Interlaboratory evaluation of endotoxin analyses in agricultural dusts--comparison of LAL assay and mass spectrometry

Endotoxin exposure is associated with wheeze and asthma morbidity, while early life exposure may reduce risk of allergy and asthma. Unfortunately, it is difficult to compare endotoxin results from different laboratories and environments. We undertook this study to determine if lipopolysaccharide (LPS) extraction efficiency could account for differences among laboratories. We generated and collected aerosols from chicken and swine barns, and corn processing. We randomly allocated side-by-side filter samples to five laboratories for Limulus assay of endotoxin. Lyophilized aliquots of filter extracts were analyzed for 3-hydroxy fatty acids (3-OHFAs) as a marker of LPS using gas chromatography-mass spectrometry. There were significant differences in endotoxin assay and GC-MS (LPS) results between laboratories for all dust types (p < 0.01). Patterns of differences between labs varied by dust type. Relationships between assay and GC/MS results also depended on dust type. The percentages of individual 3-OHFA chain lengths varied across labs (p < 0.0001) suggesting that each lab recovered a different fraction of the LPS available. The presence of large amounts of particle associated LPS and absence of a freezing thawing cycle were associated with lower correlations between LPS and bioactivity, consistent with an absence of Limulus response to cell-bound endotoxin. These data suggest that extraction methods affect endotoxin measurements. The LAL methods may be most suitable when comparing exposures within similar environments; GC-MS offers additional information helpful in optimizing sample treatment and extraction. GC-MS may be of use when comparing across heterogeneous environments and should be considered for inclusion in future studies of human health outcomes.     

An Evaluation of Selenium Concentrations in Water, Sediment, Invertebrates, and Fish from the Republican River Basin: 1997–1999

The Republican River Basin of Colorado,Nebraska, and Kansas lies in a valley which contains PierreShale as part of its geological substrata. Selenium is anindigenous constituent in the shale and is readily leached intosurrounding groundwater. The Basin is heavily irrigated throughthe pumping of groundwater, some of which is selenium-contaminated, onto fields in agricultural production. Water,sediment, benthic invertebrates, and/or fish were collected from46 sites in the Basin and were analyzed for selenium to determinethe potential for food-chain bioaccumulation, dietary toxicity,and reproductive effects of selenium in biota. Resultingselenium concentrations were compared to published guidelines orbiological effects thresholds. Water from 38% of the sites (n = 18) contained selenium concentrations exceeding 5 g L^-1, which is reported to be a high hazard for selenium accumulation into the planktonic food chain. An additional 12 sites (26% of the sites) contained selenium in water between 3–5 g L^-1, constituting a moderate hazard. Selenium concentrations in sedimentindicated little to no hazard for selenium accumulation fromsediments into the benthic food chain. Ninety-five percent ofbenthic invertebrates collected exhibited selenium concentrationsexceeding 3 g g^-1, a level reported as potentially lethal to fish and birds that consume them. Seventy-five percent of fish collected in 1997, 90% in 1998, and 64% in 1999 exceeded 4 g g^-1selenium, indicating a high potential for toxicity andreproductive effects. However, examination of weight profilesof various species of collected individual fish suggestedsuccessful recruitment in spite of selenium concentrations thatexceeded published biological effects thresholds for health andreproductive success. This finding suggested that universalapplication of published guidelines for selenium may beinappropriate or at least may need refinement for systems similarto the Republican River Basin. Additional research is needed todetermine the true impact of selenium on fish and wildliferesources in the Basin.     

The interaction between physical and social-psychological factors in indoor environmental health

Indoor environmental health is now recognized as an important factor in preventing respiratory health problems in the United States. It is also a concern in Canada due to the amount of time that Canadians spend indoors because of cold weather and the potential for increased time indoors during the summer if theclimate warms. The negative health effects are often labeled assick building syndrome, but diagnosing a building or itsoccupants as sick is complicated by the variety of symptoms, thepresence of chronic versus acute symptoms and social andpsychological (socio-psychological) factors that may reduce theeffectiveness of an engineering solution. As a case study, thecontribution of various factors to indoor environmental health,in three buildings at the University of Toronto, was examinedusing five different methods. The results indicate that theinhabitants of the buildings consider features other than airquality in considering building health such as design,maintenance, funding cuts and socio-psychological factors.     

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.     

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.     

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.