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Detection of avian leukosis virus antigens by the ELISA and its use for detecting infectious virus after cultivation of samples and partial characterization of specific pathogen-free chicken lines maintained in this laboratory.

An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.

Animals↗

Post-PCR multiplex fluorescent ligation detection assay and flow cytometry for rapid detection of gene-specific translocations in leukemia.

We describe a novel method to detect specific polymerase chain reaction (PCR) target amplicons, involving thermostable ligation of fluorescent and biotinylated oligonucleotides, microparticle bead capture of the ligated products, and flow cytometric analysis. This approach, termed fluorescent ligation detection reaction (f-LDR) is more rapid and cost-effective than oligoprobe Southern blot hybridization (SBH). A standard f-LDR protocol was developed to detect the leukemia-associated chimeric transcripts bcr-abl and promyelocytic leukemia-retinoic acid receptor a (PML-RARalpha) in 2 multiplex and multicolor assays. The f-LDR platform was 100% specific and demonstrated comparable or better sensitivity than standard oligoprobe SBH. The usefulness of f-LDR was evaluated in 94 posttherapy samples from 13 patients with acute promyelocytic leukemia with the PML-RARalpha gene fusion. The f-LDR method was highly concordant (93%) with oligoprobe SBH; essentially all discrepancies were noted to be due to the enhanced sensitivity of f-LDR. We conclude that f-LDR is a highly specific and sensitive post-PCR method with wide potential application.

Cell Line, Tumor↗

Simulation study for cloud detection with space lidars by use of analog detection photomultiplier tubes.

Output signal electrons from photomultiplier tubes (PMTs) have neither a Gaussian nor a Poisson distribution because of changes induced by multiplication when the number of input signal photons and dark electrons is fewer than approximately 100. Therefore the assumption of a Gaussian distribution of signal electrons cannot be used in simulations for space lidar observations with PMTs, for which the number of return signal photons is normally small. A theory is introduced for analog detection with PMTs that have Poisson-distributed secondary-electron emission at each dynode stage. The theory is validated by straightforward numerical simulations. It is shown that the multiplication in PMTs is a multiply stochastic Poisson process and that the distribution of output signal electrons can be interpreted basically as Neyman type A. Analysis by the threshold method of cloud detection with a space lidar shows considerable difference between a Gaussian approximation and the exact distribution. The result indicates that the threshold level must be optimized for the exact distribution. Return signals were simulated for a proposed space lidar, and cloud detection with the threshold method was demonstrated.

Journal Article↗

The early detection of second primary lung cancers by sputum immunostaining. LCEWDG Investigators. Lung Cancer Early Detection Group.

STUDY OBJECTIVE: To determine whether monoclonal antibody (Mab) detection of tumor-associated antigen expressed on sputum epithelial cells precedes clinical presentation of second primary lung cancer. DESIGN SETTING/PARTICIPANTS: Eleven oncology centers collaborate in the accrual of 1,000 patients with stage I non-small cell lung cancer (NSCLC) who had undergone resection. The Mabs examined in this study (624H12, 703D4) detect two promising oncofetal/differentiation markers (ie, a difucosylated Lewis X and a 31-Kd glycoprotein antigen). INTERVENTIONS: Induced sputum specimens are evaluated for quality, then are Papanicolaou and immunostained by independent central laboratories at enrollment and annually thereafter. The predictive value of Mab markers is compared with routine morphologic study for detection of second primary lung cancer during an anticipated 3 years of accrual and 1 year of follow-up. MEASUREMENTS AND RESULTS: Five hundred eighty of an anticipated 1,000 patients have been accrued on schedule. Patients are primarily white (88.6%), former smokers (75.9%), men (55.6%), with a median age of 66.7, and joined the study at an average of 3.7 years following resection of a stage 1 NSCLC (34.4% squamous, 43.6% adenocarcinoma). Central laboratories found less dysplasia and more unsatisfactory specimens (27.3%) than do the accrual institution laboratories. Immunostaining identifies more suspicious cells than does morphologic study. However, only two second primary lung cancers (eight total deaths) have occurred to date. CONCLUSIONS: Halfway through the accrual, we describe the study design and preliminary observations. This study illustrates rational selection of carcinogenesis markers by linkage of marker expression on preneoplastic specimens with subsequent expression on tumor tissue.

