Sample records for release assays igra

  1. Risk Assessment of Tuberculosis in Contacts by Interferon-γ Release Assays (IGRAs). A TBNET Study

    Zellweger, Jean-Pierre; Sotgiu, Giovanni; Block, Michael


    contacts in low incidence countries of tuberculosis. We analyzed IGRA results and the effect of preventive chemotherapy on tuberculosis progression rates among recent contacts. METHODS: Results from contact investigations at 26 centers in 10 European countries including testing for latent infection...... with Mycobacterium tuberculosis by the QuantiFERON-Gold in-Tube (QFT) test or the T-SPOT.TB (TSPOT) were prospectively collected and analyzed. MAIN RESULTS: Among 5020 contacts of 1023 index cases, 25 prevalent secondary cases were identified at screening. Twenty-four incident cases occurred among 4513 contacts...

  2. Role of interferon-gamma release assays (IGRAs for the screening of latent tuberculosis infection in patients candidates for TNF-α antagonist

    Ilaria Sauzullo


    Full Text Available Background: Tumor necrosis factor-α (TNF-a inhibitors are associated with an increased risk of reactivation of latent tuberculosis infection (LTBI; thus, the guidelines recommends TB screening for all patients before starting anti-TNF-α therapy.The use of tuberculin skin test (TST is controversial because of the immunosuppressive treatment may lead to false-negative TST results and previous BCG-vaccination produces false-positive. The introduction in clinical practice of the interferon-gamma release assays (IGRAs has opened new perspectives for diagnosis of LTBI.The aim of the study was to investigate the performance of QuantiFERON-TB Gold In Tube assay (QFT-GIT for the diagnosis of LTBI in patients with an immune-mediated inflammatory diseases candidates for anti-TNF-α therapy. Methods:We enrolled 195 patients with rheumatoid arthritis (n=72, psoriatic arthritis (n=40, psoriasis (n=41, ankylosing spondylitis (n=10, Crohn’s disease (n=18, and Behcet’s disease (n=8. Screening included: clinical evaluation, chest X-ray,TST and QFT-GIT. Results: Of the 195 patients, 32 (16.4%, 137 (70.2% and 26 (13.3% tested positive, negative and indeterminate with QFT-GIT test, respectively. The level of agreement between two tests was 81.6 % (k=0.55. Among the screened patients, 38 (19% were considered to have LTBI and received isoniazid treatment, while 31 patients (16% showed discordant results between two tests. Univariate analysis showed an association between BCG vaccination and discordant TST-positive/QFT-GIT-negative results (OR=6; 95%CI: 2.3-37.1; p<0.001; no association was observed between the immunosuppressive therapy and discordant TST-negative/QFT-GIT-positive results (OR=0.16; 95%CI: 0.01-1.8; p<0.09. Conclusions: Our results suggest that QFT-GIT may be helpful for the diagnosis of LTBI in patients candidates for anti-TNF-α treatment because of its performance seems to not be affected by any type of immunosuppression.

  3. Concordance between the tuberculin skin test and interferon gamma release assay (IGRA) for diagnosing latent tuberculosis infection in patients with systemic lupus erythematosus and patient characteristics associated with an indeterminate IGRA.

    Cho, H; Kim, Y W; Suh, C-H; Jung, J-Y; Um, Y-J; Jung, J-H; Kim, H-A


    We investigated the agreement between the tuberculin skin test (TST) and the QuantiFERON-TB gold (QFT-G) assay in the diagnosis of latent tuberculosis infection (LTBI) in patients with systemic lupus erythematosus (SLE). Furthermore, we evaluated the factors associated with indeterminate results in the QFT-G assay in patients with SLE. We enrolled 136 patients with SLE prospectively, and compared them to 66 patients with rheumatoid arthritis (RA). In addition to the TST, QFT-G assay, patients' medications, and Bacillus Calmette-Guérin (BCG) vaccination status were also investigated. A positive TST or QFT-G assay result without an active tuberculosis lesion on chest x-ray was considered to indicate a diagnosis of LTBI. The prevalence of LTBI was 26.5% in patients with SLE and 30.3% in patients with RA. The agreement between the TST and QFT-G assay was fair in SLE patients, but poor in RA patients. BCG vaccination was one factor associated with discordance between TST and QFT-G. Older age and higher SLE Disease Activity Index (SLEDAI) score were associated with a negative TST/positive QFT-G result in patients with SLE. Higher SLEDAI score and increased glucocorticoid dose were associated with an indeterminate result in the QFT-G assay for patients with SLE. Agreement between the QFT-G assay and TST in patients with SLE was found to be fair. However, BCG vaccination status, age, and SLEDAI score are all factors that could result in discordance between the two tests. Indeterminate results from the QFT-G assay may be caused by a higher SLEDAI score or increased glucocorticoid dose. © The Author(s) 2016.

  4. Diagnostic value of interferon gamma release assay (IGRA) in patients with tuberculous pleurisy%IGRA检测在结核性胸膜炎中的诊断价值

    李津; 潘燕玉; 张宝华; 程小星


    . 1% (16/17). Conclusion T-SPOT. TB assay with pleural effusion cells can be used in the diagnosis of TBP, and the diagnostic value will be even greater while combi-ning with the IGRA results from whole blood and pleural fluid cells.

  5. Acceptability of interferon-gamma release assays among healthcare workers who receive routine employee tuberculosis testing.

    Hirsch-Moverman, Yael; Wall, Kirsten; Weinfurter, Paul; Munk, Elizabeth; Moran, Joyce Ann; Maiuris, Allison; Khan, Amera; DeLuca, Nichlas


    Healthcare workers (HCWs) undergo annual testing for latent tuberculosis infection (LTBI). Compare acceptability of tuberculin skin test (TST) and interferon-gamma release assay (IGRA) among HCWs. HCWs at four medical centers in the US were administered an acceptability questionnaire including a brief objective description of both tests and eliciting attitudes regarding TST and IGRAs, confidence in results, and likelihood of taking LTBI treatment. Of 406 participants, 75% had never heard of IGRAs. IGRAs were preferred to TST. Belief in accuracy of hypothetical positive results of TST or IGRA and willingness to accept LTBI treatment were similar across tests. When presented with hypothetical discordant results, HCWs expressed more confidence in IGRAs. Perceived accuracy of results was the most important factor in test preferences. Although HCWs preferred and indicated more confidence in IGRAs, the likelihood that HCWs would believe LTBI diagnosis and initiate treatment based on positive results was similar for TST and IGRAs.

  6. An interferon-gamma release assay test performs well in routine screening for tuberculosis

    Vestergaard Danielsen, Allan; Fløe, Andreas; Lillebæk, Troels;


    Introduction: A positive interferon-gamma release assay (IGRA) is regarded as proof of latent Mycobacterium tuberculosis infection. We conducted an evaluation of the IGRA test “T-SPOT.TB” to test its performance during clinical routine use by analysing the positivity rate and odds, effect of season...

  7. Interferon-¿ release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis

    Sester, M; Sotgiu, G; Lange, C;


    Interferon-¿ release assays (IGRAs) are now established for the immunodiagnosis of latent infection with Mycobacterium tuberculosis in many countries. However, the role of IGRAs for the diagnosis of active tuberculosis (TB) remains unclear. Following preferred reporting items for systematic revie...

  8. Interferon-gamma release assay versus tuberculin skin test for latent tuberculosis infection among HIV patients in Brazil.

    Kussen, Gislene Maria Botão; Dalla-Costa, Libera Maria; Rossoni, Andrea; Raboni, Sonia Mara


    Patients HIV+ attending in a reference clinic, Southern Brazil. To compare the interferon-gamma-release assay (IGRA - QuantiFERON(®) TB Gold In-Tube) with the tuberculin skin test (TST - PPD-Rt 23) for latent tuberculosis infection (LTBI) in patients with HIV. Cohort study. Patients were simultaneously submitted to the TST and blood collection for the IGRA. A total of 140 subjects were included. Nine (6.4%) were IGRA+/TST+, 12 (8.6%) were IGRA+/TST-, 4 (3%) were IGRA-/TST+, and 115 (82%) IGRA-/TST-. There was poor agreement between tests (kappa=0.2), and no correlation between these results and CD4+ T lymphocyte counts. During follow-up, one patient with negative results on both tests died from sepsis, and another with discordant results (IGRA+/TST-) exhibited TST seroconversion. Compared to the TST, IGRA showed a sensitivity and specificity of 69% and 90%, respectively. The IGRA detected 8% more positive results than the TST. All patients were followed up for 2 years. The higher accuracy of the IGRA would result in LTBI treatments being administered to patients who would have otherwise been overlooked, decreasing the number of active tuberculosis cases. The long-term survival of HIV carriers requires further evaluation. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  9. Interferon-gamma release assays are a better tuberculosis screening test for hemodialysis patients: A study and review of the literature


    Diagnosing latent tuberculosis (TB) infection (LTBI) in dialysis patients is complicated by poor response to tuberculin skin testing (TST), but the role of interferon-gamma release assays (IGRAs) in the dialysis population remains uncertain. Seventy-nine patients were recruited to compare conventional diagnosis (CD) with the results of two IGRA tests in a dialysis unit. Combining TST, chest x-ray and screening questionnaire results (ie, CD) identified 24 patients as possible LTBI. IGRA testin...

  10. Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6

    Ruhwald, Morten; de Thurah, Lena; Kuchaka, Davis


    There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA...... tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens Esp...... containing vaccines and as adjunct test for latent infection....

  11. More significance of TB-IGRA except for the diagnose of tuberculosis.

    Xu, Jun-Chi; Li, Ze-Yi; Chen, Xin-Nian; Shi, Cui-Lin; Wu, Mei-Ying; Chen, Hui; Zhu, Xiao-Yan; Song, Hua-Feng; Wu, Min-Juan; Xu, Ping


    Tuberculosis (TB)-interferon gamma release assay (IGRA) test has the characteristics of short time, high specificity, and high sensitivity, but it lacks the correlation research between TB-IGRA test results and body's immune cells, disease progression and prognosis, which is explored in this study. A retrospective study was carried out on positive TB-IGRA patients who were infected with TB and diagnosed at our hospital from January 2014 to June 2015. The TB-IGRA, routine blood test, T-cell subgroup data were collected for statistical analysis. TB-IGRA results were in positive proportion to the lymphocytes, CD4(+) T cells and CD4(+) CD28(+) T cells, whereas negative to the Treg cells. Patient with unilateral pulmonary lesion had higher TB-IGRA than those with bilateral pulmonary lesions. After the stimulation of TB-specific antigen, the proportion of CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T Tcells were both increased and the CD4(+) IFN-γ(+) T had positive correlation with the value of TB-IGRA. IFN-γ was tested with TB-IGRA in patients with TB by the specific TB T cells and correlated with the lymphocytes, while the lymphocytes also closely related to the host's anti-TB immunity and disease outcome. Hence the result of TB-IGRA could reflect the specific anti-TB immunity ability of the host, disease progression and prognosis. This study further expands the application scope of TB-IGRA technology in the diagnosis of TB and lays a foundation for clinical practice to understand the immunity state of the patients with TB and the application of auxiliary clinical immunity regulators. © 2017 Wiley Periodicals, Inc.

  12. Thermostability of IFN-γ and IP-10 release assays for latent infection with Mycobacterium tuberculosis

    Blauenfeldt, Thomas; Wagner, Dirk; Aabye, Martine Grosos;


    INTRODUCTION: Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher...... accuracy of IP-10 release assay and IGRAs. RESULTS: We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90-1.36) fold and IFN-γ responses decreased 0.88 (IQR 0...

  13. Application and Interpretation of an Interferon-Gamma Release Assay: Results of an Audit in a Canadian Centre

    Sopharat Vat


    Full Text Available BACKGROUND: Interferon-gamma release assays (IGRAs are newly approved for diagnosing latent tuberculosis infection (LTBI. An internal audit was conducted to review the use of a newly implemented IGRA at the Hôpital du Sacré-Coeur de Montréal (Montréal, Québec to evaluate its concordance with Canadian recommendations and its implication on diagnosis.

  14. Evaluation of the tuberculin skin test and the interferon-γ release assay for TB screening in French healthcare workers

    Raffi Francois


    Full Text Available Abstract Introduction Using French cut-offs for the Tuberculin Skin Test (TST, results of the TST were compared with the results of an Interferon-γ Release Assay (IGRA in Healthcare Workers (HCW after contact to AFB-positive TB patients. Methods Between May 2006 and May 2007, a total of 148 HCWs of the University Hospital in Nantes, France were tested simultaneously with IGRA und TST. A TST was considered to indicate recent latent TB infection (LTBI if an increase of >10 mm or if TST ≥ 15 mm for those with no previous TST result was observed. For those with a positive TST, chest X-ray was performed and preventive chemotherapy was offered. Results All HCWs were BCG-vaccinated. The IGRA was positive in 18.9% and TST ≥ 10 mm was observed in 65.5%. A recent LTBI was believed to be highly probable in 30.4% following TST. Agreement between IGRA and TST was low (kappa 0.041. In 10 (16.7% out of 60 HCWs who needed chest X-ray following TST the IGRA was positive. In 9 (20% out of 45 HCWs to whom preventive chemotherapy was offered following TST the IGRA was positive. Of those considered TST-negative following the French guidelines, 20.5% were IGRA-positive. In a two-step strategy - positive TST verified by IGRA - 18 out of 28 (64.3% IGRA-positive HCWs would not have been detected using French guidelines for TST interpretation. Conclusion The introduction of IGRA in contact tracings of BCG-vaccinated HCWs reduces X-rays and preventive chemotherapies. Increasing the cut-off for a positive TST does not seem to be helpful to overcome the effect of BCG vaccination on TST.

