凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的作用

目的探讨x-相关凋亡抑制蛋白（XIAP）和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用，以及转染胞浆表达型Smac基因靶向下凋XIAP对化疗药物诱导的胰腺癌细胞凋亡的影响。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化，Western blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-NI／Smac真核表达载体并转染胰腺癌Panc-1细胞，流式细胞术检测转染Smac基因前后Panc-1细胞的凋亡敏感性。结果与BXPC-3细胞相比，Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性，Western blot分析显示Panc-1细胞高表达XIAP，在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞，而且凋亡的BXPC-3细胞释放入胞浆内的成熟Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞，可明显下调其XIAP表达水平，促进效应Caspase-3分子活化，显著提高顺铂、5-FU诱导的细胞凋亡率。结论胰腺癌细胞XIAP的表达水平下调与其化疗敏感性有关，XIAP是克服化疗抵抗的重要靶分子，而上调Smac活性蛋白的胞浆表达作为一种有效调节信号，通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

Sutureless re-anastomosis by laparoscopy versus microsurgical re-anastomosis by laparotomy for sterilization reversal: a matched cohort study

BACKGROUND: Sutureless re-anastomosis per laparoscopy is an alternative for microsurgical re-anastomosis by laparotomy in the treatment of sterilized women with renewed child wish. Our aim was to compare pregnancy rates after both surgical techniques. METHODS: We performed a retrospective cohort study in which consecutive women who underwent sutureless re-anastomosis per laparoscopy were compared to women who underwent microsurgical re-anastomosis by laparotomy. Both procedures were performed in neighbouring hospitals in Northern-Brabant, The Netherlands, and women were matched for age. The primary outcome was time to ongoing pregnancy. RESULTS: Overall, we included 41 women who had sutureless re-anastomosis by laparoscopy, and 41 age-matched women who underwent microsurgical re-anastomosis by laparotomy. The number of women who conceived was 20 (15 ongoing pregnancies) in the sutureless laparoscopic group versus 26 (24 ongoing pregnancies) in the laparotomic group, a difference due to a longer follow-up period in the laparotomic group. Time to ongoing pregnancy was comparable in both groups (P=0.46), with 3 year cumulative ongoing pregnancy rates of 45 and 52% respectively. After adjustment for other prognostic factors, the fecundity rate ratio was 0.97 (95% CI 0.26-3.6), indicating a similar performance of the two techniques. CONCLUSION: The simplified stitchless laparoscopic procedure for reversal of tubal sterilization with the use of a tubal splint, clip fixation of the muscularis and fibrin glue resulted in a promising pregnancy rate, which was similar to the pregnancy rate obtained with the microsurgical re-anastomosis per laparotomy.     

Translocation (1;22)(p36;q11.2) with concurrent del(22)(q11.2) resulted in homozygous deletion of <Emphasis Type="Italic">SNF5/INI1</Emphasis> in a newly established cell line derived from extrarenal rhabdoid tumor

Malignant rhabdoid tumor (MRT) is a highly malignant pediatric cancer, which arises in various sites such as the kidney, brain, and soft tissues. Cytogenetic studies have revealed alterations of 22q11 in MRT. Recently, deletions and mutations of the SNF5/INI1 locus in 22q11.2 have been reported in MRT, suggesting that SNF5/INI1 is a tumor suppressor gene for MRT. Here we report our molecular cytogenetic study for a newly established cell line from extrarenal MRT with t(1;22)(p36;q11.2). Consequently, the reciprocal translocation was associated with the interstitial deletion of a small segment including SNF5/INI1, and another, chromosome 22, showed terminal deletion, the breakpoint of which was located 70–80 kb centromeric to SNF5/INI1, resulting in homozygous deletion of SNF5/INI1 in this cell line.     

