Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Placental expression of interferon-gamma (IFN-gamma) and its receptor IFN-gamma R2 fail to switch from early hypoxic to late normotensive development in preeclampsia

The inability of the mother to switch from T helper cell type 1 (Th1) to Th2 cytokine profiles at the fetal-maternal interface has been proposed as one of the primary causes of miscarriage, intrauterine growth restriction (IUGR), and preeclampsia (PE). The Th1 [interferon-gamma (IFN-gamma), TNF-alpha, and IL-12] and Th2 (IL-4 and IL-10) cytokines have opposite effects on human pregnancy. Leukemia inhibitory factor (LIF) promotes embryo implantation and sustains pregnancy, whereas IFN-gamma and TNF-alpha are detrimental to pregnancy. Both IFN-gamma and LIF are produced by maternal cells and tissues at the fetal-maternal interface, whereas the IFN-gamma receptors (IFN-gamma R1 and IFN-gamma R2) and LIF receptor are abundantly expressed on the surface of placental trophoblasts. The effect of IFN-gamma on T lymphocyte activation is influenced by the relative membrane density of its two receptors, particularly IFN-gamma R2. In this study we report that in PE (25-40 wk gestation) and PE complicated by IUGR, IFN-gamma R2 protein expression is severely down-regulated and is similar to that observed in early placenta (7-10 wk gestation) developing under low O(2) tension. IFN-gamma production was found to be inversely related to the IFN-gamma R2 protein expression, and LIF receptor protein expression in PE mimicked that in early placental development. These results show that in PE, placental trophoblasts fail to establish an early to late switch with respect to IFN-gamma and IFN-gamma R2 expression. This supports the hypothesis that trophoblasts control the polarization of maternal immune effectors and cytokine profiles at the fetal-maternal interface that could be subject to oxidative stress in PE.     

The diagnostic effectiveness of an initial transvaginal scan in detecting ectopic pregnancy

BACKGROUND: To determine the effectiveness of an initial transvaginal ultrasound scan (TVS) in the detection of ectopic pregnancy in consecutive women attending an early pregnancy unit (EPU). METHODS: This was a prospective observational study. Unselected women attending a dedicated EPU underwent a TVS. Women were classified as having an intrauterine pregnancy (IUP), ectopic pregnancy or pregnancy of unknown location (PUL). Women with a PUL were followed up until the final location of the pregnancy was determined. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) with 95% confidence intervals (CIs) for the initial TVS in the diagnosis of ectopic pregnancy were calculated. RESULTS: During a one-year study period, 5318 consecutive women attended the EPU. Outcome data were available for 5240 (98.5%) women. Of these, the initial TVS showed an IUP in 4693 (89.6%) cases and an ectopic pregnancy in 91 (1.7%) cases. The remaining 456 (8.7%) women were classified as PUL, and of these 31 (6.8%) were subsequently found to have ectopic pregnancies. The overall sensitivity of the initial TVS in the diagnosis of ectopic pregnancy was 73.9% (95% CI: 65.1-81.6) with a specificity of 99.9% (95% CI: 99.8-100), a PPV of 96.7% (95% CI: 90.7-99.3) and an NPV of 99.4% (95% CI: 99.2-99.6). CONCLUSIONS: In unselected women attending an EPU, pregnancy location can be diagnosed accurately in over 90% of all pregnancies and in 73.9% of ectopic pregnancies with a single TVS.     

Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine

In vivo observations on the kinetics of F cells and of fetal hemoglobin (HbF) synthesis and in vitro studies of erythroid progenitors, their number, and the gamma-gene expression in their progeny were carried out in baboons (Papio cynocephalus) treated with 5-azacytidine. Maximum effect on the increase of HbF production in vivo was observed only when an expanded erythroid marrow population was present. In these animals, as well as in normal animals, treatment resulted in a significant reduction of the late erythroid progenitor cell pools (erythroid clusters and erythroid colony-forming units, CFU-E) in the marrow. This reduction was more pronounced among those progenitors grown in the absence of added erythropoietin, and it was followed by a rebound a few days after treatment cessation, reflecting the accumulation of regenerating progenitors. An early increase in the in vitro synthesis of HbF in erythroid clusters and CFU-E colonies was observed. This increase was further documented at the cellular level, with immunofluorescent labeling of colonies with monoclonal anti-gamma- globin chain antibodies. In contrast to the findings in late progenitors, the number of erythroid burst-forming unit (BFU-E) colonies and the synthesis of HbF in these colonies was not influenced significantly by 5-azacytidine treatment. It is proposed that the toxic effects of 5-azacytidine on late progenitors, leading to faster mobilization of earlier progenitors to the next more mature compartment, play a role in the in vivo augmentation of HbF synthesis by this drug. This perturbation in the progenitor cell population kinetics and the presumed hypomethylation of the surviving differentiating cells may act synergistically to produce a maximum HbF response after 5-azacytidine treatment.     