  15. Follow-Up Study of Tuberculosis-Exposed Supermarket Customers with Negative Tuberculin Skin Test Results in Association with Positive Gamma Interferon Release Assay Results▿

    Franken, Willeke P. J.; Koster, Ben F. P. J.; Bossink, Ailko W. J.; Thijsen, Steven F. T.; Bouwman, John J. M.; van Dissel, Jaap T.; Arend, Sandra M.


    We report a follow-up study of 29 subjects with negative tuberculin skin test (TST) results in association with positive gamma interferon release assay (IGRA) results, mainly due to responses to CFP-10 in the T-SPOT.TB assay, during a contact investigation. One year later, 12/29 subjects (41%) had converted to positive TST results in association with negative IGRA results. PMID:17626157

  16. Interferon-gamma release-assay ved mistanke om aktiv tuberkulose?

    Browatzki, Andrea; Meyer, Christian


    INTRODUCTION: The purpose of this retrospective study was to outline the practical use and clinical value of the immunodiagnostic interferon-gamma release assay (IGRA) on suspicion of active Mycobacterium tuberculosis (TB) infection. MATERIAL AND METHODS: A retrospective study of all patients (n=91...

  17. Interferon-Gamma Release Assay: An Effective Tool to Detect Early Toxoplasma gondii Infection in Mice.

    Qing Yin

    Full Text Available Early diagnosis of Toxoplasma gondii infection before the formation of tissue cysts is vital for treatment, as drugs available for toxoplasmosis cannot kill bradyzoites contained in the cysts. However, current methods, such as antibody-based ELISA, are ineffective for detection of early infection. Here, we developed an interferon-gamma release assay (IGRA, measuring the IFN-γ released by T lymphocytes stimulated by Toxoplasma antigen peptides in vitro, for the detection of T. gondii infection in mice. Splenocytes isolated from infected mice were stimulated by peptides derived from dense granule proteins GRA4 and GRA6 and rhoptry protein ROP7, and released IFN-γ was measured by ELISA. Results showed that both acute and chronic infection could be detected by IGRA. More importantly, IGRA detected infection as early as the third day post infection; while serum IgM and IgG were detected 9 days and 13 days post infection, respectively. Our findings demonstrated that an IGRA-positive and ELISA-negative sample revealed an early infection, indicating the combination of IGRA and ELISA can be employed for the early diagnosis of T. gondii infection in human beings, cats and livestock.

  18. Tuberculosis screening using a T-cell interferon-γ release assay in Japanese medical students and non-Japanese international students.

    Ogiwara, Takayuki; Kimura, Takao; Tokue, Yutaka; Watanabe, Rumi; Nara, Makoto; Obuchi, Toshiko; Yaegashi, Akiko; Yomoda, Sachie; Ohshima, Kihachi; Murakami, Masami


    Screening of medical students and international students for tuberculosis (TB) at the time of admission is a key strategy to control and prevent the spread of infection on university campus and teaching hospitals because of the high risk of exposure to TB patients. The Mycobacterium tuberculosis antigen-specific T-cell interferon-γ release assays (IGRAs) are specific latent tuberculosis detection methods used in such groups. Currently, in Japan, there are no guidelines and no baseline data on IGRAs to evaluate the risk of TB in these high-risk groups. In order to evaluate TB risk at the time of admission in university campus and medical schools in Japan, a retrospective study was conducted. A total of 969 students (585 Japanese students and 384 international students) were screened for TB using the IGRAs at the time of admission. Eight Japanese students (0.9%) were positive for IGRAs, but none were diagnosed with active TB at the follow-up. In contrast, 30 international students (7.8%) were positive for IGRAs, including two students diagnosed with active TB during follow up. Positive ratio of IGRAs in international students was significantly higher than that of medical students at the time of admission. Here we propose a standard approach for TB screening with IGRAs at the time of admission for medical students and international students in Japan.

  19. Interferon Gamma Release Assays in active Tuberculosis: new medical insights

    Sandro Pierdomenico


    Full Text Available Since first presentation, Interferon γ Release Assays (IGRAs have had basic and wide application to LTBI, in accordance with international consensus and CDC recommendations, leaving their use in active TB to the field of study and research.We reviewed the results of 633 patients investigated from 2004 to 2008 targeting active TB, with the objective to highlight immunological data supporting test performances.We evaluated Quantiferon TB Gold (1st generation IGRA kit in association to Culture (MGIT 960 and Lowenstein Jensen and PCR (Probetec-ET having the positivity of culture plus clinical diagnosis as the standard true value to compare. QTB Gold was studied in 69 TB positive patients (42 pulmonary and 27 extra-pulmonary, with Sensitivity, Specificity, PPV and NPV average to 61.8%, 94.5%, 54.3% and 95.9% respectively, after indeterminate results discharging. Significant statistical differences didn’t emerge between pulmonary and extra-pulmonary infections (CI 95%.The overall indeterminate ratio arose up to 20.3% in patients with active TB vs 2.7% of global population (p<0.001. In 22% of patients with active pulmonary disease, IGRA conversed to positivity after 15 days in replicated tests, in spite of current treatment. 4 patients, with pulmonary TB and Quantiferon persistent negativities, underwent 18 months follow-up as not respondent although SIRE phenotypic susceptibilities and enough DOT compliance. Molecular DST documented hetero resistance for rpoB (MUT 1, MUT 3 plus wild lines and katG (MUT 1 plus wild in association to lack of inhA wild lines (Genotype MTBDR plus, Hain Lifescience. These reports suggest a mutational relationship between Rv3874 – 3875 cassette, encoding ESAT-6 / CFP-10, and rpoB, katG, inhA genes plausibly implying weak or absent selective clonal Th 1 activation to IGRA antigens. Our data seem to point out: 1 positive results are able to match true active TB in less than 50% of patients; 2 negative results could leave

  20. Comparing interferon-gamma release assays to tuberculin skin test in Thai children with tuberculosis exposure.

    Hong-Van Tieu

    Full Text Available Data on the performance of interferon-gamma release assays (IGRAs, QuantiFERON TB Gold In-tube (QFNGIT and T-Spot.TB, in diagnosing tuberculosis (TB are limited in Southeast Asia. This study aims to compare the performances of the two IGRAs and TST in Thai children with recent TB exposure.This multicenter, prospective study enrolled children with recent exposure to active TB adults. Children were investigated for active TB. TST was performed and blood collected for T-Spot.TB and QFNGIT.158 children were enrolled (87% TB-exposed and 13% active TB, mean age 7.2 years. Only 3 children had HIV infection. 66.7% had TST≥10 mm, while 38.6% had TST≥15 mm. 32.5% had positive QFNGIT; 29.9% had positive T-Spot.TB. QFNGIT and T-Spot.TB positivity was higher among children with active TB compared with TB-exposed children. No indeterminate IGRA results were detected. No statistically significant differences between the performances of the IGRAs and TST at the two cut-offs with increasing TB exposure were detected. Concordance for positive IGRAs and TST ranged from 42-46% for TST≥10 mm and 62-67% for TST≥15 mm. On multivariable analyses, exposure to household primary/secondary caregiver with TB was associated with positive QFNGIT. Higher TB contact score and active TB were associated with positive T-Spot.TB.Both QFNGIT and T-Spot.TB performed well in our Thai pediatric study population. No differences in the performances between tests with increasing TB exposure were found. Due to accessibility and low cost, using TST may more ideal than IGRAs in diagnosing latent and active TB in healthy children in Thailand and other similar settings.

  1. Specificity of a whole blood IGRA in German nursing students

    Ringshausen Felix C


    Full Text Available Abstract Background Interferon-gamma release assays (IGRA are used for tuberculosis (TB screening in healthcare workers (HCWs. However, data on specificity of IGRA in serial testing of HCWs is sparse. Therefore the specificity and the negative predictive value of the IGRA - QuantiFERON-TB Gold In-Tube (QFT - in German nursing students was investigated. Methods 194 nursing students at the start of their professional career were tested with the QFT. 14 nursing students were excluded from the specificity analysis, due to exposure to mycobacterium tuberculosis. Two of these subjects were QFT- positive. None of them developed disease during the year of follow-up. A study group of 180 students, all with very low risk of prior TB infection, remained in the specificity analysis. Subjects were monitored for at least two years with respect to the development of active TB disease. IGRA was performed at the start of the training and after one year. Results The mean age of the study group (n = 180 was 23 years (range 18-53 with 70.9% female and 99.4% German born. The specificity of QFT was 98.9% (178/180; 95% CI 0.96-0.99; lowering the cut-off from 0.35 IU/ml to 0.1 IU/ml would have decreased specificity only slightly to 97.8% (176/180; 95% CI 0.94-0.99. Of the 154 nursing students available for re-testing, one student who initially scored positive reverted to negative, and one student initially negative converted to positive. None of the monitored group with initially negative QFT results developed TB disease, indicating a high negative predictive value of the IGRA in this population. Conclusions Following our data, QFT can serve as an effective tool in pre-employment TB screenings for HCWs. As its negative results were stable over time, specificity of the QFT in serial testing of HCWs is high. As the risk of acquiring TB infection in the German healthcare system appears to be low, our data supports the recommendation of performing TB screening only in

  2. Factors associated with the performance of a blood-based interferon-γ release assay in diagnosing tuberculosis.

    Sally Banfield

    Full Text Available BACKGROUND: Indeterminate results are a recognised limitation of interferon-γ release assays (IGRA in the diagnosis of latent tuberculosis (TB infection (LTBI and TB disease, especially in children. We investigated whether age and common co-morbidities were associated with IGRA performance in an unselected cohort of resettled refugees. METHODS: A retrospective cross-sectional study of refugees presenting for their post-resettlement health assessment during 2006 and 2007. Refugees were investigated for prevalent infectious diseases, including TB, and for common nutritional deficiencies and haematological abnormalities as part of standard clinical screening protocols. Tuberculosis screening was performed by IGRA; QuantiFERON-TB Gold in 2006 and QuantiFERON-TBGold In-Tube in 2007. RESULTS: Complete data were available on 1130 refugees, of whom 573 (51% were children less than 17 years and 1041 (92% were from sub-Saharan Africa. All individuals were HIV negative. A definitive IGRA result was obtained in 1004 (89% refugees, 264 (26% of which were positive; 256 (97% had LTBI and 8 (3% had TB disease. An indeterminate IGRA result was obtained in 126 (11% refugees (all failed positive mitogen control. In multivariate analysis, younger age (linear OR= 0.93 [95% CI 0.91-0.95], P<0.001, iron deficiency anaemia (2.69 [1.51-4.80], P = 0.001, malaria infection (3.04 [1.51-6.09], P = 0.002, and helminth infection (2.26 [1.48-3.46], P<0.001, but not vitamin D deficiency or insufficiency, were associated with an indeterminate IGRA result. CONCLUSIONS: Younger age and a number of common co-morbidities are significantly and independently associated with indeterminate IGRA results in resettled predominantly African refugees.

  3. Interferon gamma release assays for the diagnosis of latent TB infection in HIV-infected individuals in a low TB burden country.

    Cheallaigh, Clíona Ní


    Interferon gamma release assays (IGRAs) are used to diagnose latent tuberculosis infection. Two IGRAs are commercially available: the Quantiferon TB Gold In Tube (QFT-IT) and the T-SPOT.TB. There is debate as to which test to use in HIV+ individuals. Previous publications from high TB burden countries have raised concerns that the sensitivity of the QFT-IT assay, but not the T-SPOT.TB, may be impaired in HIV+ individuals with low CD4+ T-cell counts. We sought to compare the tests in a low TB burden setting.

  4. Comparison of two interferon-gamma release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB) in testing for latent tuberculosis infection among HIV-infected adults.

    Sultan, B; Benn, P; Mahungu, T; Young, M; Mercey, D; Morris-Jones, S; Miller, R F


    There is currently no 'gold standard' for diagnosis of latent tuberculosis infection (LTBI), and both the tuberculin skin test and interferon-gamma release assays (IGRAs) are used for diagnosis; the latter have a higher sensitivity than tuberculin skin tests for diagnosis of LTBI in HIV-infected individuals with lower CD4 counts. No evidence base exists for selection of IGRA methodology to identify LTBI among human immunodeficiency virus-infected patients in the UK. We prospectively evaluated two commercially available IGRA methods (QuantiFERON-TB Gold In Tube [QFG] and T-SPOT.TB) for testing LTBI among HIV-infected patients potentially nosocomially exposed to an HIV-infected patient with 'smear-positive' pulmonary tuberculosis. Among the exposed patients median CD4 count was 550 cells/µL; 105 (90%) of 117 were receiving antiretroviral therapy, of who 104 (99%) had an undetectable plasma HIV load. IGRAs were positive in 12 patients (10.3%); QFG positive in 11 (9.4%) and T-SPOT.TB positive in six (5.1%); both IGRAs were positive in five patients (4.3%). There was one indeterminate QFG and one borderline T-SPOT.TB result. Concordance between the two IGRAs was moderate (κ = 0.56, 95% confidence interval = 0.27-0.85). IGRAs were positive in only 4 (29%) of 14 patients with previous culture-proven tuberculosis. No patient developed tuberculosis during 20 months of follow-up.