Aged↗

PNA-in situ hybridization method for detection of HIV-1 DNA in virus-infected cells and subsequent detection of cellular and viral proteins.

We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.

CD4-Positive T-Lymphocytes↗

[Detection of myocardial ischemia by epicardial detection of potassium ion activity].

Early detection of myocardial ischaemia is a central problem in cardiological and cardiosurgical intensive care. A new approach is the use of ion-selective electrodes implanted directly on the myocardium, enabling detection of increased potassium activity as an indication of general hypoxia. After a comprehensive study of the electrode parameters, an animal experiment was carried out, in which it was found that respiration-induced hypoxia resulted in an increase in epicardial potassium activity (p < 0.01). Blood gas analysis performed simultaneously revealed reduced arterial pO2, but no acidosis. Haemodynamic data evidenced hypoxic depression of circulatory parameters. Histological examinations of the myocardium beneath the electrodes revealed typical lymphocytic infiltration. Electron microscopy demonstrated crystolysis in the mitochondria as an early sign of hypoxia, thus confirming the sensitivity of these electrodes. This underscores the potential of ion-selective electrodes for the detection of myocardial ischaemia, and they should now be investigated in the clinical setting.

Animals↗

Detection of growth hormone, prolactin and human beta-chorionic gonadotropin mRNA in growth hormone-secreting pituitary adenomas and in prolactin-secreting pituitary adenomas by in situ hybridization using a non-isotopic detection method.

A non-isotopic in situ hybridization method with digoxigenin-labelled probes was used to examine growth hormone (GH), prolactin (PRL) and human beta-chorionic gonadotropin (beta-hCG(LH)) gene expression in 63 pituitary tumours in acromegaly and 20 adenomas in hyperprolactinaemia. hCG and LH were detected simultaneously because of the extensive homology (more than 90%) of their mRNA sequences (1). A comparison with former results obtained with 35S-labelled probes shows the value of the easier and faster non-isotopic method. Additionally, immunohistochemical data are included to give even more evidence for the synthesis of the respective hormones by the tumour cells. In all 63 adenomas in acromegaly, GH mRNA was revealed in 59 PRL mRNA and in 36 beta-hCG(LH) mRNA. A positive immunostaining for GH was found in all, for PRL in 40, and for beta-hCG(LH) in 34 adenomas. The comparison of the two in situ hybridization methods revealed no differences concerning GH mRNA detection, but not all tumours positive after non-isotopic PRL and beta-hCG(LH) mRNA detection showed signals with the radioactive method. Referring to the 20 PRL-secreting adenomas, PRL gene expression was demonstrable in all, GH mRNA in 12, and beta-hCG(LH) mRNA in 2 cases. Comparing the positive results of immunohistochemistry with those of in situ hybridization, correspondence was found in 19 cases for PRL, in 5 cases for GH and in no case for beta-hCG(LH).

Acromegaly↗

[Dietary assessment of the patients detected with hiperglycemia in the "Detection of Diabetes in Suspect Cases Campaign" in Viçosa, MG].

The purpose of this work is to assess the current, qualitative and quantitative alimentary consumption of patients detected with hiperglycemia in the "Detection of Diabetes in Suspect Cases Campaign" in Viçosa, MG. 256 patients were detected with hiperglycemia in 2001; however, 156 (60.9%) were assessed in 2004. The remaining people were dead, had changed their address, did not accept to participate in the study or did not have diagnosed diabetes. The hiperglycemic patients answered an alimentary frequency questionnaire for assessing their dietary intake and their usual diet recall, both previously tested in pilot-population. The most daily consumed foods were vegetable oil (99%), bean (94%), rice (90%), artificial sweetener (80%), leaf vegetables (63%), milk (61%), and French bread (54%). Most of the patients said they avoid sugar, sugary foods and animal fats. The largest percentiles of nutrients inadequacy happened for energy (85%), fibers (87%), calcium (94%), carbohydrates (82%), monounsaturated fats (91%) and proteins (58%). These last three happened in relation to total energy intake values. The current data allowed us to know about the alimentary habits of these people and also to stimulate nutrition intakes, being useful to the nutritional counseling, suggesting specific orientations in order to provide a more appropriate feeding, contributing to better health conditions and well-being.

