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A systematic review of interventions to prevent childhood farm injuries.

OBJECTIVE: The goal of the study was to systematically review the global body of evidence surrounding the effectiveness of interventions for the prevention of acute pediatric agricultural injuries. A specific focus was the effectiveness of the North American Guidelines for Children's Agricultural Tasks. METHODS: Two reviewers independently screened studies and applied inclusion criteria on the basis of searches of 17 bibliographic databases (eg, Medline and Embase). We also screened reference lists of relevant studies and contacted experts in the area. Studies were included if they represented primary research, a comparison group was used, the study population included children or the intervention was directly applicable to children, and objective outcomes were reported. Two reviewers independently assessed the methodologic quality of included studies with the Downs and Black checklist. A qualitative analysis was performed because of extensive heterogeneity among studies. RESULTS: We included 23 controlled studies, ie, 4 randomized, controlled trials, 5 controlled trials, and 14 quasiexperimental or observational studies. Only 8 of the relevant studies were published in peer-reviewed journals. School-based programs appeared to be effective at increasing short-term knowledge acquisition; outcomes were enhanced with active, hands-on participation, as opposed to passive activities. Safety day camps showed positive results for knowledge acquisition. Tractor training programs and community- and farm-based interventions showed mixed results. Studies examining the North American Guidelines for Children's Agricultural Tasks suggested that uptake improves if dissemination is accompanied by a farm visit from a safety specialist or if information about child development principles is provided in conjunction with the guidelines. CONCLUSIONS: There is a lack of randomized, controlled trials and community-based trials in this area. Studies primarily examined intermediate outcomes, such as knowledge acquisition; few studies evaluated changes in injury rates. The interventions targeted at children and youths that were evaluated focused on educational interventions. There is both the need and potential for the development and evaluation of injury control interventions for children, particularly programs addressing lethal injuries to young/preschool-aged children.

Accident Prevention↗

Cell remodeling in the fat body of an insect.

The fat body of the Lepidopteran, Calpodes ethlius, undergoes major functional changes during larval-adult metamorphosis. These changes occur in conjunction with extensive cell remodeling - a process whereby one population of cellular organelles is destroyed and replaced by another during development. Fat body organelles including mitochondria, microbodies, and RER are destroyed on a massive scale shortly before pupation (Locke and Collins, 1965; Locke and McMahon, 1971) a new populations of each are regenerated shortly after emergence of the adult. In addition, protein, lipid and RNA reserves formed shortly before pupation and multivesicular bodies formed shortly before emergence are secreted into the haemocoel during the first few days of adult life. Electron microscopic studies using tracer techniques, cytochemical and enzyme localization procedures, and sterological analyses have been undertaken to determine the time course and mechanism of organelle regeneration and the fate of reserves stored in the fat body.

Acid Phosphatase↗

Effectiveness of different types of clay for reducing the detrimental effects of aflatoxin-contaminated diets on performance and serum profiles of weanling pigs.

Three trials were conducted with recently weaned pigs (n = 198) to determine the effects of feeding different types of clay in conjunction with aflatoxin-contaminated diets. In Trial 1, pigs (n = 54; trial length 4 wk) were assigned to either an uncontaminated treatment (NC), 800 ppb of aflatoxin from contaminated corn (AC), or AC with one of four clays. In Trial 2 (n = 81; trial length 5 wk), pigs were assigned to NC, AC (500 ppb of aflatoxin from rice starch), or AC with one of seven types of clay. In both trials, pigs fed AC had decreased ADG and gain:feed ratios (P < .05) compared with controls. The clays differed in their ability to produce gains similar to those of controls. The clays did reduce changes in the serum measurements normally affected by aflatoxin, including albumin, total protein, gamma glutamyltransferase (GGT), and alkaline phosphatase (ALP) levels, in a manner similar to their effect on ADG. In Trial 3, pigs (n = 63) were assigned to one of seven diets for 4 wk: NC, AC (800 ppb of aflatoxin) with no clay, AC with one of four levels of a treated Ca bentonite (.25, .5, 1, and 2%), or AC and .5% hydrated sodium calcium aluminosilicate. The addition of treated Ca bentonite to AC improved ADG (P < .05) and ADFI (P < .01) linearly. Gain:feed ratios were not affected by treatments. The inclusion of treated Ca bentonite to the AC diet linearly decreased aspartate aminotransferase (AST) levels and quadratically decreased ALP and GGT levels (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗

Sublingual immunotherapy for allergic rhinoconjunctivitis--the seeming and the real.