  5. The impact of HIV infection and CD4 cell count on the performance of an interferon gamma release assay in patients with pulmonary tuberculosis

    Aabye, Martine G.; Ravn, Pernille; PrayGod, George;


    BACKGROUND: The performance of the tuberculosis specific Interferon Gamma Release Assays (IGRAs) has not been sufficiently documented in tuberculosis- and HIV-endemic settings. This study evaluated the sensitivity of the QuantiFERON TB-Gold In-Tube (QFT-IT) in patients with culture confirmed...

  6. The Effectiveness of Screening with Interferon-Gamma Release Assays in a University Health Care Setting with a Diverse Global Population

    Birch, Samantha J.; Golbeck, Amanda L.


    Objective: This analysis examined the effectiveness of utilizing interferon-gamma release assay (IGRA) technology in a TB (TB) screening program at a university. Participants: Participants were 2299 students at a Montana university who had presented to the university health center for TB screening during 2012 and 2013. Methods: A retrospective…

  7. Tuberculosis screening in patients with psoriasis before antitumour necrosis factor therapy : comparison of an interferon-gamma release assay vs. tuberculin skin test

    Laffitte, E.; Janssens, J. P.; Roux-Lombard, P.; Thielen, A. M.; Barde, C.; Marazza, G.; Panizzon, R. G.; Saurat, J-H.


    Background Antitumour necrosis factor (anti-TNF) treatments may reactivate latent tuberculosis infection (LTBI). For detecting LTBI, the tuberculin skin test (TST) has low sensitivity and specificity. Interferon-gamma release assays (IGRA) have been shown to be more sensitive and specific than TST.

  8. Tuberculosis contact investigation using interferon-gamma release assay with chest x-ray and computed tomography.

    Fujikawa, Akira; Fujii, Tatsuya; Mimura, Satoshi; Takahashi, Ryota; Sakai, Masao; Suzuki, Shinya; Kyoto, Yukishige; Uwabe, Yasuhide; Maeda, Shinji; Mori, Toru


    Between September 2009 and January 2010, 6 members of the Japanese Eastern Army, who had completed the same training program, were diagnosed with active tuberculosis (TB) on different occasions. The Ministry of Defense conducted a contact investigation of all members who had come into contact with the infected members. The purpose of this study was to verify the efficacy of the TB screening protocol used in this investigation. A total of 884 subjects underwent interferon-gamma release assay (IGRA) and chest X-ray. The 132 subjects who were IGRA positive or with X-ray findings suggestive of TB subsequently underwent chest computer tomography (CT). Chest CT was performed for 132 subjects. Based on CT findings, 24 (2.7%) subjects were classified into the active TB group, 107 (12.1%) into the latent tuberculosis infection (LTBI) group, and 753 (85.2%) into the non-TB group. The first 2 groups underwent anti-TB therapy, and all 3 groups were followed for 2 years after treatment. Although one subject in the active TB group experienced relapse during the follow-up period, no patient in the LTBI or non-TB groups developed TB. IGRA and chest X-ray, followed by chest CT for those IGRA positive or with suspicious X-ray findings, appears to be an effective means of TB contact screening and infection prevention.

  9. Comparison of interferon-γ release assays, QuantiFERON TB-GIT and T-Spot.TB, in renal transplantation.

    Ishikawa, Satoru; Igari, Hidetoshi; Akutsu, Naotake; Tsuyuzaki, Mizue; Aoyama, Hiromichi; Hasegawa, Masayuki; Otsuki, Kazunori; Maruyama, Michihiro; Saigo, Kenichi; Suzuki, Kiminori; Yamagishi, Fumio


    Renal transplant recipients are at increased risk of reactivating latent tuberculosis infection (LTBI) and progressing to active tuberculosis (TB). This study was conducted in National hospital for tuberculosis and renal transplantation (RT) in Japan. The purpose is to compare two interferon-γ release assays (IGRAs), QuantiFERON(®)-TB Gold in Tube (QFT) and T-SPOT(®).TB (TSPOT), in patients after renal transplantation for detecting latent TB infection (LTBI). Total 92 renal transplant recipients (median age 46 years, range 17-75) were prospectively enrolled, and QFT and TSPOT were concurrently examined. Total subjects were 92 patients (median age 46 years, range 17-75). The positive rate in QFT and TSPOT were 6.5% (95% confidence interval (CI) 3.0-13.5) and 2.2% (95% CI 1.0-7.6), respectively. There was a significant difference in IGRAs positivity (P TB during median follow-up of 994 days. Neither QFT nor TSPOT reaches estimated TB infection rate in Japan, especially elderly recipients aged 60 year-old or more. Therefore, both IGRAs might underestimate LTBI owing to immune suppressive therapy and aging. Physicians for renal transplantation need to understand the characteristics of both IGRAs and pay attention to the possibility of developing active TB even in patients of negative IGRAs results. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  10. Usefulness of interferon-γ release assay for the diagnosis of sputum smear-negative pulmonary and extra-pulmonary TB in Zhejiang Province, China.

    Ji, Lei; Lou, Yong-Liang; Wu, Zhong-Xiu; Jiang, Jin-Qin; Fan, Xing-Li; Wang, Li-Fang; Liu, Xiao-Xiang; Du, Peng; Yan, Jie; Sun, Ai-Hua


    Quick diagnosis of smear-negative pulmonary tuberculosis (TB) and extra-pulmonary TB are urgently needed in clinical diagnosis. Our research aims to investigate the usefulness of the interferon-γ release assay (IGRA) for the diagnosis of smear-negative pulmonary and extra-pulmonary TB. We performed TB antibody and TB-IGRA tests on 389 pulmonary TB patients (including 120 smear-positive pulmonary TB patients and 269 smear-negative pulmonary TB patients), 113 extra-pulmonary TB patients, 81 patients with other pulmonary diseases and 100 healthy controls. Blood samples for the TB-Ab test and the TB-IGRA were collected, processed, and interpreted according to the manufacturer's protocol. The detection ratio of smear-positive pulmonary TB patients and smear-negative pulmonary TB patients were 90.8% (109 of 120) and 89.6% (241 of 269), respectively. There was no statistically significant difference of its performance between these two sample sets (P > 0.05). The detection ratio of positive TB patients and extra-pulmonary TB patients were 90.0% (350 of 389) and 87.6% (99 of 113), respectively, which was not significantly different (P > 0.05). In this work, the total detection ratio using TB-IGRA was 89.4%, therefore TB-IGRA has diagnostic values in smear-negative pulmonary TB and extra-pulmonary TB diagnosis.

  11. Tuberculin skin test and interferon-γ release assay show better correlation after the tuberculin 'window period' in tuberculosis contacts.

    Anibarro, Luis; Trigo, Matilde; Villaverde, Carlos; Pena, Alberto; González-Fernández, Africa


    T-cell interferon-gamma release assays (IGRAs) have been shown to be effective tools for the detection of Mycobacterium tuberculosis infection, offering an enhanced specificity compared to the tuberculin skin test (TST). Most tuberculosis (TB) contact studies have shown a better correlation of IGRA with the intensity of M. tuberculosis exposure than that obtained using the TST. However, the correlation between tests performed before and after the tuberculin 'window period' (time between infection and when the immunological response becomes measurable) remains to be studied. A longitudinal prospective analysis was performed in TB contacts. We analyzed the correlation between a commercially available IGRA (QuantiFERON®-TB Gold in-Tube, QFT) and the TST before and after the tuberculin window period (2 months). Concordance between both tests was assessed using the Kappa coefficient (κ). Correlation of both tests with the degree of TB exposure was also analyzed. One hundred and fifty-two TB contacts were included in the study. Agreement between the TST and IGRA was better after the window period (κ = 0.60 at the first visit and κ = 0.73 after 2 months), especially for non-BCG vaccinated subjects (κ = 0.81). Both a positive TST and QFT were correlated, after the window period, with the size of place of contact (the smaller the place of contact, the higher the probability of having a positive test) (p = 0.022 and p = 0.02, respectively) and with the total numbers of hours spent with the index case (p = 0.006 for TST and p = 0.007 for QFT). IGRAs are a good alternative to the TST in contact tracing studies, especially after the tuberculin window period.

  12. Contribution of a heparin-binding haemagglutinin interferon-gamma release assay to the detection of Mycobacterium tuberculosis infection in HIV-infected patients: comparison with the tuberculin skin test and the QuantiFERON-TB Gold In-tube.

    Wyndham-Thomas, Chloé; Dirix, Violette; Schepers, Kinda; Martin, Charlotte; Hildebrand, Marc; Goffard, Jean-Christophe; Domont, Fanny; Libin, Myriam; Loyens, Marc; Locht, Camille; Van Vooren, Jean-Paul; Mascart, Françoise


    The screening and treatment of latent tuberculosis (TB) infection reduces the risk of progression to active disease and is currently recommended for HIV-infected patients. The aim of this study is to evaluate, in a low TB incidence setting, the potential contribution of an interferon-gamma release assay in response to the mycobacterial latency antigen Heparin-Binding Haemagglutinin (HBHA-IGRA), to the detection of Mycobacterium tuberculosis infection in HIV-infected patients. Treatment-naïve HIV-infected adults were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the HBHA-IGRA in parallel to a classical method consisting of medical history, chest X-ray, tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT). Prospective clinical and biological follow-up ensued, with repeated testing with HBHA-IGRA. A group of HIV-infected patients with clinical suspicion of active TB was also recruited and tested with the HBHA-IGRA. Multiplex analysis was performed on the culture supernatants of this in-house assay to identify test read-outs alternative to interferon-gamma that could increase the sensitivity of the test. Among 48 candidates enrolled for screening, 9 were identified with latent TB by TST and/or QFT-GIT results. Four of these 9 patients and an additional 3 screened positive with the HBHA-IGRA. This in-house assay identified all the patients that were positive for the TST and showed the best concordance with the presence of a M. tuberculosis exposure risk. During follow-up (median 14 months) no case of active TB was reported and HBHA-IGRA results remained globally constant. Fourteen HIV-infected patients with clinical suspicion of active TB were recruited. Active TB was confirmed for 6 of them among which 3 were HBHA-IGRA positive, each with very high interferon-gamma concentrations. All patients for whom active TB was finally excluded, including 2 non-tubercular mycobacterial infections, had negative HBHA-IGRA

  13. Comparison of interferon {gamma} release assays and conventional screening tests before tumour necrosis factor {alpha} blockade in patients with inflammatory arthritis.

    Martin, J


    OBJECTIVE: To compare the performance of two interferon gamma release assays (IGRAs) and conventional screening tests in patients with inflammatory arthritis undergoing screening for latent tuberculosis infection (LTBI) before treatment with anti-tumour necrosis factor alpha (anti-TNFalpha) compounds. METHODS: Successive patients were subjected to conventional LTBI screening, including a tuberculin skin test (TST). The T-SPOT.TB test was performed on all patients and the QuantiFERON-TB Gold test was performed on a large subset. The results of the IGRAs were compared with the results of conventional screening tests. RESULTS: A total 150 patients were evaluated. The majority (57.9%) had rheumatoid arthritis. Previous vaccination with Bacille Calmette-Guerin was confirmed in 82% of patients. No patient had received prior anti-TB treatment. A total of 57 patients (38.0%) had at least one positive conventional risk factor. In contrast, an unequivocally positive T-SPOT.TB test was seen in only 14\\/143 (9.8%). There was 98.2% agreement between the two IGRAs. Statistically significant associations were found between each of the IGRAs and both TST and risk history, but not chest x-ray (CXR). A positive IGRA result was significantly associated with increased age. TB was not reactivated in any patient during the follow-up period. Interpretation: This study suggests that IGRAs may be useful when screening for LTBI before anti-TNFalpha therapy in patients with immune-mediated inflammatory diseases. The observations reported here also highlight the inadequate performance of CXR as a marker of LTBI.

  14. In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

    Rajiv L Gaur

    Full Text Available BACKGROUND: Activation of innate immunity via pathogen recognition receptors (PRR modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ release assays (IGRAs are functional T cell assays used to diagnose latent tuberculosis infection (LTBI; however, novel approaches are needed to improve their sensitivity. METHODS: In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube with Toll-like receptor agonists poly(I:C, LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls. RESULTS: In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells. CONCLUSIONS: In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

  15. Specificity of the Tuberculin Skin Test and the T-SPOT."TB" Assay among Students in a Low-Tuberculosis Incidence Setting

    Talbot, Elizabeth A.; Harland, Dawn; Wieland-Alter, Wendy; Burrer, Sherry; Adams, Lisa V.