Adhesion dependent release of hepatocyte growth factor and interleukin-1 receptor antagonist from human blood granulocytes and monocytes: Evidence for the involvement of plasma IgG,complement C3 and β2 integrin

Objective:Evolving evidence of anti-inflammatory effects is observed in patients with rheumatoid arthritis or ulcerative colitis following periodic adsorptive granulocyte and monocyte (GM) apheresis with a column containing cellulose acetate (CA) beads as apheresis carriers. This study was undertaken to obtain insights into mechanisms of anti-inflammatory actions of adsorptive GM apheresis with CA beads. Methods:In a series of in-vitro experiments, we investigated the effects of plasma proteins and the leucocytes 2 integrin (CD18) on granulocyte adsorption to CA beads. Results:Granulocyte adsorption to CA beads required plasma IgG, the complement C3 and was inhibited by an antibody to leucocytes CD18. Further, hepatocyte growth factor (HGF) and interleukin-1 receptor antagonist (IL-1ra) which have strong anti-inflammatory actions were released by granulocytes that adhered to CA beads. Conclusions:Plasma IgG, C3 derived complement activation fragments and leucocytes CD18 are involved in granulocyte adhesion to CA beads and hence the release of HGF and IL-1ra.Received 27 October 2003; returned for revision 16 December 2003; accepted by M. J. Parnham 8 January 2004     

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.     

Follicular thyroid lesions coexisting with Hashimoto's thyroiditis: incidence and possible sources of diagnostic errors

Our objectives were to study the types and incidence of thyroid follicular lesions coexisting with Hashimoto's thyroiditis (HT), the pitfalls in their cytodiagnosis, and the effect on management. All cases of HT diagnosed by fine-needle aspiration (FNA) and/or histology over a 7-yr period were retrospectively studied. HT coexisted with follicular adenoma (FA) in 6 cases, follicular variant of papillary carcinoma (FVPC) in 1 case, and goitrous nodule (GN) in 2 cases. The overall incidence rates of thyroid neoplasm and goitrous nodules coexistent with HT were 15% and 3.5%, respectively. A preoperative FNA diagnosis was available in 10 histologically proven cases of HT. A false-positive diagnosis of follicular neoplasm (FN) that led to unnecessary thyroidectomies was given in 3 cases. In 2 of these, the cytological diagnosis was HT with the possibility of coexisting FN, and in the third case, the cytological finding of HT was misinterpreted as FN. The main causes of these diagnostic pitfalls were the presence of hyperplastic follicular cells with nuclear pleomorphism, a paucity of lymphoid cells in burned-out HT, and lack of ones exposure. Nuclear pleomorphism was observed in none of the follicular adenomas. FNA diagnosed accurately the coexisting lesions in 6 cases; 3 FA, 1 FVPC, and 2 GN, but it did not sample HT. In one case, FNA diagnosed correctly both HT and the coexisting FA. Therefore, the presence of a coexistent neoplasm or goitrous nodule reduced the chances of sampling HT by 85.7%, with no false-negative results. Indeed, aspiration on and around the thyroid nodule helps in sampling HT. However, HT may dominate the smear and obscure neoplasia. This can be avoided if the procedure is performed by the pathologist and the aspiration is done on the nodule only. The overlapping cytological features of FN and HT were the main causes of false-positive results. This can be reduced by avoiding the diagnosis of FN in the presence of follicular-cell pleomorphism and/or moderate to excessive numbers of lymphoid cells, provided proper aspiration technique is maintained.     

Abnormal IL-2 receptor levels in non-pregnant women with a history of recurrent miscarriage