BACKGROUND: Subcutaneous specific immunotherapy (SIT) has been shown to be a causal treatment with long-term efficacy for allergic rhinoconjunctivitis and asthma, and a preventive measure against the development of asthma and new sensitizations. As it is associated with several inconveniences and serious side effects, sublingual immunotherapy (SLIT) has been developed to evade these problems. METHODS: The present review of previously published studies on allergic rhinoconjunctivitis aimed to determine the efficacy of SLIT in comparison with subcutaneous treatment and to summarize long-term results of immunotherapy and its effects on the prevention and treatment of asthma and the prevention of new sensitizations. RESULTS: The effect of SLIT on allergic rhinoconjunctivitis is low to moderate, depends on the allergen used and is more pronounced in adults than in children, in whom a consistent effect has not been demonstrated. Direct comparison with SIT shows conflicting and inconsistent results. Detailed studies on the prevention of asthma and new sensitizations are not available. Consistent effects of asthma treatment on both symptom and medication scores and lung function have not been reported. A quantitative evaluation is not possible due to indistinct inclusion criteria and different outcome criteria. In summary, currently SLIT plays no significant role in the treatment of asthma, apart from children monosensitized to house dust mites in whom it may have low-moderate effects. Only one study deals with the long-term efficacy of SLIT, which demonstrated a persistent positive effect on asthma, whereas data on rhinoconjunctivitis are completely lacking. CONCLUSIONS: Primary and secondary targets of specific immunotherapy have not been answered satisfactorily for the sublingual route. To date, SLIT cannot be recommended as an adequate alternative to the subcutaneous form. Questions regarding the cumulative dose, duration of therapy and immunological mechanisms have also not been answered. The indication should thus be limited to adult patients with pollen-induced rhinoconjunctivitis being unable to perform SIT, e.g. due to significant side effects.

Administration, Sublingual↗

Vital staining properties of neutral red. Vital staining of cornea and conjunctiva.

Vital staining by instillation in the conjunctival sac of 1% neutral red in 54 normal and 153 differently affected eyes was studied by slit lamp examination. Of these, 134 eyes were after-stained by a mixture of 1% tetrazolium and 1/4% alcian blue and 73 eyes were after-stained by a mixture of 1% rose bengal and 1% fluorescein. Microscopy of a further 10 conjunctival scrapings and 40 mucous conjunctival threads disclosed neutral-red-stained inclusion bodies in the cytoplasm of the epithelial cells and the granulocytes. In the slit lamp the cornea and the conjunctiva were seen to be stained only rarely and, if so, poorly by neutral red, both in normal and affected eyes. Better staining was obtained with tetrazolium, and the best with rose bengal. The plica semilunaris, Marx' line, and the mucous thread were stained by neutral red, more intensely by rose bengal, and the least by tetrazolium. The inferior fornix and the tarsus were rarely stained by any of the three dyes. Neutral red seems to offer no diagnostic advantages over the vital stains with which the dye has been compared in the present study. More particularly we found no pathological processes that were stained more intensely by neutral red than by the other vital stains.

Cataract↗

Interventions for replacing missing teeth: bone augmentation techniques for dental implant treatment.

BACKGROUND: Dental implants require sufficient bone to be adequately stabilised. For some patients implant treatment would not be an option without bone augmentation. A variety of materials and surgical techniques are available for bone augmentation. OBJECTIVES: General objectives: To test the null hypothesis of no difference in the success, function, morbidity and patient satisfaction between different bone augmentation techniques for dental implant treatment. SPECIFIC OBJECTIVES: (A) to test whether and when augmentation procedures are necessary; (B) to test which is the most effective augmentation technique for specific clinical indications. Trials were divided into three broad categories according to different indications for the bone augmentation techniques: (1) major vertical or horizontal bone augmentation or both; (2) implants placed in extraction sockets; (3) fenestrated implants. SEARCH STRATEGY: The Cochrane Oral Health Group's Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE were searched. Several dental journals were handsearched. The bibliographies of review articles were checked, and personal references were searched. More than 55 implant manufacturing companies were also contacted. Last electronic search was conducted on 1 October 2005. SELECTION CRITERIA: Randomised controlled trials (RCTs) of different techniques and materials for augmenting bone for implant treatment reporting the outcome of implant therapy at least to abutment connection. DATA COLLECTION AND ANALYSIS: Screening of eligible studies, assessment of the methodological quality of the trials and data extraction were conducted independently and in duplicate. Authors were contacted for any missing information. Results were expressed as random-effects models using weighted mean differences for continuous outcomes and odd ratios for dichotomous outcomes with 95% confidence intervals. The statistical unit of the analysis was the patient. MAIN RESULTS: Thirteen RCTs out of 29 potentially eligible trials reporting the outcome of 330 patients were suitable for inclusion. Since different techniques were evaluated in different trials, no meta-analysis could be performed. Six trials evaluated different techniques for vertical or horizontal bone augmentation or both. Four trials evaluated different techniques of bone grafting for implants placed in extraction sockets and three trials evaluated different techniques to treat bone dehiscence or fenestrations around implants. AUTHORS' CONCLUSIONS: Major bone grafting procedures of extremely resorbed mandibles may not be justified. Bone substitutes (Bio-Oss or Cerasorb) may replace autogenous bone for sinus lift procedures of extremely atrophic sinuses. Both guided bone regeneration (GBR) procedures and distraction osteogenesis can augment bone vertically, but it is unclear which is the most efficient technique. It is unclear whether augmentation procedures at immediate single implants placed in fresh extraction sockets are needed, and which is the most effective augmentation procedure, however, sites treated with barrier + Bio-Oss showed a higher position of the gingival margin, when compared to sites treated with barriers alone. Non-resorbable barriers at fenestrated implants regenerated more bone than no barriers, however it remains unclear whether such bone is of benefit to the patient. It is unclear which is the most effective technique for augmenting bone around fenestrated implants. No bone promoting molecule has been shown to be effective or necessary in conjunction with dental implant treatment. The use of particulated autogenous bone from intraoral locations, also taken with dedicated aspirators, might be associated with an increased risk of infective complications. These findings are based on few trials including few patients, having sometimes short follow up, and being often judged to be at high risk of bias.