    Objective: Interferon-[gamma] release assays (IGRAs) are an important tool for detecting latent "Mycobacterium tuberculosis" infection (LTBI). Insufficient data exist about IGRA specificity in college health centers, most of which screen students for LTBI using the tuberculin skin test (TST). Participants: Students at a low-TB incidence college…

  16. How Methodologic Differences Affect Results of Economic Analyses: A Systematic Review of Interferon Gamma Release Assays for the Diagnosis of LTBI


    INTRODUCTION: Cost effectiveness analyses (CEA) can provide useful information on how to invest limited funds, however they are less useful if different analysis of the same intervention provide unclear or contradictory results. The objective of our study was to conduct a systematic review of methodologic aspects of CEA that evaluate Interferon Gamma Release Assays (IGRA) for the detection of Latent Tuberculosis Infection (LTBI), in order to understand how differences affect study results. ME...

  17. Positive tuberculin skin test or interferon-gamma release assay in patients with radiographic lesion suggesting old healed tuberculosis.

    Jeong, Yun-Jeong; Yoon, Soonho; Koo, Hyeon-kyoung; Lim, Hyo-Jeong; Lee, Ji Sun; Lee, Sang-Min; Yang, Seok-Chul; Yoo, Chul-Gyu; Kim, Young-Whan; Han, Sung-Koo; Yim, Jae-Joon


    Radiographic lesions suggesting old healed tuberculosis (TB) is considered a risk factor for the subsequent development of active TB. The aim of this study was to estimate the positive rates of tuberculin skin test (TST) and interferon-gamma release assay (IGRA) in persons with old healed TB. Participants with lesions suggesting old healed TB on chest images and controls without such lesions were prospectively enrolled between January 1, 2010, and January 31, 2011. TST and the QuantiFERON-TB Gold In-Tube test (QFT-GIT) were performed. In total, 193 participants with old healed TB and 126 controls were recruited. The rates of positive TST and QFT-GIT among patients with old healed TB were 54.6% and 77.7%, respectively. The rates of positive TST and QFT-GIT among patients without old healed TB were 38.9% and 61.9%. Sixteen percent of participants with old healed TB showed negative results by both TST and QFT-GIT. The positive rate of TST waned among participants with old healed TB who were older than 60 yr, whereas QFT-GIT positivity was unaffected by age. The positive rates of TST and IGRA among participants with radiographic lesions suggesting old healed TB was higher than without those lesions. In addition, IGRA may be more accurate than TST for the detection of latent TB infection, especially in populations of individuals older than 60 yr.

  18. 不同国家γ干扰素释放试验临床应用指南的概述%Outline of interferon gamma release assays clinical application guidelines in different countries

    尹青琴; 申阿东


    γ干扰素释放试验(interferon gamma release assays,IGRAs)是一种新型的快速诊断结核分枝杆菌感染的免疫学方法.目前已经有16个国家制定了IGRAs诊断结核分枝杆菌感染的应用指南.各国IGRAs应用指南主要针对以下情况进行制定的:成人活动性结核病、成人潜伏结核感染、医疗工作者潜伏结核感染、免疫抑制者结核分枝杆菌感染筛查、儿童结核分枝杆菌感染.不同国家对不同人群的IGRAs应用指南不尽相同,故我国应根据本国国情针对不同人群进行大样本研究以得出IGRAs诊断结核分枝杆菌感染的最科学、最有效的应用原则.%Interferon gamma release assays (IGRAs) are new and rapid immunodiagnostic methods of Mycobacterium tuberculosis infection.Recently 16 countries have already made guidelines of IGRAs used to diagnose Mycobacterium tuberculosis infection.The guidelines of IGRAs are made mainly by following different situations:active tuberculosis in adults,latent tuberculosis infection in adults,Mycobacterium tuberculosis infection in medical workers and immunosuppressive individuals,Mycobacterium tuberculosis infection in children.Different countries have different IGRAs guidelines for different subjects,so according to our national condition,large studies for different subjects should be done in order to get the most scientific and the most effective application principles of IGRAs used to diagnose tuberculosis.

  19. Cost-effectiveness of interferon-gamma release assay for entry tuberculosis screening in prisons.

    Kowada, A


    The incidence of active tuberculosis (TB) and latent tuberculosis infection (LTBI) in inmates and prison staff is higher than that in the general population. Mycobacterium tuberculosis-specific interferon-gamma release assays (IGRAs) provide more accurate diagnosis of M. tuberculosis infection with higher specificity than the tuberculin skin test (TST). To assess the cost effectiveness of QuantiFERON®-TB Gold In-Tube (QFT) compared to TST, TST followed by QFT and chest X-ray, we constructed Markov models using a societal perspective on the lifetime horizon. The main outcome measure of effectiveness was quality-adjusted life-years (QALYs) gained. The incremental cost-effectiveness was compared. The QFT-alone strategy was the most cost-effective for entry TB screening in prisons in developed countries. Cost-effectiveness was not sensitive to the rates of BCG vaccination, LTBI, TB, HIV infection and multidrug-resistant TB. Entry TB screening using an IGRA in prisons should be considered on the basis of its cost-effectiveness by public health intervention.

  20. Interferon-γ release assays versus tuberculin skin test for targeting people for tuberculosis preventive treatment: an evidence-based review.

    Muñoz, Laura; Santin, Miguel


    To assess whether Interferon-γ release assays (IGRAs) reduce the number of people considered for tuberculosis (TB) preventive treatment without increasing subsequent active disease. Longitudinal studies with both tuberculin skin test (TST) and IGRAs were identified through a PubMed search. Reductions in diagnosis of TB infection and increases in incident TB in people considered not infected, using IGRAs either instead of TST or as a confirmatory test (two-step approach), were assessed. In comparison with TST alone, the pooled reductions in diagnosis of TB infection obtained with IGRAs were 16.7% and 5.8% at 5 and 10 mm cut-offs respectively, and 24.5% and 12.4% at 5 and 10 mm respectively with the two-step approach. Compared with TST alone, incident TB among people considered not infected increased with the two-step approach (0.94% with T-SPOT(®).TB and 1.1% with QuantiFERON(®)-TB Gold In-Tube) in one of seven studies in high-income countries. In middle- and low-income countries, two of four studies presented increases (0.08 and 0.03 per 100 patient-years respectively) with the two-step approach. In high-income countries, the use of IGRAs, either instead of TST or as confirmatory test reduces the number of people considered for preventive treatment, without a significant risk of subsequent active disease. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  1. Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment.

    Park, I-Nae; Shim, Tae Sun


    The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB. From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment. A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range.

  2. Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

    Park, I-Nae; Shim, Tae Sun


    Background/Aims The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB. Methods From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment. Results A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p < 0.05 for all). Conclusions Although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range. PMID:27951621

  3. Performance of interferon-gamma and IP-10 release assays for diagnosing latent tuberculosis infections in patients with concurrent malaria in Tanzania

    Drabe, Camilla H; Vestergaard, Lasse S; Helleberg, Marie


    Interferon-gamma (IFN-γ) release assays (IGRAs) are used to detect cellular immune recognition of Mycobacterium tuberculosis. The chemokine IFN-γ-inducible protein 10 (IP-10) is an alternative diagnostic biomarker to IFN-γ. Several conditions interfere with IGRA test performance. We aimed to assess......-infected patients without malaria infection. Malaria was treated with artemether-lumefantrine (Coartem(®)). QFT testing was performed before initiation of malaria treatment and at days 7 and 42. In total, 172 patients completed follow-up. IFN-γ and IP-10 was measured in QFT supernatants. We found that during...... malaria infection IFN-γ and IP-10 levels in the unstimulated samples were elevated, mitogen responsiveness was impaired, and CD4 cell counts were decreased. These alterations reverted after malaria treatment. Concurrent malaria infection did not affect QFT test results, whereas there were more...

  4. Prevalence of latent tuberculosis infection in persons with and without human immunodeficiency virus infection using two interferon-gamma release assays and tuberculin skin test in a low human immunodeficiency virus prevalence, intermediate tuberculosis-burden country.

    Lin, Wei-Cheng; Lin, Hsi-Hsun; Lee, Susan Shin-Jung; Sy, Cheng-Len; Wu, Kuan-Sheng; Chen, Jui-Kuang; Tsai, Hung-Chin; Chen, Yao-Shen


    The risk of tuberculosis (TB) is higher in human immunodeficiency virus (HIV)-infected patients and intravenous drug users (IDUs). We determined the prevalence and risk factors of latent TB infection (LTBI) in individuals with or without HIV infection, including IDUs, in a country with a low HIV prevalence, an intermediate TB burden, and a high Bacillus Calmette-Guérin (BCG) vaccine coverage using two interferon-gamma release assays (IGRAs) and the tuberculin skin test (TST). For this prospective, cross-sectional study, HIV-infected and -uninfected patients from a regional hospital and medical center in Taiwan were enrolled. Results of the two IGRAs [QuantiFERON-TB Gold (QFT-G) and QuantiFERON-TB Gold In-Tube (QFT-GIT)] and the TST were compared. Risk factors for positivity were analyzed. We recruited 233 patients [198 (85%) men; mean age, 39.4 years]. Most patients (74%) were BCG vaccinated. The prevalence of LTBI was estimated to be 22.8% by TST, 15.9% by QFT-G, and 20.6% by QFT-GIT. HIV-infected individuals had fewer positive QFT-GIT [7.0% vs. 28.6%, p < 0.001, adjusted odds ratio (aOR) = 0.28, p = 0.05] and TST results, and more indeterminate QFT-G responses (9.3% vs. 0.7%, p = 0.002). Concordance between IGRAs and TST was very poor in HIV-infected patients (κ < 0.05). Independent risk factors for IGRA positivity were increasing age (QFT-G: aOR = 1.98, p = 0.03; QFT-GIT: aOR = 2.00, p = 0.01) and IDUs (aOR = 4.33, p = 0.05 by QFT-G). HIV-infected persons had a significantly lower response to both IGRAs and TST. High discordance was found between the two generations of IGRAs and between IGRAs and TST. Increasing age, a known risk factor for LTBI, was significantly associated with IGRAs, but not with TST. Copyright © 2014. Published by Elsevier B.V.

  5. Methods Used in Economic Evaluations of Tuberculin Skin Tests and Interferon Gamma Release Assays for the Screening of Latent Tuberculosis Infection: A Systematic Review.

    Koufopoulou, Maria; Sutton, Andrew John; Breheny, Katie; Diwakar, Lavanya


    Latent tuberculosis infection (LTBI) provides a constant pool of new active tuberculosis cases; a third of the earth's population is estimated to be infected with LTBI. The objective of this systematic review was to assess the quality and summarize the available evidence from published economic evaluations reporting on the cost-effectiveness of tuberculin skin tests (TSTs) compared with interferon gamma release assays (IGRAs) for the screening of LTBI. An extensive systematic review of the published literature was conducted. A two-step process was adopted to identify relevant articles: information was extracted into evidence tables and then analyzed. The quality of the publications was assessed using a 10-item checklist specific for economic evaluations. Twenty-eight studies were identified for inclusion in this review. Most of the studies found IGRAs to be more cost-effective than TSTs; however, the conclusions from the studies varied significantly. Most studies scored highly on the checklist although only one fulfilled all the stipulated criteria. A wide variety of methodological approaches were documented; identified differences included the type of economic evaluation and model, time horizon, perspective, and outcomes measures. The lack of consistent methods across studies makes it difficult to draw any firm conclusions about the most cost-effective option between TSTs and IGRAs. This problem can be solved by improving the quality of economic evaluation studies in the field of LTBI screening, through adherence to quality checklists. Copyright © 2016. Published by Elsevier Inc.

  6. How methodologic differences affect results of economic analyses: a systematic review of interferon gamma release assays for the diagnosis of LTBI.

    Olivia Oxlade

    Full Text Available INTRODUCTION: Cost effectiveness analyses (CEA can provide useful information on how to invest limited funds, however they are less useful if different analysis of the same intervention provide unclear or contradictory results. The objective of our study was to conduct a systematic review of methodologic aspects of CEA that evaluate Interferon Gamma Release Assays (IGRA for the detection of Latent Tuberculosis Infection (LTBI, in order to understand how differences affect study results. METHODS: A systematic review of studies was conducted with particular focus on study quality and the variability in inputs used in models used to assess cost-effectiveness. A common decision analysis model of the IGRA versus Tuberculin Skin Test (TST screening strategy was developed and used to quantify the impact on predicted results of observed differences of model inputs taken from the studies identified. RESULTS: Thirteen studies were ultimately included in the review. Several specific methodologic issues were identified across studies, including how study inputs were selected, inconsistencies in the costing approach, the utility of the QALY (Quality Adjusted Life Year as the effectiveness outcome, and how authors choose to present and interpret study results. When the IGRA versus TST test strategies were compared using our common decision analysis model predicted effectiveness largely overlapped. IMPLICATIONS: Many methodologic issues that contribute to inconsistent results and reduced study quality were identified in studies that assessed the cost-effectiveness of the IGRA test. More specific and relevant guidelines are needed in order to help authors standardize modelling approaches, inputs, assumptions and how results are presented and interpreted.

