An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.     

Prenatal diagnosis of herlitz junctional epidermolysis bullosa by amniocentesis

Herlitz junctional epidermolysis bullosa (HJEB) is a severe blistering disorder which usually results in death during infancy. We have previously shown that the anchoring filament protein laminin-5 (kalinin/nicein), which mediates keratinocyte attachment and dermal–epidermal cohesion, is abnormally expressed in individuals with HJEB. Laminin-5 was detected by Western blot analysis in amniotic fluid from 44 consecutive normal secondtrimester control pregnancies, but was undetectable in second-trimester amniotic fluid from four pregnancies with fetuses affected by HJEB. In one case of severe non-Herlitz JEB, laminin-5 was detected in both amniotic fluid and skin. In human amniotic fluid, the laminin-5 a3 subunit was processed to a major 165 kD species and a minor 145 kD species and the β2 subunit was partially processed to 105 kD. Although laminin-5 was covalently associated with laminin-6 (K-laminin) in amniotic membrane, no covalent interaction was detected in amniotic fluid. Laminin-5 from amniotic fluid strongly supported keratinocyte attachment. These results suggest that Western blot analysis of second-trimester amniotic fluid is useful in determining the prenatal diagnosis of HJEB and that laminin-5 may serve a physiologically important function in amniotic fluid.     

Transvaginal chorionic villus sampling

Chorionic villus sampling (CVS) with either transcervical catheters or transabdominal needles is a widely-accepted method for prenatal diagnosis. However, there exists a small subset of patients in whom sampling is difficult or impossible with either route because of individual anatomic variations. A new method of chorionic villus biopsy has been developed to circumvent these problems, utilizing transvaginal chorionic needle aspiration guided by an intravaginal ultrasound probe. This technique was performed successfully in 15 patients in whom villi could not be obtained by either of the conventional methods. This method now makes CVS possible in essentially all women regardless of their uterine anatomy or placental placement; it may also prove useful for very early chorionic sampling.     

Prenatal diagnosis of cystinosis utilizing chorionic villus sampling

The prenatal diagnosis of cystinosis is currently based on the increased amount of free-cystine present in amniotic fluid cells. Amniocyte cultures must be grown for at least 2 weeks to obtain sufficient cells for such measurements. Thus, the diagnosis cannot be made until close to 20 weeks gestational age by this method. We report a case in which chorionic villi were used for direct cystine measurement resulting in the in utero diagnosis of cystinosis at 9 weeks gestational age. The diagnosis was confirmed by the study of cultured chorionic villus cells, and of the 10-week abortus.     

Rapid prenatal diagnosis of Patau's syndrome in a fetus with an abdominal wall defect by 72 hour culture of cells from amniotic fluid

A woman in the 32nd week of pregnancy was referred for investigation because of fetal abnormalities, including an abdominal wall defect, detected by ultrasonography. In view of the increased risk of chromosome abnormality, amniocentesis was performed to enable informed decisions about the management of the pregnancy and delivery to be taken. Cells from the liquor were inoculated into standard lymphocyte culture medium and incubated for 72 h. Slides with a high mitotic index and good quality metaphases, comparable to those from a blood culture, were obtained after harvesting. Cytogenetic analysis showed the karyotype to be 46,XY,—14,+t(13ql4q), which is consistent with Patau's syndrome. This technique appears to be an option for rapid karyotyping in cases of abdominal wall defect, where a chromosomal abnormality is suspected.     

喜马拉雅中部药用植物的商业评估

本研究对尼泊尔全国药用植物的年产量及商业价值进行了评估.我们对尼泊尔15个地区的当地药用植物交易者(n=149)和印度的地区批发商(n=53)进行了无限制问卷调查,借以收集数据.据估计,年交易额的范围为7000～27 000t,案例年份1997～1998年的产量为14 500t.按印度主要市场的地区批发商购买价格计算,相应的年出口额估计为700～3000万美元,1997～1998年为1600万美元.约有10%的农户参与了商品化采集.低海拔生态区在产量和价值两方面均占优势;就价值而言,药草是最重要的生活型.约有36%的产量和51%的价值来自破坏性采集.有人认为,年产量和价值数据仍有可能属于保守估计.本文还简短讨论了本研究结果对喜马拉雅药用植物保护及贸易的意义.     

Can Forest-protection carbon projects improve rural livelihoods? Analysis of the Noel Kempff Mercado climate action project,Bolivia

We studied the Noel KempffMercado Climate Action Project (NKMCAP),Bolivia, to assess whether forestprotection carbon (C) projects cansignificantly benefit local people. Wehypothesized that forest protection canonly securely deliver C if significantstakeholders are meaningfully andtransparently involved, traditional orcustomary rights are recognized and theirloss compensated for, and there are directlinkages between conservation anddevelopment objective. Our researchfocused on 53 members of the communities ofFlorida, Porvenir and Piso Firme and 36secondary stakeholders. In each of thevillages we held half-day meetings withcommunity leaders, complemented bysemi-structured one-hour interviews with 5,10, and 7 families, representing 20%, 10%and 8% of each community. The long-termimpact of the NKMCAP on the localcommunities may well be positive. However,in the short run, certain sections of thelocal communities are financially poorer. Forest protection projects clearly have thepotential to sequester C, protectbiodiversity and simultaneously contributeto sustainable rural development, but ifthey really are to improve rurallivelihoods, they must be designed andimplemented carefully and participatively.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Urban Flood Damage and Greenhouse Scenarios - The Implications for Policy: An Example from Australia

Urban flooding is often used as an illustration of the potentially adverse effects of greenhouse-induced climate change on extreme events. There is however, a paucity of studies that convert climate scenarios into changes in flood damage. This account summarises the use of modelling techniques, for three flood prone urban catchments in south eastern Australia, to assess changes to urban flood losses for the 'most wet' and 'most dry' scenarios for the year 2070. The most wet scenario indicates that annual average flood damage could increase within the range of 2.5 to 10 times, under the most dry scenario flood regimes would be similar to those experienced at present. The socio-economic scenarios based on the changes to flood losses are used to consider policy responses. It is unlikely that many local government authorities will respond because of lack of interest and because of major changes to the climate scenarios proposed over the last decade. Any response is likely to be incremental and accord with the 'no regrets' and the precautionary principle'.