Animals↗

Comparison of the effectiveness of inhaler devices in asthma and chronic obstructive airways disease: a systematic review of the literature.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) are common diseases of the airways and lungs that have a major impact on the health of the population. The mainstay of treatment is by inhalation of medication to the site of the disease process. This can be achieved by a number of different device types, which have wide variations in costs to the health service. A number of different inhalation devices are available. The pressurised metered-dose inhaler (pMDI) is the most commonly used and cheapest device, which may also be used in conjunction with a spacer device. Newer chlorofluorocarbons (CFC)-free inhaler devices using hydrofluoroalkanes (HFAs) have also been developed. The drug is dissolved or suspended in the propellant under pressure. When activated, a valve system releases a metered volume of drug and propellant. Other devices include breath-actuated pMDIs (BA-pMDI), such as Autohaler and Easi-Breathe. They incorporate a mechanism activated during inhalation that triggers the metered-dose inhaler. Dry powder inhalers (DPI), such as Turbohaler, Diskhaler, Accuhaler and Rotahaler, are activated by inspiration by the patient. The powdered drug is dispersed into particles by the inspiration. With nebulisers oxygen, compressed air, or ultrasonic power is used to break up solutions or suspensions of medication into droplets for inhalation. The aerosol is administered by mask or by a mouthpiece. There has been no previous systematic review of the evidence of clinical effectiveness and cost-effectiveness of these different inhaler devices. OBJECTIVES: To review systematically the clinical effectiveness and cost-effectiveness of inhaler devices in asthma and COPD. METHODS: The different aspects of inhaler devices were separated into the most clinically relevant comparisons. Methods involved systematic searching of electronic databases and bibliographies for randomised controlled trials (RCTs) and systematic reviews. Pharmaceutical companies and experts in the field were contacted for further information. Trials that met the inclusion criteria were appraised and data extraction was under-taken by one reviewer and checked by a second reviewer, with any discrepancies being resolved through agreement. RESULTS--IN VITRO CHARACTERISTICS VERSUS IN VIVO TESTING AND CLINICAL RESPONSE: There is evidence that when comparative testing is performed on inhaler devices using the same methods, there is some correlation between particle size measurements and clinical response. However, the measurements are dependent upon the methods used, and a single measure of a device in isolation is of limited value. Also, there is little data on comparing devices of different types. There is currently insufficient data to verify the ability of in vitro assessments to predict inhaler performance in vivo. RESULTS--EFFECTIVENESS OF METERED-DOSE INHALERS FOR THE DELIVERY OF CORTICOSTEROIDS IN ASTHMA: The review of three trials in children and 21 trials in adults demonstrated no evidence to suggest clinical benefits of any other inhaler device over a pMDI in corticosteroid delivery. RESULTS--EFFECTIVENESS OF METERED-DOSE INHALERS FOR THE DELIVERY OF BETA-AGONISTS IN STABLE ASTHMA: In children, 11 studies were reviewed, of which seven compared the Turbohaler with the pMDI. One study found a significant treatment difference in peak expiratory flow rate, although there were differences in the patients' baseline characteristics. In adults, a review of 70 studies found no demonstrable difference in the clinical bronchodilator effect of short-acting b2-agonists delivered by the standard pMDI compared with that produced by any other DPI, HFA-pMDI or the Autohaler device. The finding that HFA-pMDIs may reduce treatment failure and oral steroid requirement in beta-agonist delivery needs further confirmatory research in adequately randomised clinical trials. RESULTS--EFFECTIVENESS OF NEBULISERS VERSUS METERED-DOSE INHALERS FOR THE DELIVERY OF BRONCHODILATORS IN STABLE ASTHMA: In children, three included trials compared different devices with a nebuliser and demonstrated no evidence of clinical superiority of nebulisers over inhaler devices in bronchodilator delivery. A total of 23 studies in adults found no equivalence for the main pulmonary outcomes and no evidence of difference in other outcomes. RESULTS--EFFECTIVENESS OF METERED-DOSE INHALERS FOR THE DELIVERY OF BETA-AGONISTS IN COPD: Only two studies were included in this review. No evidence of clinical difference was found in beta-agonist delivery. RESULTS--EFFECTIVENESS OF NEBULISERS VERSUS METERED-DOSE INHALERS FOR THE DELIVERY OF BRONCHODILATORS IN COPD: Evidence from 14 trials demonstrated equivalence for the main outcomes of pulmonary function. For other outcomes there was no evidence of treatment difference in bronchodilator delivery. RESULTS--PATIENTS' ABILITY TO USE METERED-DOSE INHALERS: Differences among studies and the heterogeneity of the results make it difficult to draw conclusions about inhaler technique differences between device types. The review of technique after teaching the correct technique suggests that there is no difference in patients' ability to use DPI or pMDIs. RESULTS--ECONOMIC ANALYSIS: The total number of NHS prescriptions for inhaler therapy for asthma in 1998 was over 31 million, with a net ingredient cost in excess of 392 million GB pounds. This economic assessment uses decision analysis to estimate the relative cost-effectiveness of inhaler devices for the delivery of bronchodilator and corticosteroid inhaled therapy. Overall, there were no differences in patient outcomes among the devices. On the assumption that the devices were clinically equivalent, pMDIs were the most cost-effective devices for asthma treatment. CONCLUSIONS: This systematic review examined the evidence from clinical trials evaluating the clinical effectiveness of different inhaler devices in the delivery of inhaled corticosteroids and beta2-bronchodilators for patients with asthma and COPD. The evidence from the published clinical literature demonstrates no difference in clinical effectiveness between nebulisers and alternative inhaler devices compared to standard pMDI with or without a spacer device. The cost-effectiveness evidence therefore favours pMDIs (or the cheapest inhaler device) as first-line treatment in all patients with stable asthma unless other specific reasons are identified. Patients can use pMDIs as effectively as other inhaler devices as long as the correct inhalation technique is taught. CONCLUSIONS--RECOMMENDATIONS FOR RESEARCH: Further clinical trials are required to demonstrate any differences in the clinical effectiveness and cost-effectiveness of inhaler devices and nebulisers compared with pMDIs. These should be of sufficient statistical power and methodological rigour to demonstrate any clinical benefit. Trials should be undertaken in community settings to ensure the generalisability of results. Outcome measures should be more patient-centred and report adverse effects more completely. Reporting of data from trials should be improved.