  7. The impact of HIV infection and CD4 cell count on the performance of an interferon gamma release assay in patients with pulmonary tuberculosis

    Aabye, Martine G.; Ravn, Pernille; PrayGod, George


    BACKGROUND: The performance of the tuberculosis specific Interferon Gamma Release Assays (IGRAs) has not been sufficiently documented in tuberculosis- and HIV-endemic settings. This study evaluated the sensitivity of the QuantiFERON TB-Gold In-Tube (QFT-IT) in patients with culture confirmed...... pulmonary tuberculosis (PTB) in a TB- and HIV-endemic population and the effect of HIV-infection and CD4 cell count on test performance. METHODOLOGY/PRINCIPAL FINDINGS: 161 patients with sputum culture confirmed PTB were subjected to HIV- and QFT-IT testing and measurement of CD4 cell count. The QFT...

  8. [Up-to-date applicability of interferon-γ release assays for the diagnosis of tuberculosis].

    Domínguez, José; Latorre, Irene


    Utility of the in-vitro immunodiagnostic methods, based on the detection of interferon-γ released by T-cells after specific Mycobacterium tuberculosis antigen stimulation (IGRA), has been an improvement in the accuracy of the latent tuberculosis infection diagnosis. IGRA have a well-known higher specificity than the tuberculin skin testing (TST). Moreover, they can obtain a larger number of positive results than the TST in immunocompromised patients. IGRA have shown a high correlation with M. tuberculosis exposure, but their positive and negative predictive value are similar than those obtained by TST. Nevertheless, given their high specificity, they allow reducing number of unnecessary preventive treatments. In addition, these in-vitro techniques are less affected than TST by the different immunosuppressing status. In this review is discussed up-to-date applicability of IGRA in different patient groups: contact studies, pediatric population, immunosuppressed patients, health care workers and active tuberculosis patients. Furthermore, it has been included possible future directions for latent tuberculosis infection and active tuberculosis diagnosis. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  9. Comparing interferon-gamma release assays with tuberculin skin test for identifying latent tuberculosis infection that progresses to active tuberculosis: systematic review and meta-analysis.

    Auguste, Peter; Tsertsvadze, Alexander; Pink, Joshua; Court, Rachel; McCarthy, Noel; Sutcliffe, Paul; Clarke, Aileen


    Timely and accurate identification of people with latent tuberculosis infection (LTBI) is important for controlling Mycobacterium tuberculosis (TB). There is no gold standard for diagnosis of LTBI. Screening tests such as interferon gamma release assays (IGRAs) and tuberculin skin test (TST) provide indirect and imperfect information. This systematic review compared two types of IGRAs QuantiFERON®-TB Gold In-Tube test (QFT-GIT) and T-SPOT.TB with TST for identification of LTBI by predicting progression to a diagnosis of active TB in three subgroups: children, immunocompromised people, and those recently arrived from countries with high TB burden. Cohort studies were eligible for inclusion. We searched MEDLINE, EMBASE, the Cochrane Library and other databases from December 2009 to June 2015. One reviewer screened studies, extracted data, and assessed risk of bias with cross checking by a second reviewer. Strength of association between test results and incidence of TB was summarised using cumulative incidence ratios (CIRs with 95% CIs). Summary effect measures: the ratio of CIRs (R-CIR) with 95% CIs. R-CIRs, were pooled using a random-effects model. Heterogeneity was assessed using Chi-squared and I(2) statistics. Seventeen studies, mostly of moderate or high risk of bias (five in children, 10 in immunocompromised people, and two in those recently arrived) were included. In children, while in two studies, there was no significant difference between QFT-GIT and TST (≥5 mm) (pooled R-CIR = 1.11, 95% CI: 0.71, 1.74), two other studies showed QFT-GIT to outperform TST (≥10 mm) in identifying LTBI. In immunocompromised people, IGRA (T-SPOT.TB) was not significant different from TST (≥10 mm) for identifying LTBI, (pooled R-CIR = 1.01, 95% CI: 0.65, 1.58). The forest plot of two studies in recently arrived people from countries with high TB burden demonstrated inconsistent findings (high heterogeneity; I(2) = 92%). Prospective studies comparing IGRA

  10. Trajectories of tuberculosis-specific interferon-gamma release assay responses among medical and nursing students in rural India.

    Zwerling, Alice; Joshi, Rajnish; Kalantri, S P; Dakshinamoorthy, Gajalakshmi; Reddy, Maryada Venkatarama; Benedetti, Andrea; Schwartzman, Kevin; Menzies, Dick; Pai, Madhukar


    Interferon gamma release assays (IGRAs) have been shown to be highly dynamic tests when used in serial testing for TB infection. However, there is little information demonstrating a clear association between TB exposure and IGRA responses over time, particularly in high TB incidence settings. To assess whether QuantiFERON-TB Gold In-Tube (QFT) responses are associated with occupational TB exposures in a cohort of young health care trainees in India. All medical and nursing students at Mahatma Gandhi Institute of Medical Sciences were approached. Participants were followed up for 18 months; QFT was performed 4 times, once every 6 months. Various modeling approaches were used to define IFN-gamma trajectories and correlations with TB exposure. Among 270 medical and nursing trainees, high rates of conversions (6.3-20.9%) and reversions (20.0-26.2%) were found depending on the definitions used. Stable converters were more likely to have had TB exposure in hospital pre-study. Recent occupational exposures were not consistently associated with QFT responses over time. IFN-gamma responses and rates of change could not be explained by occupational exposure investigated. High conversion and subsequent reversion rates suggest many health care workers (HCWs) would revert in the absence of treatment, either by clearing the infection naturally or due to fluctuations in the underlying immunological response and/or poor assay reproducibility. QFT may not be an ideal diagnostic test for repeated screening of HCWs in a high TB incidence setting. Copyright © 2013 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.

  11. Tuberculin Skin Tests versus Interferon-Gamma Release Assays in Tuberculosis Screening among Immigrant Visa Applicants

    Stella O. Chuke


    Full Text Available Objective. Use of tuberculin skin tests (TSTs and interferon gamma release assays (IGRAs as part of tuberculosis (TB screening among immigrants from high TB-burden countries has not been fully evaluated. Methods. Prevalence of Mycobacterium tuberculosis infection (MTBI based on TST, or the QuantiFERON-TB Gold test (QFT-G, was determined among immigrant applicants in Vietnam bound for the United States (US; factors associated with test results and discordance were assessed; predictive values of TST and QFT-G for identifying chest radiographs (CXRs consistent with TB were calculated. Results. Of 1,246 immigrant visa applicants studied, 57.9% were TST positive, 28.3% were QFT-G positive, and test agreement was 59.4%. Increasing age was associated with positive TST results, positive QFT-G results, TST-positive but QFT-G-negative discordance, and abnormal CXRs consistent with TB. Positive predictive values of TST and QFT-G for an abnormal CXR were 25.9% and 25.6%, respectively. Conclusion. The estimated prevalence of MTBI among US-bound visa applicants in Vietnam based on TST was twice that based on QFT-G, and 14 times higher than a TST-based estimate of MTBI prevalence reported for the general US population in 2000. QFT-G was not better than TST at predicting abnormal CXRs consistent with TB.

  12. Interferon-γ release assays for the diagnosis of latent Mycobacterium tuberculosis infection: a systematic review and meta-analysis

    Diel, R; Goletti, D; Ferrara, G;


    We conducted a systematic review and meta-analysis to compare the accuracy of the QuantiFERON-TB® Gold In-Tube (QFT-G-IT) and the T-SPOT®.TB assays with the tuberculin skin test (TST) for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). The Medline, Embase and Cochrane databases...... of IGRAs varied 98-100%. In immunocompetent adults, NPV for progression to tuberculosis within 2 yrs were 97.8% for T-SPOT®.TB and 99.8% for QFT-G-IT. When test performance of an immunodiagnostic test was not restricted to prior positivity of another test, progression rates to tuberculosis among IGRA...... tuberculosis cases. IGRAs may have a relative advantage over the TST in detecting LTBI and allow the exclusion of M. tuberculosis infection with higher reliability....

  13. Interferon-gamma release assays for the diagnosis of active tuberculosis in HIV-infected patients: a systematic review and meta-analysis.

    Jun Chen

    Full Text Available BACKGROUND: Interferon-gamma release assays (IGRAs have provided a new method for the diagnosis of Mycobacterium tuberculosis infection. However, the role of IGRAs for the diagnosis of active tuberculosis (TB, especially in HIV-infected patients remains unclear. METHODS: We searched PubMed, EMBASE and Cochrane databases to identify studies published in January 2001-July 2011 that evaluated the evidence of using QuantiFERON-TB Gold in-tube (QFT-GIT and T-SPOT.TB (T-SPOT on blood for the diagnosis of active TB in HIV-infected patients. RESULTS: The search identified 16 eligible studies that included 2801 HIV-infected individuals (637 culture confirmed TB cases. The pooled sensitivity for the diagnosis of active TB was 76.7% (95%CI, 71.6-80.5% and 77.4% (95%CI, 71.4-82.6% for QFT-GIT and T-SPOT, respectively, while the specificity was 76.1% (95%CI, 74.0-78.0% and 63.1% (95%CI, 57.6-68.3% after excluding the indeterminate results. Studies conducted in low/middle income countries showed slightly lower sensitivity and specificity when compared to that in high-income countries. The proportion of indeterminate results was as high as 10% (95%CI, 8.8-11.3% and 13.2% (95%CI, 10.6-16.0% for QFT-GIT and T-SPOT, respectively. CONCLUSION: IGRAs in their current formulations have limited accuracy in diagnosing active TB in HIV-infected patients, and should not be used alone to rule out or rule in active TB cases in HIV-infected patients. Further modification is needed to improve their accuracy.

  14. Performance of the tuberculin skin test and interferon-γ release assay for detection of tuberculosis infection in immunocompromised patients in a BCG-vaccinated population

    Kim Young


    Full Text Available Abstract Background Interferon-γ release assay (IGRA may improve diagnostic accuracy for latent tuberculosis infection (LTBI. This study compared the performance of the tuberculin skin test (TST with that of IGRA for the diagnosis of LTBI in immunocompromised patients in an intermediate TB burden country where BCG vaccination is mandatory. Methods We conducted a retrospective observational study of patients given the TST and an IGRA, the QuantiFERON-TB Gold In-Tube (QFT-IT, at Severance Hospital, a tertiary hospital in South Korea, from December 2006 to May 2009. Results Of 211 patients who underwent TST and QFT-IT testing, 117 (55% were classified as immunocompromised. Significantly fewer immunocompromised than immunocompetent patients had positive TST results (10.3% vs. 27.7%, p 0.001, whereas the percentage of positive QFT-IT results was comparable for both groups (21.4% vs. 25.5%. However, indeterminate QFT-IT results were more frequent in immunocompromised than immunocompetent patients (21.4% vs. 9.6%, p 0.021. Agreement between the TST and QFT-IT was fair for the immunocompromised group (κ = 0.38, but moderate agreement was observed for the immunocompetent group (κ = 0.57. Indeterminate QFT-IT results were associated with anaemia, lymphocytopenia, hypoproteinemia, and hypoalbuminemia. Conclusion In immunocompromised patients, the QFT-IT may be more sensitive than the TST for detection of LTBI, but it resulted in a considerable proportion of indeterminate results. Therefore, both tests may maximise the efficacy of screening for LTBI in immunocompromised patients.

  15. Evaluation of the prognostic value of IFN-gamma release assay and tuberculin skin test in household contacts of infectious tuberculosis cases in Senegal.

    Christian Lienhardt

    Full Text Available BACKGROUND: Chemoprophylaxis of contacts of infectious tuberculosis (TB cases is recommended for TB control, particularly in endemic countries, but is hampered by the difficulty to diagnose latent TB infection (LTBI, classically assessed through response to the Tuberculin Skin Test (TST. Interferon-gamma release assays (IGRA are proposed new tools to diagnose LTBI, but there are limited data on their ability to predict the development of active TB disease. To address this, we investigated the response to TST and IGRA in household contacts of infectious TB cases in a TB high-burden country and the potential correlation with development of TB. METHODOLOGY/PRINCIPAL FINDINGS: Prospective household contacts study conducted in two health centres in Dakar, Senegal. A total of 2679 household contacts of 206 newly detected smear and/or culture positive index TB cases aged 18 years or greater were identified A TST was performed in each contact and an ESAT6/CFP10 ELISPOT assay performed in a random sample of those. Contacts were followed-up for 24 months. TB was diagnosed in 52 contacts, an incidence rate of 9.27/1000 person-years. In univariable analysis, the presence of positive TST (> or = 10 mm and ELISPOT (>32 SFC/million PBMC responses at baseline were associated with active TB during follow-up: Rate Ratio [RR] = 2.32 (95%CI:1.12-4.84 and RR = 2.09 (95%CI:0.83-5.31, respectively. After adjustment for age, sex and proximity to index case, adjusted RRs were 1.51 (95%CI:0.71-3.19 and 1.98 (95%CI:0.77-5.09, respectively. Restricting analysis to the 40 microbiologically confirmed cases, the adjusted RR for positive ELISPOT was 3.61 (95%CI:1.03-12.65. The median ELISPOT response in contacts who developed TB was 5-fold greater than in those who did not develop TB (p = 0.02. CONCLUSIONS/SIGNIFICANCE: TST and IGRAs are markers of a contact of the immune system with tubercle bacilli. In a TB endemic area, a high ELISPOT response may reflect increased

  16. Screening for latent tuberculosis in Norwegian health care workers: high frequency of discordant tuberculin skin test positive and interferon-gamma release assay negative results.