Adult↗

A handy detection cell for end-column electrochemical detection in capillary electrophoresis.

A handy and simple detection cell was constructed using a mixing joint for end-column electrochemical detection in capillary electrophoresis (CE). The cell allows for positioning of the working electrode at the end of the separation capillary without the aid of micropositioners. The design facilitates the exchange of electrodes and capillaries without the need to refabricate the entire capillary-electrode setup. The cell can be assembled in a short period of time. Alignment with the joint screw proved to be reproducible for working electrodes of copper and gold. The advantages of reduced time and low cost make the device very attractive for the routine analysis of electroactive species, such as carbohydrates and their derivatives, purine bases and nucleosides, amino acids, and catecholamines etc. by CE with electrochemical detection.

Journal Article↗

Infectious pathogen detection arrays: viral detection in cell lines and postmortem brain tissue.

A unique array-based pathogen chip has been developed for the detection of viral RNA or DNA relevant to pathologies of the central nervous system. A total of 715 unique oligonucleotides (60-mer) representing approximately 100 pathogens were designed based on open reading frames (ORFs) from highly conserved and heterogenic regions within viral families. In addition, viral genes reflecting different stages of pathogen infection were also included to potentially define the stage of the viral infection. Viruses (double-stranded DNA, double- or single-stranded RNA, delta, retroid), parasites, and bacteria were included. Test samples labeled with Cy5 were examined by cohybridization with a reference RNA, labeled with Cy3, to the pathogen microarray chip. Good reproducibility of experiments was observed, based on data generated from duplicate hybridizations and duplicate spots on the microarray platform. A viral transcript detection sensitivity of 1 x 10(3) plaque-forming units (pfus) was achieved using selected cell lines and viruses. These findings suggest that the array-based platform described here is capable of detecting a broad spectrum of viruses in a single assay with relatively high sensitivity, specificity, and reproducibility. This method may be used to provide evidence of viral infection in postmortem tissue from psychiatric patients as well as a wide range of other diagnostic categories.

Autopsy↗

Gastric cancer detected by mass survey. Comparison between mass survey and outpatient detection.

Although the incidence of stomach cancer in Japan has decreased only slightly, the mortality has decreased markedly. The main reason for this success is the early diagnosis of cancer, to which mass survey has contributed. From 1964 to 1985, 290,914 screening examinations were done at the Mass Survey Center of the Cancer Institute Hospital, Tokyo. In 474 people (0.16%) cancer was detected, and of those, 52% were in an early stage of disease. For the mass survey group, the 5-year survival rate was 80%, and the 10-year survival rate 78.5%. The outpatient group rates were 56.2% and 55.1% for 5 and 10 years, respectively. This difference could be explained by the higher percentage of early gastric cancer and less extensive lymph node metastasis in patients with serosal involvement, in the outpatient group. The 10-year survival results show that early treatment results in an absolute better long-term survival. This refutes the hypothesis that 5-year survival rates of early gastric cancer patients are biased because of lead time of early diagnosis. Mass survey, even for a small district, provides an excellent opportunity to detect gastric cancer in people without symptoms. The high percentage of cases detected with cancer in an early stage reduces stomach cancer mortality.

Adult↗

Estrus detection and estrus detection aids.