Adolescent↗

Radiosurgery alone or in combination with whole-brain radiotherapy for brain metastases.

PURPOSE: Evaluation of the treatment outcome after radiosurgery (RS) alone or in combination with whole-brain radiotherapy (WBRT) with special attention to prescribed dose and its influence on local control and survival. PATIENTS AND METHODS: Between September 1984 and January 1997, 236 patients with 311 brain metastases treated with radiosurgery met the following inclusion criteria: one to three brain metastases per patient; no previous WBRT; and Kamofsky performance status (KPS) > or = 50%. One hundred fifty-eight patients treated only with RS received a median dose of 20 Gy prescribed to the 80% isodose line; 78 patients received RS with a median dose of 15 Gy/80% and an additional course of WBRT. RESULTS: For the entire series, overall median survival was 5.5 months, with control of CNS disease achieved in 92% of the treated brain metastases; the results were not significantly different between patients treated by RS with or without WBRT. However, in patients without evidence of extracranial disease, median survival was increased for patients who received WBRT (15.4 vs 8.3 months; P=.08). Additionally, there was a suggestion that increased doses for patients treated with RS only resulted in improved outcome. Four lesions were suspicious for radiation necrosis by magnetic resonance imaging (MRI); in one of the four lesions, radiation necrosis was confirmed histologically. The incidence of transient low-grade toxicity was 18%; symptoms could be treated by the temporary administration of steroids. CONCLUSION: RS is an effective, noninvasive means of controlling brain metastases when used alone or in combination with WBRT. There is a trend for superior local control and especially in patients without extracranial disease for superior survival when RS is used in conjunction with WBRT. Randomized trials would seem to be warranted, comparing the benefit of RS with or without additional WBRT.

Brain Neoplasms↗

The histopathology of experimental trachoma: ultrastructural changes in the conjunctival epithelium.

Experimental acute conjunctivitis was produced in cynomolgus monkeys by ocular inoculation with serovar B of Chlamydia trachomatis. The cellular responses to chlamydial conjunctivitis infection were examined by light, transmission, and scanning electron microscopy. A self-limited, acute conjunctivitis resulted from a single primary inoculation. A moderate lymphocytic infiltrate was present in the conjunctiva. After repeated inoculation, a chronic conjunctivitis (trachoma) developed. Prominent lymphoid follicles with distinct germinal centers were present in these tissues. Scanning electron microscopy revealed patchy areas of cellular alteration and loss of microvilli. Intracellular injury, documented by transmission electron microscopy, included disruption of the plasmalemmal membranes and rupture of the cytoplasmic organelles. The inflammatory infiltrate consisted of plasma cells, polymorphonuclear cells, lymphocytes, eosinophils, and degranulating mast cells. The immune response following single and repeated chlamydial infections was characterized by immunoperoxidase staining with monoclonal antibodies to pan-leukocytes, macrophages, and B cells. The center of the follicle was comprised of B cells, with T cells in the cap region. Large macrophages were also found in the germinal center. Further study is required to determine which cellular mechanisms are involved in the histopathologic and immunologic alterations induced in the conjunctiva after chlamydial infection.