    Gran, Gerd; Aßmus, Jörg; Dyrhol-Riise, Anne Ma


    Tuberculosis (TB) presents globally a significant health problem and health care workers (HCW) are at increased risk of contracting TB infection. There is no diagnostic gold standard for latent TB infection (LTBI), but both blood based interferon-gamma release assays (IGRA) and the tuberculin skin test (TST) are used. According to the national guidelines, HCW who have been exposed for TB should be screened and offered preventive anti-TB chemotherapy, but the role of IGRA in HCW screening is still unclear. A total of 387 HCW working in clinical and laboratory departments in three major hospitals in the Western region of Norway with possible exposure to TB were included in a cross-sectional study. The HCW were asked for risk factors for TB and tested with TST and the QuantiFERON®TB Gold In-Tube test (QFT). A logistic regression model analyzed the associations between risk factors for TB and positive QFT or TST. A total of 13 (3.4%) demonstrated a persistent positive QFT, whereas 214 (55.3%) had a positive TST (≥ 6 mm) and 53 (13.7%) a TST ≥ 15 mm. Only ten (4.7%) of the HCW with a positive TST were QFT positive. Origin from a TB-endemic country was the only risk factor associated with a positive QFT (OR 14.13, 95% CI 1.37 - 145.38, p=0.026), whereas there was no significant association between risk factors for TB and TST ≥ 15 mm. The five HCW with an initial positive QFT that retested negative all had low interferon-gamma (IFN-γ) responses below 0.70 IU/ml when first tested. We demonstrate a low prevalence of LTBI in HCW working in hospitals with TB patients in our region. The "IGRA-only" seems like a desirable screening strategy despite its limitations in serial testing, due to the high numbers of discordant TST positive/IGRA negative results in HCW, probably caused by BCG vaccination or boosting due to repetitive TST testing. Thus, guidelines for TB screening in HCW should be updated in order to secure accurate diagnosis of LTBI and offer proper

  17. Serial interferon-gamma release assays during treatment of active tuberculosis in young adults

    Lee Choon-Taek


    Full Text Available Abstract Background The role of interferon-γ release assay (IGRA in monitoring responses to anti-tuberculosis (TB treatment is not clear. We evaluated the results of the QuantiFERON-TB Gold In-tube (QFT-GIT assay over time during the anti-TB treatment of adults with no underlying disease. Methods We enrolled soldiers who were newly diagnosed with active TB and admitted to the central referral military hospital in South Korea between May 1, 2008 and September 30, 2009. For each participant, we preformed QFT-GIT assay before treatment (baseline and at 1, 3, and 6 months after initiating anti-TB medication. Results Of 67 eligible patients, 59 (88.1% completed the study protocol. All participants were males who were human immunodeficiency virus (HIV-negative and had no chronic diseases. Their median age was 21 years (range, 20-48. Initially, 57 (96.6% patients had positive QFT-GIT results, and 53 (89.8%, 42 (71.2%, and 39 (66.1% had positive QFT-GIT results at 1, 3, and 6 months, respectively. The IFN-γ level at baseline was 5.31 ± 5.34 IU/ml, and the levels at 1, 3, and 6 months were 3.95 ± 4.30, 1.82 ± 2.14, and 1.50 ± 2.12 IU/ml, respectively. All patients had clinical and radiologic improvements after treatment and were cured. A lower IFN-γ level, C-reactive protein ≥ 3 mg/dl, and the presence of fever (≥ 38.3°C at diagnosis were associated with negative reversion of the QFT-GIT assay. Conclusion Although the IFN-γ level measured by QFT-GIT assay decreased after successful anti-TB treatment in most participants, less than half of them exhibited QFT-GIT reversion. Thus, the reversion to negativity of the QFT-GIT assay may not be a good surrogate for treatment response in otherwise healthy young patients with TB.

  18. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers


    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (pcultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (pculture population, the concentration of cells being cultured, and the plastic ware used for the in vitro culture. These findings stress the importance of documenting the precise assay conditions when publishing results of in vitro IFN-γ release assays.

  19. Application of interferon-gamma release assay in diagnosis of tuberculous meningitis%γ-干扰素体外释放试验在结核性脑膜炎诊断中的应用

    钱福初; 何建方; 沈轶群; 秦基取; 王伟洪; 杨水新; 戴利成


    Objective;To evaluate the diagnosis value of Mycobacterium tuberculosis infection related interferon -gamma (IFN -γ) in vitro release assay(TB -IGRA) in patients with tuberculous meningitis(TBM). Methods: The level of interferon - gamnia( IFN - -γ) release in vitro in whole blood were detected in 32 patients with tuberculous meningitis, 33 patients with bacterial meningitis and 23 patients with virus meningitis. Also the results were compared with the tuberculosis antibody, acid - fast staining, TB - DNA PCR, bacterial culture of cerebrospinal fluid and PPD skin test. Results: The sensitivity of TB - IGRA was more higher than other tests in diagnosis of tuberculous meningitis, furthermore, TB -IGRA also had high specificity. The positive likelihood ratio of this assay was 9.83, the Youden's index of this assay was 78.6%. Conclusion; The TB - IGRA will be a convenient, quick and effective tool for diagnosis of TBM patients.%目的:探讨体外干扰素释放试验对结核性脑膜炎诊断的意义.方法:检测结核性脑膜炎组32例,非结核性脑膜炎组56例(包括化脓性脑膜炎33例,病毒性脑膜炎23例)病人外周血中体外干扰素释放水平,同时与脑脊液抗结核抗体、脑脊液抗酸染色、核酸检测、PPD及脑脊液抗酸杆菌培养结果进行比较.结果:经比较体外干扰素释放试验对结核性脑膜炎检测敏感度达87.5%,均高于其它几种方法,并且具有较高特异性,且阳性似然比为9.83,正确诊断指数达78.6%.结论:体外干扰素释放试验是一种可辅助结核性脑膜炎诊断的方便、快速、有效的方法.

  20. Qualitative Comparison of IGRA and ESRL Radiosonde Archived Databases

    Walker, John R.


    Multiple databases of atmospheric profile information are freely available to individuals and groups such as the Natural Environments group. Two of the primary database archives provided by NOAA that are most frequently used are those from the Earth Science Research Laboratory (ESRL) and the Integrated Global Radiosonde Archive (IGRA). Inquiries have been made as to why one database is used as opposed to the other, yet to the best of knowledge, no formal comparison has been performed. The goal of this study is to provide a qualitative comparison of the ESRL and IGRA radiosonde databases. For part of this analyses, 14 upper air observation sites were selected. These sites all have the common attribute of having been used or are planned for use in the development of Range Reference Atmospheres (RRAs) in support of NASA's and DOD's current and future goals.

  1. Improving T-cell assays for the diagnosis of latent TB infection: potential of a diagnostic test based on IP-10

    Ruhwald, Morten; Petersen, Janne; Kofoed, Kristian


    BACKGROUND: There is a need for simple tools such as the M.tuberculosis specific IFN-gamma release assays (IGRA) to improve diagnosis of M.tuberculosis-infection in children. The aim of the study was to evaluate the performance of an IP-10 and IL-2 based tests for the diagnosis of M.tuberculosis-...

  2. Evaluation of clinical diagnostic performance of interferon-gamma release assays for tuberculosis%γ-干扰素释放试验对结核病患者临床诊断性能评价研究

    秦建成; 徐军; 戴亮; 赵静; 王卫东; 李想; 张岚; 毛远丽


    Objective To evaluate the diagnostic performance and application value of enzyme-linked immunosorbent assay (ELISA)-based interferon-gamma release assays (IGRAs) with A.TB kit for patients with tuberculosis. Methods Of 108 patients en-rolled in the study, 60 were definitely diagnosed with tuberculosis (tuberculosis group), and 48 were controls (non-tuberculosis group). ELISA-based IGRAs with A.TB kit and enzyme-linked immunosorbent spot (ELISPOT)-based IGRAs with T-SPOT.TB kit were car-ried out simultaneously. Results The sensitivity and specificity of ELISA-based IGRAs with A.TB kit were 86.67% and 85.42%, and the area under a receiver-operating characteristic (ROC) curve was 0.822 [95% confidence interval (CI), 0.733-0.912]. The sensitivity and specificity of ELISPOT-based IGRAs with T-SPOT.TB kit were 91.67% and 87.50%, and the area under ROC curve was 0.884 (95%CI, 0.806-0.962). The difference in the area under ROC curve was not significant between the two assays (χ2=0.081, P=0.776). The consistency of the two assays was 87.04%(94/108) with the Kappa value of 0.738. Conclusions ELISA-based IGRAs with A.TB kit and ELISPOT-based IGRAs with T-SPOT.TB kit have a similar overall diagnostic performance, but ELISA-based IGRAs with A.TB kit is superior to ELISPOT-based IGRAs with T-SPOT.TB kit in the operation and interpretation of the test results.%目的:评价采用A.TB试剂[酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)]进行结核杆菌特异性γ-干扰素释放试验(interferon-gamma release assays, IGRAs)对结核病的临床诊断性能及应用价值。方法108例研究对象中,临床明确诊断结核病60例(结核组),非结核病对照者48例(非结核对照组)。同时采用A.TB试剂(ELISA法)和T-SPOT. TB试剂[酶联免疫斑点试验(enzyme-linked immunosorbent spot, ELISPOT)]进行IGRAs。结果 A.TB试剂(ELISA法)检测的灵敏度为86.67%,特异度为85.42%,受试

  3. IP-10 release assays in the diagnosis of tuberculosis infection

    Ruhwald, Morten; Aabye, Martine G; Ravn, Pernille


    The current state-of-the-art tests for infection with Mycobacterium tuberculosis - the IFN-γ release assays - rely on accurate measurement of the cytokine IFN-γ. Many other potential biomarkers are expressed in concert with IFN-γ, and IP-10 in particular has shown promising results. IP-10...

  4. Stabilnost ugovora vs. mobilnost igrača

    Andrijana Bilić


    Full Text Available U radu autorica razmatra problem usklađivanja ugovorne stabilnosti profesionalnih nogometaša i njihove slobode kretanja kao radnika. Nakon prikaza i analize koncepta međunarodne mobilnosti profesionalnih nogometaša, a posebice njegovog ograničenja u vidu sustava transfera uspostavljenog od strane FIFA-e, autorica ukazuje na najčešće probleme koji se javljaju, a vezani su uz definiranje statusa igrača, opciju jednostranog produljenja ugovora te jednostrani raskid ugovora i u tom smislu daje rješenja de lege ferenda.

  5. γ-干扰素释放试验对活动性肺结核的诊断价值%Diagnostic value of interferon gamma release assay to active pulmonary tuberculosis

    吴静; 徐建; 张映铭; 王彩英


    Objective To evaluate the diagnostic value of interferon gamma release assay to active pulmonary tuberculosis. Methods The interferon gamma release assay based on enzyme linked immunospot assay was performed in 75 patients with suspected active pulmonary tuberculosis. And the result was compared with tuberculosis culture. Results The sensitivity, specificity and accuracy of interferon gamma release assay were 81.1%, 90.9% and 84. 0%, respectively. The sensitivity of interferon gamma release assay was higher than that of tuberculosis culture (P<0. 01). Among 23 cases af negative culture results, 16 cases were detected positive by interferon gamma release assay. Conclusion Interferon gamma release assay is highly sensitive, specific and helpful to the early and rapid detection of active pulmonary tuberculosis.%目的 评价γ-干扰素释放试验(interferon gamma release assay,IGRA)对活动性肺结核的诊断价值.方法 采用IGRA检测75例肺结核疑似病例,与结核菌培养结果进行比较.结果 IGRA诊断肺结核灵敏度为81.1%,特异度为90.9%,准确性为84.0%;IGRA灵敏度明显高于结核菌培养(P<0.01);在23例培养阴性的肺结核确诊病例中,16例IGRA阳性.结论 IGRA灵敏度高,特异度强,有助于活动性肺结核的早期、快速诊断.

  6. Cytokine release assays: current practices and future directions.

    Finco, D; Grimaldi, C; Fort, M; Walker, M; Kiessling, A; Wolf, B; Salcedo, T; Faggioni, R; Schneider, A; Ibraghimov, A; Scesney, S; Serna, D; Prell, R; Stebbings, R; Narayanan, P K


    As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.