There is no substitute for good management. Each animal should be identified carefully. The herds man should know all signs of estrus or impending estrus. In addition, for visual detection of estrus to be highly effective, sufficient time must be taken at least twice a day to catch animals with a short estrus period. Presently there are two visual aids to estrus detection which provide potential 24-h surveillance. One aid is a pressure sensitive device mounted on the back of each cow which can be triggered when the cow stands for mounting. The second one is a marking device worn by sexually aggressive animals which will stripe with colored ink the back of estrous animals as the marker animal mounts and dismounts. Both devices are effective when used properly. Other tests of changes in cervical mucus, vaginal characteristics, temperature, blood flow, and hormone changes in blood and milk are either not suffciently reliable or else simple enough yet to be practical aids for routine detection of estrus in dairy cattle. Milk progesterone can assist in characterizing problem cows.

Animals↗

Doctors detected self-administering opioids in New South Wales, 1985-1994: characteristics and outcomes.

OBJECTIVE: To describe the characteristics and outcomes of doctors whose drug authorities were withdrawn as a result of self-administering opioids for non-medical purposes. DESIGN: Retrospective review of New South Wales Health Department information relating to all doctors whose authorities to possess, supply, prescribe or administer drugs of addiction had been withdrawn in the period 1985 to 1994 as a result of confirmed self-administration of opioids. OUTCOME MEASURES: Age, sex, geographical location and practice category at the time of intervention; drugs used; period of opioid use before authority withdrawal; means of detection; and registration status as at August 1995. RESULTS: From 1985 to 1994, 79 doctors had their drug authorities withdrawn (0.4% of the NSW medical profession in 1994). The groups significantly over-represented were general practitioners and those aged 30-39 years. Pethidine was the main drug used (66 doctors; 84%). Drug use for more than two years before detection was reported by 34 (43%) doctors. Community pharmacists were the source of reports leading to detection of 28 (35%) doctors. As at August 1995, 27 (34%) of the study group were not practising; 10 (13%) had died. CONCLUSION: Outcomes for these doctors were poor. There was substantial attrition from practice and a high mortality rate.

Adult↗

[DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences].

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.

Base Sequence↗

Sensitivity and specificity of QCA in detecting coronary arterial remodeling after intracoronary brachytherapy: a comparison to serial volumetric three-dimensional intravascular ultrasound analysis. Can we detect positive remodeling by luminography?

BACKGROUND: After treatment with intracoronary brachytherapy, enlargement of lumen (negative late loss) at follow-up has been demonstrated. The aim of the study is to analyze the sensitivity and specificity of quantitative coronary angiography (QCA) parameters to detect a positive vessel remodeling after intracoronary -radiation as compared to intravascular ultrasound (IVUS). METHODS: Twenty-seven patients (27 vessels) treated with balloon angioplasty followed by catheter-based intracoronary -radiation with a (90)Strontium/(90)Yttrium source were assessed by both QCA and three-dimensional IVUS with electrocardiogram-gated pullback. Irradiated segments were analyzed over the total treatment length and in subsegment lengths of 5 mm. RESULTS: Change in minimum luminal diameter (MLD) was not a predictor for the positive remodeling in either total irradiated segments or 5 mm subsegments, with a 54.3% ROC curve area [95% confidence interval (CI), 30-79%], sensitivity of 39% and specificity of 44% (p = not significant) in the total irradiated segments and a 55.9% ROC curve area (95% CI, 46-66%), sensitivity of 55% and specificity of 54% (p = not significant) in the 5 mm subsegments. Changes in mean and maximal lumen diameter were not significant parameters to detect positive vessel remodeling. When only central subsegments were analyzed, change in MLD was a significant predictor, with a 63.3% ROC curve area (95% CI, 52-75%), sensitivity of 55% and specificity of 64% (p = 0.029). CONCLUSION: Lumen enlargement detected by QCA does not reliably indicate a positive vessel remodeling after intracoronary radiation. IVUS analysis may be necessary to investigate the mechanism of restenosis after balloon angioplasty followed by catheter-based radiation.

Angioplasty, Balloon, Coronary↗

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids.

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.

Buffers↗