Animals↗

Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study).

BACKGROUND: Children with allergic rhinitis are likely to develop asthma. OBJECTIVE: The purpose of this investigation was to determine whether specific immunotherapy can prevent the development of asthma and reduce bronchial hyperresponsiveness in children with seasonal allergic rhinoconjunctivitis. METHODS: From 6 pediatric allergy centers, 205 children aged 6 to 14 years (mean age, 10.7 years) with grass and/or birch pollen allergy but without any other clinically important allergy were randomized either to receive specific immunotherapy for 3 years or to an open control group. All subjects had moderate to severe hay fever symptoms, but at inclusion none reported asthma with need of daily treatment. Symptomatic treatment was limited to loratadine, levocabastine, sodium cromoglycate, and nasal budesonide. Asthma was evaluated clinically and by peak flow. Methacholine bronchial provocation tests were carried out during the season(s) and during the winter. RESULTS: Before the start of immunotherapy, 20% of the children had mild asthma symptoms during the pollen season(s). Among those without asthma, the actively treated children had significantly fewer asthma symptoms after 3 years as evaluated by clinical diagnosis (odds ratio, 2.52; P <.05). Methacholine bronchial provocation test results improved significant in the active group (P <.05). CONCLUSION: Immunotherapy can reduce the development of asthma in children with seasonal rhinoconjunctivitis.

Adolescent↗

Timed intercourse versus intra-uterine insemination with or without ovarian hyperstimulation for subfertility in men.

BACKGROUND: Although intra-uterine insemination (IUI) is widely used, however its effectiveness remains a matter of debate. Although IUI is less invasive and expensive than IVF or GIFT, it should only be applied if the probability of conception is improved significantly as compared to the natural chance of conceiving. To increase the number of available oocytes at the site of fertilization, controlled ovarian hyperstimulation (COH) can be applied in conjunction with IUI. Uncontrolled studies suggest a beneficial effect of COH in combination with IUI, also when a male factor is present. To be able to draw firm conclusions whether IUI and/or COH improve the probability of conception, several comparisons should be performed in randomized controlled trials (RCTs). OBJECTIVES: To determine for male subfertility whether intrauterine insemination (IUI) improves the probability of conception compared with timed intercourse and whether the addition of controlled ovarian hyperstimulation influences the results. SEARCH STRATEGY: 1. The specialist database of the Cochrane Menstrual Disorders and Subfertility Group. 2. Medline search. 3. Embase search. 4. DDFU search. 5. BIOSIS search. 6. SCIsearch. 7. Manual searching of references mentioned in the obtained studies. 8. Personal communication and write letters to experts (14) in the field. 9. Abstracts of The American Society for Reproductive Medicine and European Society for Human Reproduction and Embryology Meetings. When important information is lacking from the original publications the authors will be contacted. SELECTION CRITERIA: Randomized controlled trials only. DATA COLLECTION AND ANALYSIS: Independently by the first 2 authors: 1. Trial design characteristics. 2. Baseline characteristics of participants. 3. Types of intervention. 4. Outcomes where pregnancy is the outcome of main interest. Number of multiple pregnancies and number of cycles with ovarian hyperstimulation syndrome (OHSS) are secondary outcomes. Analysis of agreement between the two observers was determined for the following items: inclusion or exclusion of a trial, method of randomization, definition of male subfertility, design of the trial, number of pregnancies and completed cycles. Sensitivity analysis is performed. MAIN RESULTS: Seventeen trials fulfilled the selection criteria for this review and were included. Four trials are pending. Crude agreement concerning inclusion or exclusion of trials occurred for 41 of 43 (95%) trials reviewed (kappa 0.90). The included trials comprised 3,662 completed cycles. In natural cycles intrauterine insemination (IUI) significantly improved the probability of conception compared with timed intercourse (TI) (combined odds ratio with 95% confidence intervals: 2.43, 1.54 - 3.83). In cycles with controlled ovarian hyperstimulation (COH) IUI significantly improved the probability of conception also compared with TI (combined odds ratio with 95% confidence intervals: 2.14, 1.30 - 3.51). Despite clinical heterogeneity, these results are based on strong evidence. Intrauterine insemination in cycles with COH improved the probability of conception compared with IUI in natural cycles but significance was not reached (combined odds ratio with 95% confidence intervals: 1.79, 0.98 - 3.25). Comparing IUI in COH-cycles with TI in natural cycles the first treatment modality significantly improved the probability of conception (combined odds ratio with 95% confidence intervals: 6.23, 2.35 - 16.52). REVIEWER'S CONCLUSIONS: Intra-uterine insemination offers couples with male subfertility benefit over timed intercourse, both in natural cycles and in cycles with COH. In the case of a severe semen defect (with more than 1 million motile sperm after semen preparation and no triple sperm defect) IUI in natural cycles should be the treatment of first choice. The value of COH need to be further investigated in RCTs. Mild ovarian hyperstimulation with gonadotrophins is advised in cases with less sever

Coitus↗

Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep.