  7. 胸水中干扰素释放测定在结核性胸膜炎诊断中的价值%Value of interferon-γ release assay of pleural effusion in the diagnosis of tuberculous pleuritis

    石瑞如; 张曼林; 韩云港; 包惠圆; 王亚丽; 刘欣欣; 王伟; 张国龙; 苑星


    目的 探讨胸水中体外诱发的γ-干扰素释放测定(IGRA)在结核性胸膜炎诊断中的价值.方法 收集本院2009年11月至2012年1月呼吸内科连续收治的62例病理确诊的结核性胸膜炎患者和30例其他原因如肿瘤、炎性反应的胸膜炎患者,治疗前两周内收集胸水,结核分枝杆菌早期分泌抗原靶6(ESAT-6)刺激后应用ELISA法进行γ-干扰素浓度测定.结果 结核性胸膜炎患者胸水经ESAT-6抗原刺激后,γ-干扰素增加的百分率为16%~ 53%,IGRA的阳性率为90.3%(56/62);非结核性胸水患者经ESAT-6抗原刺激后,γ-干扰素增加6%~8%,30例中有2例IGRA为临界状态,其余均为阴性.结核性胸膜炎患者胸水γ-干扰素基础值水平也高于非结核性胸水患者[(1825±69) ng/L比(967±105) ng/L,P<0.05].结论 胸水IGRA检测有望成为鉴别结核性胸膜炎的一种准确可靠的方法.%Objective To explore the value of interferon-γ (IFN-γ) release assay (IGRA) of pleural effusion in the diagnosis of tuberculous pleuritis.Methods Between November 2009 and January 2012,62 patients with pathologically confirmed diagnosis of tuberculous pleuritis and 30 with miscellaneous diseases (i.e.tumor and inflammatory pleuritis) from the Department of Respiratory Internal Medicine,Henan Provincial Chest Hospital were enrolled in the study.The pleural effusion was sampled 2 weeks prior to the treatment.IFN-γ concentration was measured by enzyme-linked immunosorbent assay following stimulation with early secretory antigen target-6 (EAST-6) for Mycobacterium tuberculosis.Results Positire rate of IGRA was 90.3% (56/62) in tuberculous pleuritis patients,with an increase by 16%-53% in IFN-γ production.Though patients with non-tuberculous pleuritis yielded a 6%-8% increment in IFN-γ release following EAST-6 stimulation,30 cases tested negatively to IGRA with exception of 2 with borderline positive results.Pleuralfluid of tuberculous pleuritis

  8. Decay kinetics of an interferon gamma release assay with anti-tuberculosis therapy in newly diagnosed tuberculosis cases.

    Ifedayo M O Adetifa

    Full Text Available BACKGROUND: Qualitative and quantitative changes in IGRA response offer promise as biomarkers to monitor Tuberculosis (TB drug therapy, and for the comparison of new interventions. We studied the decay kinetics of TB-specific antigen T-cell responses measured with an in-house ELISPOT assay during the course of therapy. METHODS: Newly diagnosed sputum smear positive TB cases with typical TB chest radiographs were recruited. All patients were given standard anti-TB treatment. Each subject was followed up for 6 months and treatment outcomes were documented. Blood samples were obtained for the ESAT-6 and CFP-10 (EC ELISPOT at diagnosis, 1-, 2-, 4- and 6-months. Qualitative and quantitative reversion of the ELISPOT results were assessed with McNemar test, conditional logistic regression and mixed-effects hierarchical Poisson models. RESULTS: A total of 116 cases were recruited and EC ELISPOT was positive for 87% (95 of 109 at recruitment. There was a significant decrease in the proportion of EC ELISPOT positive cases over the treatment period (p<0.001. Most of the reversion occurred between the start and first month of treatment and at completion at 6 months. ESAT-6 had higher median counts compared to CFP-10 at all time points. Counts for each antigen declined significantly with therapy (p<0.001. Reverters had lower median SFUs at the start of treatment compared to non-Reverters for both antigens. Apart from the higher median counts for non-Reverters, no other risk factors for non-reversion were found. CONCLUSIONS: TB treatment induces qualitative and quantitative reversion of a positive in-house IGRA in newly diagnosed cases of active TB disease. As this does not occur reliably in the majority of cured individuals, qualitative and quantitative reversion of an IGRA ELISPOT has limited clinical utility as a surrogate marker of treatment efficacy.

  9. Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment.

    Gaucher, Sonia; Jarraya, Mohamed


    MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

  10. 21 CFR 864.7040 - Adenosine triphosphate release assay.


    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  11. The Clinical Usefulness of Tuberculin Skin Test versus Interferon-Gamma Release Assays for Diagnosis of Latent Tuberculosis in HIV Patients: A Meta-Analysis

    Ayubi, Erfan; Doosti-Irani, Amin; Sanjari Moghaddam, Ali; Sani, Mohadeseh; Nazarzadeh, Milad; Mostafavi, Ehsan


    Background Accurate diagnosis of latent tuberculosis infection (LTBI) is becoming increasingly concerning due to the increasing the HIV epidemic, which have increased the risk for reactivation to active tuberculosis (TB) infection. LTBI is diagnosed by tuberculin skin test (TST) and interferon-gamma release assays (IGRAs). Objectives The aim of the present study was to conduct a meta-analysis of published papers on the agreement (kappa) between TST and QuantiFERON-TB Gold In-Tube (QFT-GIT) tests for diagnosis of LTBI in HIV patient. Methods Electronic databases including PubMed/Medline, Elsevier/Scopus and Embase/Ovid were reviewed up Jan. 2016. We performed a random effect model meta-analysis for estimation of pooled Kappa between the two methods of diagnosis. Meta regression was used for assessing potential heterogeneity and Egger’s test was used for assessing small study effect and publication bias. Results The initial search strategy produced 6744 records. Of them, 23 cross-sectional studies met the inclusion criteria and 20 studies entered in meta-analysis. The pooled kappa was and prevalence-adjusted and bias-adjusted kappa (PABAK) were 0.37 (95% CI: 0.28, 0.46) and 0.59 (0.49, 0.69). The discordance of TST-/QFT-GIT+ was more than TST+/QFT-GIT-. Kappa estimate between two tests was linearly associated with age and prevalence index and inversely associated with bias index. Conclusion Fair agreement between TST and QFT-GIT makes it difficult to know whether TST is as useful as the QFT-GIT in HIV-infected patients. The higher discordance of TST-/QFT-GIT+ in compared to TST+/QFT-GIT- can induce the higher sensitivity of QFT-GIT for diagnosis LTBI in HIV patients. Disagreement between two tests can be influenced by error in measurements and prevalence of HIV. PMID:27622293

  12. Interferon Gamma Release Assay versus Tuberculin Skin Testing among Healthcare Workers of Highly Diverse Origin in a Moderate Tuberculosis Burden Country.

    Al Hajoj, Sahal; Varghese, Bright; Datijan, Alria; Shoukri, Mohammed; Alzahrani, Ali; Alkhenizan, Abdallah; AlSaif, Abdulaziz; Althawadi, Sahar; Fernandez, Grace; Alrajhi, Abdulrahman


    Health care workers (HCW's) are always at an increased risk of contracting tuberculosis (TB) infection. In Saudi Arabia, Interferon Gamma Release Assay (IGRA) has not been evaluated as a screening tool for latent TB infection (LTBI) among HCW's considering their high demographic diversity. During February 2012 to January 2015 a cross sectional study has been conducted in a tertiary care center with maximum demographically diverse staff population in the capital city-Riyadh. After a short interview and consenting, all the candidates were subjected to tuberculin skin test (TST) and QuantiFERON TB gold In-tube test (QFT). A logistic regression analysis was carried out for establishing the associations between putative risk factors and the diagnostic tests. The candidates were classified according to geographical origin and a detailed analysis was conducted on the impact of their origin towards the results of TST and QFT. Of the 1595 candidates enrolled, 90.6% were BCG vaccinated, female (67.9%) and mainly nurses (53.2%). Candidates with high risk of suspected or confirmed TB patient exposure were 56.1% and 76.5% of them had origin were associated with TST positivity while Occupational TB exposure and geographical origin of the candidates were associated with QFT positivity. A regular follow up on recently TST converted candidates showed no progression to active TB. The putative factors associated with the discordance were origin of the candidate (p value Origin of the candidates has strong association with the results of TST and QFT. The discordant results particularly TST negative and QFT positive needs more detailed analysis.

  13. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity

    Baseler Michael


    Full Text Available Abstract Background The interferon-γ (IFN-γ ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. Methods We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY and an in vitro stimulated anti-Flu matrix peptide (FMP-specific CTL. Results When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Conclusion Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT

  14. Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.

    Mobin A Karimi

    Full Text Available The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

  15. Evaluation of the Erythrocyte Malondialdehyde (MDA) Release Assay.


    Jolla, CA 92037) (95). Triglycerides, in this assay, undergo lipase hydrolysis , phosphorylation, and oxidation/reduction reactions. The final product...tocopherol in the guinea pig . J Nutr 109, 105-109(1979). 82. Mino M, Kasugai 0, Nagita A. Relationship between red blood cell and liver tocopherol...blood cell tocopherol and liver tocopherol in hyperlipemic rats as compared with plasma tocopherol. Lipids 1985;20:488-491. 6. Moudgil KD, Narang BS

  16. Diabetes is associated with lower tuberculosis antigen-specific interferon gamma release in Tanzanian tuberculosis patients and non-tuberculosis controls

    Faurholt-Jepsen, Daniel; Aabye, Martine Grosos; Jensen, Andreas Vestergaard


    Abstract Background: Diabetes is increasingly common in TB endemic regions and plays a role as a possible risk factor for increased progression from latent TB infection (LTBI) to active TB disease. Although the pathophysiological mechanisms are not fully understood, the immune system is weakened...... in diabetes patients and therefore the validity of interferon gamma release assays (IGRA) may be compromised. The aim of the present study was to assess the association between diabetes and Mycobacterium tuberculosis (Mtb) antigen-specific interferon gamma (IFN-γ) release in a TB endemic area among culture......-confirmed TB patients and non-TB controls. Methods: Culture-confirmed pulmonary TB patients (n = 187) and healthy non-TB neighbourhood controls (n = 190) from Mwanza, Tanzania were tested for the presence of circulating T cells recognizing Mtb antigens using an IGRA. The diabetes status of all participants...

  17. Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials

    D'Souza, F.; Bruin, A.; Biersteker, R.; Donnelly, G.T.; Klijnstra, J.W.; Rentrop, C.H.A.; Willemsen, P.R.


    An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and relea

  18. γ-干扰素释放试验对HIV感染者合并菌阴肺结核辅助诊断价值的探讨%Auxiliary diagnostic value of interferon-γrelease assay in HIV and pulmonary tuberculosis with negative cul-ture

    张林; 李琦


    value of interferon-γ release assays ( IGRAs) for human immunodeficiency virus infection and pulmonary tuberculosis with negative culture. Methods The clinical data of 128 cases of negative HIV pulmonary tuberculosis, HIV/AIDS and pulmonary tuberculosis by IGRAs were ret-rospectively analyzed. Rate within the group and between groups were compared by chi-square test. T test was used for measurement data. Taking positive or negative results of IGRAs as the dependent variable factor, it used logistic regression (parameter estimation based on the condition of backward method) to analyze the results. Results (1) The level of blood interferon-γ and the positive detection rate were higher in HIV and pulmonary tuberculosis with negative culture patients than in patients with HIV/AIDS (U=7. 813, P=0. 005;chi-square=8. 486, P=0. 004), but it was close to pulmonary tuberculosis with negative culture patients ( U =1. 109, P =0. 292; chi-square =2. 459, P=0. 117). (2) Compared with negative HIV/ pulmonary tuberculosis patients, the level of of CD4+, lymphocyte count and CD4+ /CD8+ increased obviously in IGRAs positive HIV/ pulmonary tuberculosis patients ( t=5. 349, P=0. 002;t=2. 834, P =0. 009; t =5. 576, P <0. 001), and blood sedimentation decreased (t = -2. 317, P=0. 029). (3) Logistic regression analysis showed that CD4+ was independent factor for HIV/ pulmonary tuberculosis with negative culture (Waldχ2 =4. 434, P=0. 035, OR=13. 5, 95% CI=1. 197 -152. 211). (4) The sensitivity of IGRAs detection was 35. 7%, and the diagnosis accuracy was 72. 7%, which were higher than tu-berculosis antibody (21. 4%, 63. 6%) and PPD (14. 2%, 67. 0%) in HIV/ pulmonary tuberculosis with negative culture. Its specific degree was 90. 0%, which was similar with PPD (91. 7%) and slightly higher than tuberculosis antibody (83. 3%). The detection sensitivity of IGRAs joint PPD and tuberculosis antibody was up to 60. 7%. Con-clusion IGRAS can help to sieve out early from HIV patients or find HIV

  19. Using IGRA in the diagnosis of tuberculosis or latent tuberculosis infection in HIV-positive persons

    Ravn, P.


    Persons with HIV and LTBI are at very high risk of developing active TB. The CD4 count drops in persons during the natural course of HIV/AIDS and at the same time the risk of TB increases. It is clear that there is a TB protective effect when HIV patients are placed on highly active antiretroviral......-positive individuals, and our learning curve is going up with new knowledge and circumstantial evidence. There is clearly an effect on the performance of the IGRA test when persons with active TB are co-infected with HIV. Studies looking at the performance of QFT-GIT in HIV-positive/negative TB patients show...... a positive IGRA response. There are, however, important unresolved issues that require further studies. What is the added value of using IGRAs in a clinical setting to correctly diagnose patients and prevent new cases? Are the differences between QFT-GIT and T-SPOT clinically relevant? Will we ever have...