Uterine artery Doppler in the prediction of adverse pregnancy outcome

PURPOSE OF REVIEW: To review publications, published during the past year, that have examined uterine artery Doppler findings in women with adverse pregnancy outcome. RECENT FINDINGS: Almost two-thirds of stillbirths that occur in the early preterm period (up to 32 weeks) can be predicted by uterine artery Doppler at 23 weeks. First trimester screening studies have shown that an abnormal result increases the risk of subsequent fetal growth restriction, and such women are at particularly high risk when indices remain abnormal in the second trimester. Studies combining uterine artery Doppler with maternal serum markers have demonstrated that measurement of first-trimester maternal serum pregnancy-associated plasma protein A and free beta human chorionic gonadotrophin improve sensitivities of second-trimester Doppler. As these are frequently measured in Down syndrome screening and they lend themselves in screening for pre-eclampsia. Women with abnormal first and second-trimester serum markers constitute a high-risk group. Maternal serum placental protein 13 remains a promising method for early screening, although a recent study suggests lower sensitivities than initially reported. SUMMARY: Uterine artery Doppler screening identifies women at high risk for developing adverse pregnancy outcomes. Detection rates may be increased and false positive rates reduced by combination with maternal characteristics or serum markers.     

Maternal uterine artery Doppler flow velocimetry and the risk of stillbirth

OBJECTIVE: We sought to relate the risk of antepartum stillbirth to uterine artery Doppler flow velocimetry at 22-24 weeks. METHODS: Data were available from 30,519 unselected women from seven units in the UK who had uterine artery Doppler performed between 22 and 24 weeks of gestation. The risk of stillbirth (n=109) was assessed using time to event and logistic regression analysis. Stillbirths were subdivided into placental (due to abruption, preeclampsia, or growth restriction) or unexplained. RESULTS: The risk of placental stillbirth was increased among women with a mean pulsatility index in the top decile (adjusted hazard ratio [HR] 5.5, 95% confidence interval [CI] 2.8-10.6) and those with a bilateral notch (adjusted HR 3.9, 95% CI 2.0-7.8). The relationship between a mean pulsatility index in the top decile and the risk of unexplained stillbirth was weaker (adjusted HR 2.5, 95% CI 1.1-5.6) and there was no association with a bilateral notch. Placental stillbirths occurred at earlier gestations than unexplained stillbirths (median [interquartile range] 30 [26-36] compared with 38 [36-40], P<.001). Consequently, being in the top 5% of predicted risk of stillbirth on the basis of the combination of mean pulsatility index and notching was a good predictor (sensitivity, specificity, and positive likelihood ratio) of all cause stillbirth up to 32 weeks (58%, 95%, and 12.1, respectively) but a poor predictor of stillbirth at later gestations (7%, 95%, and 1.3, respectively). CONCLUSION: Abnormal uterine artery Doppler was a better predictor of the risk of stillbirth due to placental causes than unexplained stillbirth. Consequently, abnormal uterine artery Doppler was a good predictor of stillbirth at extreme preterm gestations but a poor predictor of stillbirth at term. LEVEL OF EVIDENCE: II.     

Severe twin-twin transfusion syndrome: outcome after fetoscopic laser ablation of the placental vascular equator

Objective  To assess the safety and efficacy of a modified fetoscopic laser ablation technique for the management of severe twin–twin transfusion syndrome (TTTS) in a large series of pregnancies.
Design  Prospective cohort study.
Setting  Tertiary referral fetal medicine unit.
Population  Women with pregnancies complicated by severe TTTS (Quintero stage III or IV), before 26 weeks of gestation.
Methods  Fetoscopic laser ablation of placental anastomoses was performed. The sonoendoscopic approach was used to identify the placental vascular equator and to photocoagulate crossing vessels.
Main outcome measures  Overall survival, fetal and perinatal mortalities, gestational age at delivery, birthweight, operating time and recurrence of TTTS.
Results  A total of 77 women underwent the procedure. The mean gestational age at treatment was 20 (range 16–26) weeks. On average, four vessels were ablated during each procedure, with a mean operative time of 15 (range 5–25) minutes. None of the women required a repeat fetoscopic laser treatment for recurrence of the TTTS. There was at least one survivor in 74% (57/77) of pregnancies, and the overall survival rate was 57% (88/154).
Conclusions  Fetoscopic laser ablation is a safe and effective form of treatment in the management of severe TTTS. The technique of identifying the common villous district of the placenta by ultrasound and photocoagulating any vessels crossing the vascular equator appears to be an acceptable alternative to both the nonselective and highly selective methods described so far. This approach is associated with a short operating time, low likelihood of TTTS recurrence or fetal anaemia and with survival results that are equivalent to previously reported techniques.     

Human Calcitonin Delivery in Rats by Iontophoresis

Abstract— In-vitro iontophoresis (0·33 mA cm⁻²) of calcitonin (50 μg mL⁻¹, pH 4) was performed with the hairless rat skin model. Direct current was as potent as pulse current (2·5 kHz on/off 1/1) iontophoresis in promoting transdermal permeation of calcitonin. Increase in duration of current application from 20 min to 1 h did not increase calcitonin flux. Results suggest that calcitonin can be blocked in the skin pores through which it travels or can accumulate in the skin and be progressively released from the depot. Invivo experiments showed that transdermal iontophoretic administration of calcitonin induced a hypocalcaemic effect in rats.     

The efficacy of postoperative pain management with ketorolac for day case oral surgery

The efficacy of the non-steroidal anti-inflammatory analgesic, ketorolac (Toradol), was investigated in 52 day case patients undergoing removal of impacted third molar teeth under intravenous sedation and local analgesia. The study was double-blind, randomized and placebo-controlled. A single 30 mg dose of ketorolac was administered intravenously just prior to induction of sedation with midazolam. Ketorolac was well tolerated and provided good postoperative analgesia. It is suggested that ketorolac is a useful addition to the analgesic armamentarium and appropriately prescribed, provides good pain relief following day case oral surgery.