BACKGROUND: Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres. RESULTS: Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype--VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171--allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002-2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study. CONCLUSION: The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.

Animals↗

The alternatively spliced V region contributes to the differential incorporation of plasma and cellular fibronectins into fibrin clots.

During blood clot formation in vivo, plasma fibronectin (pFN) is cross-linked to fibrin by coagulation factor XIIIa. Cellular FN (cFN), which localizes to connective tissue, is distinguished from pFN by the inclusion of alternatively spliced segments. To determine if these two FNs are functionally equivalent in blood clotting, the cross-linking of rat pFN and cFN to fibrin was compared in an in vitro clotting assay. Fibrinogen and FN were incubated at physiological ratios in the presence of thrombin and factor XIIIa. Cross-linking of FN to fibrin was monitored by SDS-PAGE and immunoblotting. Over 24 h, cFN was incorporated at a significantly slower rate than pFN and was not completely cross-linked to fibrin at a temperature that favors this interaction (0 degrees C). This difference was observed with purified fibrinogens from human, rat, and bovine and with rat plasma and was maintained even after incubation of pFN with rat fibroblasts for several days. Using the same assay, purified recombinant V(+)-V0 and V(+)-V+ FN dimers resembling pFN and cFN, respectively, showed a similar difference in cross-linking kinetics. These results suggest that the asymmetric distribution of the V region among pFN dimers plays a role in regulating its incorporation into blood clots. In fibrin clots, cFN was converted into a set of cross-linked intermediates distinct from those of pFN. For example, while pFN was initially cross-linked into a pFN-fibrin alpha heterodimer, this product was not a major intermediate in clots formed with cFN. This finding, in conjunction with evidence for the formation of factor XIIIa-catalyzed cFN-cFN cross-links, indicated that cFN molecules interact with each other, and with fibrin, differently from pFN. Together, these results show an important functional distinction between pFN and cFN.

Alternative Splicing↗

Measurement of IgG antibodies to Chlamydia trachomatis by commercial enzyme immunoassays and immunofluorescence in sera from pregnant women and patients with infertility, pelvic inflammatory disease, ectopic pregnancy, and laboratory diagnosed Chlamydia psittaci/Chlamydia pneumoniae infection.

BACKGROUND: Screening for Chlamydia trachomatis specific antibodies is valuable in diagnosing asymptomatic pelvic inflammatory disease (PID) and tubal damage following repeated episodes of PID. The assays in current use are unsuitable for screening large numbers of samples so there is a need to develop more suitable assays. AIMS: To compare the performance of several commercial C trachomatis enzyme immunoassays (EIAs) (SeroCT, C tracho(pep), Medac p-EIA, Vircell and Labsystems C trachomatis IgG EIAs) using major outer membrane protein (MOMP), an inactivated organism EIA (Genzyme Virotech EIA), and a genus specific EIA (Platelia Chlamydia IgG) with the whole cell inclusion immunofluorescence (WIF) assay. In addition, to adapt, using time resolved fluorescence technology, the assay showing the highest correlation with WIF. METHODS: Ninety sera from patients presenting with ectopic pregnancies, 187 sera from those with a variety of types of infertility, 33 sera from cases of PID where a fourfold rise in WIF titre occurred, and 90 sera from antenatal clinic attenders were tested. A panel of 36 sera from laboratory diagnosed cases of Chlamydia psittaci/Chlamydia pneumoniae infection was also tested. RESULTS: The Genzyme Virotech EIA showed the highest rank correlation coefficient (0.82) with WIF, particularly at high WIF titres. The MOMP specific assays varied in their correlation with WIF, with rank correlation coefficients ranging from 0.70 (Medac p-EIA) to 0.80 (Vircell EIA). The Genzyme Virotech assay showed poor specificity (5.6%; 95% confidence interval (CI), 0.68% to 18.7%)--it was reactive with 34 of the panel of 36 C psittaci/C pneumoniae positive sera. The MOMP based EIAs showed high specificity, particularly the Medac p-ELISA (97.2%; 95% CI, 85.5% to 99.9%)--only one serum was reactive. In view of the good correlation between WIF and the Genzyme Virotech EIA, a time resolved fluorescence immunoassay (TRFIA) was developed using the Genzyme Virotech antigen. Using an appropriate cut off the TRFIA assay showed excellent correlation with WIF. CONCLUSIONS: The TRFIA assay may be useful as a screening assay, possibly in conjunction with one of the highly specific EIAs studied (for example, Medac p-EIA) to confirm the antibody specificity of sera selected by the screening assay.

