigated whether this model could provide us with more information on what happens after implantation in the first trimester of pregnancy. METHODS: The outcome of 370 consecutive single top quality embryo transfers in patients younger than 38 years was analysed for pregnancy and first-trimester pregnancy loss (FTPL) before 13 weeks of gestation. Analysis was done on each cohort of embryos from which the transferred top quality embryo was selected. Serum HCG levels were measured on day 8 and day 12 after day 3 embryo transfer. The HCG index was calculated as the level of HCG on day 12/HCG on day 8. RESULTS: The pregnancy rate after single top quality embryo transfer was 51.9%. This was independent of the patients' age. FTPL, however, appeared to be age dependent: 15.4% for the whole group, 9% in patients younger than 30 years and 19% in patients above 30 years. The pregnancy rate was 50% in IVF cycles and 52% in ICSI cycles; FTPL was 19% in IVF cycles and 10% in ICSI cycles. Multiple regression analysis showed that these differences originated from age differences between both populations rather than from technique-related factors. An HCG level >or=45 IU/l on day 12 was predictive for ongoing pregnancy with 75.6% sensitivity and 100% specificity; an HCG index >or=3.5 similarly predicted ongoing pregnancy with 72.3% sensitivity and 100% specificity. CONCLUSIONS: These data show that embryo selection for transfer on day 3 can be used as an excellent tool for prediction of pregnancy but not for prediction of FTPL. The pregnancy rate of a single top quality embryo is not related to age, whereas FTPL is age dependent.     

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.     

逆转录病毒介导的人促红细胞生成素基因在淋巴细胞中的表达

本实验设计和构建了人促红细胞生成素（Epo）重组逆转录病毒载体（LXSN-Epo）,载体中小鼠白血病病毒长末端调控序列翻译调控人Epo基因的表达。通过重组逆转录病毒,将Epo基因转导到大鼠T和B淋巴细胞中,DNA-PCR分析证明带有人Epo基因的逆转录病毒已整合到T和B淋巴细胞基因中,RT-PCR分析显示了在两种细胞中均表达了 Epo mRNA。体外生物学活性测定显示,在转导的T淋巴细胞培养上清中Epo表达水平高达243mU/ml,在感染的B淋巴细胞培养上清中Epo活性达43mU/ml。在感染的B细胞中,Epo表达在mRNA和蛋白水平均证明了逆转录病毒能有效地和迅速地转导Epo基因到新培养的B细胞中,而不需要药物选择。这些研究开始了逆转录病毒介导的基因转移走向体内Epo基因治疗研究的过程。     

Meta-analysis of gross insertions causing human genetic disease: novel mutational mechanisms and the role of replication slippage

Although gross insertions (>20 bp) comprise <1% of disease-causing mutations, they nevertheless represent an important category of pathological lesion. In an attempt to study these insertions in a systematic way, 158 gross insertions ranging in size between 21 bp and approximately 10 kb were identified using the Human Gene Mutation Database (www.hgmd.org). A careful meta-analytical study revealed extensive diversity in terms of the nature of the inserted DNA sequence and has provided new insights into the underlying mutational mechanisms. Some 70% of gross insertions were found to represent sequence duplications of different types (tandem, partial tandem, or complex). Although most of the tandem duplications were explicable by simple replication slippage, the three complex duplications appear to result from multiple slippage events. Some 11% of gross insertions were attributable to nonpolyglutamine repeat expansions (including octapeptide repeat expansions in the prion protein gene [PRNP] and polyalanine tract expansions) and evidence is presented to support the contention that these mutations are also caused by replication slippage rather than by unequal crossing over. Some 17% of gross insertions, all >or=276 bp in length, were found to be due to LINE-1 (L1) retrotransposition involving different types of element (L1 trans-driven Alu, L1 direct, and L1 trans-driven SVA). A second example of pathological mitochondrial-nuclear sequence transfer was identified in the USH1C gene but appears to arise via a novel mechanism, trans-replication slippage. Finally, evidence for another novel mechanism of human genetic disease, involving the possible capture of DNA oligonucleotides, is presented in the context of a 26-bp insertion into the ERCC6 gene.     

臀肌重建术的解剖学和生物力学优化分析

在六侧新鲜儿童标本上,对臀肌重建术进行了应用解剖学和生物力学优化分析,比较了骶棘肌、腰大肌、背阔肌和腹外斜肌重建臀肌的效果,得到了移位肌的最优移置部位和移位后重建臀肌功能的估算。结果表明骶棘肌重建臀肌的效果最好,以下依次是腰大肌、背阔肌和胸外斜肌。