BACKGROUND: Immunological abnormalities have been found in pregnant women with a history of recurrent miscarriage. This study compared interleukin-2 receptor (IL-2R) levels in non-pregnant women with a history of recurrent miscarriage with those found in serum from a non-pregnant group with no such history. METHODS: Group 1 comprised 49 non-pregnant women with a history of recurrent miscarriage (at least three consecutive miscarriages). Group 2 comprised 22 non-pregnant women with no history of miscarriage. Serum IL-2R levels were measured in all patients. RESULTS: The results obtained showed that although all women were not pregnant at the time of sampling, IL-2R levels were significantly higher in women in Group 1 compared with those in Group 2 (1589 +/- 1289 versus 1082 +/- 823 pg/ml; P < 0.05). Follow-up data were available for 21 women from Group 1. The next pregnancy ended successfully for 14 of these women, while seven miscarried again. The IL-2R levels obtained pre-pregnancy were not significantly different between the two groups (1480 +/- 910 versus 1356 +/- 716 pg/ml). CONCLUSION: This study has shown that non-pregnant women with a history of recurrent miscarriage have raised IL-2R levels. These increased pre-pregnancy IL-2R levels did not necessarily predict miscarriage for the next pregnancy.     

Investigation of the role of the serotonergic activity of certain subtype-selective alpha1A antagonists in the relaxant effect on the pregnant rat uterus in vitro

Results from recent studies have shown that alpha(1A)-adrenergic receptor (alpha(1A)-AR) antagonists could offer a new alternative in the treatment of preterm delivery. However, members of this group [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil (5-MU)] are known to influence serotonin (5-hydroxy-tryptamine) (5-HT(1A)) receptors, too. Our objective was to clarify the role of their 5-HT(1A) activities in the uterus relaxant effect. RT-PCR was used to determine mRNA expression of the receptor subtypes in 22 day pregnant rat uteri. Isolated uteri were stimulated by 5-HT or electrical field to investigate the contraction-inhibiting effect and the 5-HT(1A) activity of the alpha(1A) antagonists. Both receptor subtypes are present in rat myometrium. 5-HT induced contractions were inhibited by the alpha(1A) antagonists. Besides shifting the dose-response curve of 5-HT to the right, 5-MU decreased its maximal effect. The alpha(1A) antagonists inhibited electrical field stimulation-induced contractions. 5-HT(1A) blockade increased the maximal effect of 5-MU but did not change that of WB4101. These results suggest that the contraction increase caused by 5-HT is mediated by alpha(1A) receptors. Serotonergic activity of alpha(1) antagonists and especially alpha(1A) antagonists should be investigated as it may alter their efficacy and could interfere with their side-effects. It is proposed that novel alpha(1A) antagonists should be designed with no 5-HT(1A) activity to achieve maximal relaxant effect.     

S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.     

Predicting pregnancy and spermatogenesis by survival analysis during gonadotrophin treatment of gonadotrophin-deficient infertile men

BACKGROUND: Predictors of fertility or spermatogenesis during gonadotrophin therapy of gonadotrophin-deficient men remain poorly defined. METHODS AND RESULTS: In order to evaluate potential predictors, this study evaluated 29 consecutive gonadotrophin-deficient men all desiring paternity who received 43 courses of therapy in one centre between 1982 and 1998. The Kaplan-Meier survival analysis estimates of median (SE) time to a sperm concentration of >0, >5 and >20 x 10(6)/ml were 5.5 (1.1), 12.4 (2.3) and 29.1 (1.9) months respectively. Conception occurred in 22/43 cycles (with eight men achieving two pregnancies) with a median (SE) Kaplan-Meier estimate of 20.5 (4.7) months. The median sperm concentration at conception was 5.0 (SE 2.0; range 0.0-59.5) x 10(6)/ml. Multivariate correlated Cox proportional hazards models predicting these same sperm thresholds and conception were developed by forward stepwise variable selection with verification of the model by backward stepping. Larger testicular volume, prior gonadotrophin therapy, completion of puberty, older age, the absence of adverse fertility factors and the absence of multiple pituitary hormone deficiency predicted a favourable response. Multivariate modelling suggests that the two most important predictors of sperm output are testicular volume and pubertal status. The most important potentially modifiable predictor was prior gonadotrophin therapy. The efficacy of recombinant and urinary FSH were similar. Prior androgen therapy and partner's age did not appear to be significant. CONCLUSIONS: Since prolonged treatment may be required to induce spermatogenesis, attention to these predictors may allow appropriate early use of advanced reproductive technologies.