Antibodies, Bacterial↗

HLAMatchmaker-based strategy to identify acceptable HLA class I mismatches for highly sensitized kidney transplant candidates.

HLAMatchmaker determines HLA compatibility at the level of polymorphic amino acid triplets in antibody-accessible sequence positions. Recent studies have shown that among HLA-DR-matched kidney transplants, the HLA-A,B antigen mismatches which are compatible at the triplet level have almost identical graft survival rates as the zero-HLA-A,B antigen mismatches. This finding provides the basis of a new strategy to identify HLA-mismatched organs that have similar success rates as the zero-HLA-antigen mismatches. This report describes how in conjunction with the Acceptable Mismatch program in Eurotransplant, HLAMatchmaker can expand the pool of potential donors for highly sensitized patients, for whom it is very difficult to find a compatible transplant. Sera from 35 highly sensitized kidney transplant candidates with an average PRA of 96% were screened by lymphocytotoxicity with HLA-typed panels that included cells that were selectively mismatched for one or two HLA antigens for each patient. Acceptable and unacceptable HLA-A,B antigen mismatches were determined from the serum reactivity with the cell panel. HLAMatchmaker analysis was applied to identify additional HLA class I antigens that were matched at the triplet level. For each patient, we calculated the probability of finding a donor (PFD) in the different match categories from HLA gene frequencies in the kidney donor population. The median PFD for a zero-antigen mismatch was 0.025%. Matching at the triplet level increased the median PFD to 0.037% ( P = 0.008). The median PFD was 0.058% for a 0-1-triplet mismatch and 0.226% for a 0-2-triplet mismatch. Serum screening identified acceptable antigen mismatches for 28 of 35 highly sensitized patients, and the median PFD increased to 0.307% for a zero/acceptable antigen mismatch. The application of HLAMatchmaker permitted for 33 patients (or 92%) the identification of additional antigens that were acceptable at the triplet level, and the median PFD for a zero/acceptable triplet mismatch went up to 0.425%. Inclusion of one-triplet mismatches increased the median PFD to 1.112%. Validation studies have shown that patient sera reacted with none of the zero-triplet-mismatched antigens, 8-13% of the one-triplet mismatches, and 12-19% of the two-triplet mismatches. Although most antigens with one or two mismatched triplets appear acceptable to highly sensitized patients, a serum analysis must ascertain that the patient's antibodies do not recognize such mismatched triplets. HLAMatchmaker offers a useful strategy of identifying more donors with acceptable HLA mismatches and could alleviate the problem of accumulation of highly sensitized patients on the transplant waiting list.

Algorithms↗

Effectiveness of in situ air sparging for removing NAPL gasoline from a sandy aquifer near Perth, Western Australia.

In situ air sparging has the potential to augment the removal of nonaqueous phase liquid (NAPL) contaminants in soil vapour extraction (SVE) systems when the NAPL is present in the capillary fringe or below the water table. NAPL removal can also be enhanced from above the water table by improving air access. Results are presented from a pilot-scale field trial aimed at evaluating the performance of such a remediation strategy where in situ air sparging was used in conjunction with a soil vapour extraction system to remove weathered gasoline NAPL from an unconfined sandy aquifer. A simple analysis that partitions extracted soil vapour between air injected through the sparge well and air drawn from the atmosphere across the soil surface, together with petroleum hydrocarbon concentrations in the extracted soil vapour, was used to interpret the effectiveness of air sparging. The composition and mass of the NAPL in the aquifer were also monitored along with observations on the distribution of air in the aquifer. Results showed that sparged air constituted 42% of the extracted soil vapour but contributed the majority of the petroleum hydrocarbons removed. For the first 5 days of sparging, hydrocarbon concentrations in the sparged air were in equilibrium with the NAPL in the aquifer leading to total petroleum hydrocarbon concentrations in the combined system being three to four times greater than for soil vapour extraction alone. Petroleum hydrocarbon concentrations in the extracted soil vapour decreased over time as a result of the depletion of the more volatile constituents from the NAPL, possible development of mass transfer limitations and increased fraction of clean air from depleted zones. Overall, 65% of the 673 kg of petroleum hydrocarbons extracted in soil vapour over a period of 30 days was carried in sparged air from the single sparge well. Percentages of the mass carried in the sparged air were even higher (median 70%) for individual aromatic hydrocarbons. Inclusion of air sparging increased the mass extracted by a factor of 1.9 (more for individual petroleum hydrocarbons) over and above that for soil vapour extraction alone for the 30 days of sparging. Air sparging was also effective in removing residual NAPL from below the water table. The mass of petroleum hydrocarbons removed from the site was not reflected in changes to the mass of NAPL in the aquifer. This result is apparently due to lateral inflow of NAPL to the site although evidence of a depleted source of volatilised hydrocarbons suggests the possible development of mass transfer limitations.

Air↗

Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium.

The ovarian surface epithelium (OSE) is the precursor of common epithelial ovarian carcinomas. In the present study, we examined the molecular mechanisms and possible physiological basis for the propensity of OSE cells to undergo epithelio-mesenchymal transition (EMT) in response to environmental influences. We hypothesized that EMT may be a homeostatic mechanism that permits displaced OSE to assume a stromal phenotype within the ovarian cortex. We report that EGF in conjunction with hydrocortisone is the EMT-inducing factor of OSE as shown by changes to a fibroblast-like morphology and growth pattern. EGF increased cell motility, enhanced the activities of secreted pro-matrix metalloproteinase (MMP)-2 and -9, and enhanced expression and activation of Erk and integrin-linked kinase (ILK). Increased ILK expression correlated with the activation of PKB/Akt, the phosphorylation of GSK-3beta, and the increased expression of cyclin E and cdk2 kinase. EGF withdrawal resulted in a more epithelial morphology and reversal of the EGF-induced activation of signaling pathways and pro-MMP activity. In contrast, treatment of EGF-treated cells with specific inhibitors of phosphatidylinositol 3-kinase, Mek, or ILK inhibited the inhibitor-specific pathways. The inhibitors caused suppression of EGF-induced migration and pro-MMP-2/-9 activities but did not lead to any change in EGF-induced mesenchymal morphology. ILK small interfering RNA inhibited Akt phosphorylation and reduced pro-MMP-2/-9 activities but had no effect on Erk activation or cell morphology. These results indicate that the EGF-induced morphological and functional changes in OSE cells are controlled by distinct signaling mechanisms working in concert. EMT of OSE cells displaced by ovulation likely permits their survival and integration with a fibroblast-like identity within the stroma. Failure to do so may lead to the formation of epithelium-derived inclusion cysts, which are known preferential sites of malignant transformation.

Adult↗

Nelfinavir. A review of its therapeutic efficacy in HIV infection.

UNLABELLED: Nelfinavir is a selective inhibitor of HIV protease, the enzyme responsible for post-translational processing of HIV propeptides. In the presence of the drug, immature, noninfectious virus particles are produced. Nelfinavir in combination with nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors and/or other protease inhibitors profoundly suppresses viral replication. Plasma HIV RNA levels (viral loan) rapidly fall below the limit of detection (LOD; usually 400 or 500 copies/ml in the majority of patients. When used in combination with NRTIs, nelfinavir 1250mg twice daily produced similar results to 3-times-daily nelfinavir at a range of total daily dosages. In an ongoing study > 70% of adults receiving a nelfinavir based combination regimen had plasma HIV RNA levels below the LOD (< 400 copies/ml) after 84 weeks. In addition, 73% of paediatric patients receiving nelfinavir plus at least 1 new NRTI had viral loads below the LOD (< 400 copies/ml) after 34 weeks. Furthermore, CD4+ cell counts generally increased in conjunction with reductions in viral load. Combination therapy with nelfinavir and saquinavir results in higher saquinavir plasma concentrations, make twice-daily administration of saquinavir feasible and may delay the emergence of resistant viral strains. A unique mutation at codon 30 (D30N) of the protease gene confers resistance to nelfinavir, but HIV with D30N mutation remains fully susceptible to indinavir, ritonavir and saquinavir in vitro. Nonetheless, in clinical use, significant cross-resistance is seen with all currently available protease inhibitors. Diarrhoea is the most frequently reported adverse event in patients receiving nelfinavir-based combination therapy and has been reported in up to 32% of nelfinavir recipients in randomised trials. Diarrhoea is generally of mild to moderate severity and does not result in weight loss. Rash, nausea, headache and asthenia were each reported in < or = 5% of patients. Approximately 5% of patients enrolled in an expanded access programme in the US discontinued nelfinavir because of adverse events. Nelfinavir is metabolised by the cytochrome P450 system. Several clinically significant pharmacokinetic drug interactions between nelfinavir and other drugs (i.e. ketoconazole, rifabutin, rifabutin, rifampicin), including other protease inhibitors (i.e. indinavir, ritonavir, saquinavir) have been documented. As with other available protease inhibitors, hyperglycaemia, hyperlipidaemia and abnormal fat distribution have been reported, albeit infrequently, in association with nelfinavir. CONCLUSION: Nelfinavir-based combination regimens are well tolerated and produce profound and prolonged suppression of HIV replication in adult and paediatric patients. Hence, nelfinavir is suitable for inclusion in antiretroviral regimens for initial therapy for HIV infection and, alternatively, in regimens for patients unable to tolerate other protease inhibitors.

Adult↗