  20. Concordancia de las pruebas de tuberculina e Interferón gamma en población reclusa Concordance of tuberculin tests and Interferon gamma release assays in the prison population

    A. Marco Mouriño


    Full Text Available Objetivos: Estudiar en población penitenciaria la concordancia de la prueba de la tuberculina (PT y las pruebas de interferón gamma (IFG. Material y métodos: Estudio prospectivo realizado en una prisión en mayo-junio de 2009. Se estudian los ingresos sin antecedente de tuberculosis (TB o con PT previa negativa o no realizada. Se realizó IDR de Mantoux (positivo ³ 10 mm y extracción sanguínea para prueba de IFG (QuantiFERON®-TB Gold. En los infectados, se realizó despistaje de TB. Se pasó un cuestionario y se solicitó consentimiento informado. El estudio fue aprobado por un Comité ético ajeno a instituciones penitenciarias. La concordancia entre PT e IFG se basó en el índice Kappa. Resultados: Se incluyeron 181 casos. El 62% eran extranjeros, el 17% vacunados por BCG, el 8,4% UDI y el 4% VIH+. En los extranjeros había más vacunados, menos UDI y menos infectados por VIH que en autóctonos (p=0,02, p=0,02, y p=0,01, respectivamente. La PT fue positiva en el 24% y la IFG en el 26%. Hubo información de ambas en 149 (82% casos. El 15,8% fueron discordantes. El índice Kappa fue de 0,6 (0,4-0,7. La concordancia varió según subgrupos, siendo mayor en autóctonos (kappa= 0,8 y menor en vacunados (kappa=0,4 e inmigrantes (kappa=0,5. Conclusión: La concordancia global fue moderada-buena, pero en vacunados e inmigrantes fue menor. El nivel de discordancia aconseja ampliar el estudio, así como evaluar que prueba predice mejor el riesgo de progresión a TB y el coste-beneficio de ambas en la población reclusa de nuestro país.Objective: To study the agreement of Tuberculin Skin Tests (TST and Interferon Gamma Release Assays (IGRA when screening tuberculosis infection amongst inmates recently admitted to prison. Materials and methods: Prospective study conducted in a prison during the months of May and June 2009. Inmates without a TB history, with previous TST negatives or without prior TSTs were included. Participants signed an

  1. Indirect assays for deoxyhypusine hydroxylase using dual-label ratio changes and oxidative release of radioactivity

    Abbruzzese, A.; Park, M.H.; Folk, J.E.


    Two procedures for rapid assay of deoxyhypusine hydroxylase activity are described. One of these assays measures changes in the /sup 3/H:/sup 14/C ratio of dual-labeled protein that results from the release of tritium from a specific position in the side chain of the /sup 3/H, /sup 14/C-labeled constituent amino acid deoxyhypusine upon its conversion to (/sup 3/H, /sup 14/C)hypusine. The other assay relies upon release of radioactivity from product protein by periodate oxidation of the radiolabeled side chain of component hypusine. The good correspondence of each of these assays with the ion exchange chromatographic method which measures hypusine and deoxyhypusine in acid hydrolysates of protein indicates that each provides a valid means of determining deoxyhypusine hydroxylase activity.

  2. A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity.

    Wagner, Bettina; Childs, Bronwen A; Erb, Hollis N


    Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the

  3. The impact of HIV infection and CD4 cell count on the performance of an interferon gamma release assay in patients with pulmonary tuberculosis.

    Martine G Aabye

    Full Text Available BACKGROUND: The performance of the tuberculosis specific Interferon Gamma Release Assays (IGRAs has not been sufficiently documented in tuberculosis- and HIV-endemic settings. This study evaluated the sensitivity of the QuantiFERON TB-Gold In-Tube (QFT-IT in patients with culture confirmed pulmonary tuberculosis (PTB in a TB- and HIV-endemic population and the effect of HIV-infection and CD4 cell count on test performance. METHODOLOGY/PRINCIPAL FINDINGS: 161 patients with sputum culture confirmed PTB were subjected to HIV- and QFT-IT testing and measurement of CD4 cell count. The QFT-IT was positive in 74% (119/161; 95% CI: 67-81%. Sensitivity was higher in HIV-negative (75/93 than in HIV-positive (44/68 patients (81% vs. 65%, p = 0.02 and increased with CD4 cell count in HIV-positive patients (test for trend p = 0.03. 23 patients (14% had an indeterminate result and this proportion decreased with increasing CD4 cell count in HIV-positive patients (test for trend p = 0.03. Low CD4 cell count (<300 cells/microl did not account for all QFT-IT indeterminate nor all negative results. Sensitivity when excluding indeterminate results was 86% (95% CI: 81-92% and did not differ between HIV-negative and HIV-positive patients (88 vs. 83%, p = 0.39. CONCLUSIONS/SIGNIFICANCE: Sensitivity of the QFT-IT for diagnosing active PTB infection was reasonable when excluding indeterminate results and in HIV-negative patients. However, since the test missed more than 10% of patients, its potential as a rule-out test for active TB disease is limited. Furthermore, test performance is impaired by low CD4 cell count in HIV-positive patients and possibly by other factors as well in both HIV-positive and HIV-negative patients. This might limit the potential of the test in populations where HIV-infection is prevalent.

  4. Simultaneous radioenzymatic assay of dopamine and dihydroxyphenylacetic acid: an index of in vivo dopamine release

    Cheng, C.H.; Wooten, G.F.


    The relative brain tissue concentrations of dopamine (DA) and its deaminated metabolite, dihydroxyphenylacetic acid (DOPAC), appears to be a reliable index of the functional activity of dopaminergic neurons. In order to apply this approach to the assessment of dopaminergic neuronal activity in small regions of brain, we have developed a sensitive radioenzymatic assay for simultaneous measurement of DA and DOPAC. The sensitivity of the assay for DA is approximately 10 pg and for DOPAC 100 pg. In addition, the assay is highly specific, simple, and relatively inexpensive. The concurrent estimation of tissue DA and DOPAC concentrations seems to be a reliable means of evaluating the rate of DA turnover or release in behavioral, electrical stimulation, and certain drug paradigms. However, the release or turnover of DA as induced by D-amphetamine (and perhaps other indirectly-acting dopaminemimetic drugs) cannot be meaningfully assessed by measurement of DA and DOPAC alone.

  5. Dječja igra u humanizaciji boravka djece u bolnici

    KOLAK, Željka; ŠEČIĆ, Ana; Matijević, Valentina; BARTOLOVIĆ, JELENA; KOVAČIĆ-PERCELA, Daniela; HAJDAŠ, Mirjana; MLINAR, Milena


    Prilikom bolničkog liječenja (hospitalizacije) potrebno je voditi brigu o zadovoljenju djetetovih potreba. Odlazak u bolnicu velika je promjena za dijete, i često praćena neugodnim emocionalnim stanjima koja se mogu opisati kao sindrom hospitalizma. Većina djece prolazi sljedeće faze kojima se opisuje ovaj sindrom: faza prosvjeda, faza očajanja i faza prividne prilagodbe. Mnogo je teorija o prirodi i smislu igre, a ono u čemu se one generalno slažu jest da je dječja igra puno više od same ...

  6. Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials.

    D'Souza, Fraddry; Bruin, Anouk; Biersteker, Rens; Donnelly, Glen; Klijnstra, Job; Rentrop, Corne; Willemsen, Peter


    An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and release properties of many surfaces when compared to a reference. The method is based upon the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent SYTO 13 stain. A strong linear correlation exists between the fluorescence of the bacterial suspension measured (RFU) using a plate reader and the total bacterial count measured with epifluorescence microscopy. This relationship allows the fluorescent technique to be used for the quantification of bacterial cells attached to surfaces. As the bacteria proliferate on the surface over a period of time, the relative fluorescence unit (RFU) measured using the plate reader also shows an increase with time. This was observed on all three test surfaces (glass, Epikote and Silastic T2) over a period of 4 h of bacterial growth, followed by a release assay, which was carried out by the application of hydrodynamic shear forces using a custom-made rotary device. Different fixed rotor speeds were tested, and based on the release analysis, 12 knots was used to provide standard shear force. The assay developed was then applied for assessing three different antifouling coatings of different surface roughness. The novel assay allows the rapid and sensitive enumeration of attached bacteria directly on the coated surface. This is the first plate reader assay technique that allows estimation of irreversibly attached bacterial cells directly on the coated surface without their removal from the surface or extraction of a stain into solution.

  7. A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

    Wolf, Babette; Morgan, Hannah; Krieg, Jennifer; Gani, Zaahira; Milicov, Adriana; Warncke, Max; Brennan, Frank; Jones, Stewart; Sims, Jennifer; Kiessling, Andrea


    The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine

  8. Significance of interferon-gamma release assay in child tuberculosis infection%干扰素-γ释放试验在儿童结核感染中的应用价值

    钟华清; 徐梦华; 卢丽娟; 杜亚娟; 张洁琼; 徐锦


    目的:评价干扰素-γ(IFN-γ)释放试验(IGRA)对儿童结核感染的诊断价值。方法采用以结核分枝杆菌特异性抗原——早期分泌抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)为基础,基于酶联免疫斑点法(ELISPOT)的 IGRA(即 T-SPOT)检测了2200例非结核病患儿和34例活动性结核病患儿体内特异性 T 淋巴细胞分泌的 IFN-γ水平,评价其在儿童活动性结核病和结核分枝杆菌潜伏感染中的敏感性和特异性,同时分析了该实验的各项影响因素。结果T-SPOT 诊断活动性结核病的敏感性为73.5%、特异性为99.0%。T-SPOT检测结果与性别、年龄、卡介苗接种史、结核病接触史、机体免疫状态和感染部位无关。结论T-SPOT 敏感性较高,特异性强,有助于儿童活动性结核病的早期快速诊断。%Objective To evaluate the significance of interferon-gamma(IFN-γ)release assay(IGRA)for diagnosis of tuberculosis infection in child. Methods Mycobacterium tuberculosis-specific antigen [early-secreted antigenic target-6 (ESAT-6)and culture filtrate protein-10(CFP-10)] was used. By IGRA enzyme-linked immunospot assay(ELISPOT) (T-SPOT),34 active tuberculosis children and 2 200 non-tuberculosis children were enrolled and determined for IFN-γlevels,and the sensitivity and specificity were evaluated. Some of influential factors were analyzed. Results The sensitivity and specificity of T-SPOT for the diagnosis of active tuberculosis were 73.5% and 99.0%,respectively. The results of T-SPOT had no relationship with sex,age,Bacillus Calmette-Guerin vaccination status,the history of exposure to source case,immunity state and infection position. Conclusion T-SPOT is sensitive,specific and helpful for early and rapid diagnosis of active tuberculosis in children.

  9. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G


    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  10. Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

    El-Hawiet, Amr; Shoemaker, Glen K; Daneshfar, Rambod; Kitova, Elena N; Klassen, John S


    Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

  11. Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

    Asther Marcel


    Full Text Available Abstract Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol, it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the

  12. Didaktična računalniška igra na temo osnov matematične logike

    Zupančič, Katja


    V magistrskem delu Didaktična računalniška igra na temo osnov matematične logike smo predstavili nekaj osnovnih opredelitev pojma igra ter teorijo računalniških iger in izobraževalnih računalniških iger. Pregledali smo učne načrte (splošni učni načrt za matematiko, učni načrt za izbirna predmeta logika in matematična delavnica) in učbenike za osnovno šolo, kjer se pojavljajo teme osnov matematične logike, ter glavne učne cilje tekmovanja iz logike, ki ga organizira Zveza za tehnično kulturo S...

  13. Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine-release assay.

    Pfeffer, A; Phegan, M D; Bany, J


    The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.

  14. γ-干扰素释放试验对活动性肺结核的诊断价值%Clinical value of interferon-gamma release assay in the diagnosis of active pulmonary tuberculosis

    任斐; 岳英; 苗风济; 孙晓方; 严文; 苟超伦


    目的:评价γ-干扰素释放试验( interferon-gamma release assay,IGRA, T-SPOT. TB)对活动性肺结核的诊断价值。方法选取活动性肺结核组(观察组)120例和非结核病组(对照组)84例共计204例,两组患者均行结核菌素试验( tuberculin skin test,TST),血清结核抗体检测( TB-Ab),T-SPOT. TB检测,比较三种方法对活动性肺结核诊断的敏感度、特异度、阳性预测值、阴性预测值。结果外周血 T-SPOT. TB 诊断活动性肺结核的敏感度为90.0%,明显高于 TST (50.0%)和 TB-Ab (55.8%)(P0.05);T-SPOT. TB诊断活动性肺结核的阳性预测值明显高于TST(81.2% vs 67.4%,P0.05);T-SPOT. TB诊断活动性肺结核的阴性预测值为83.0%,明显高于TST(47.8%)和TB-Ab(51.4%)(P0. 05). The positive prediction value of T-SPOT. TB in the diagnosis of active pulmonary tuberculosis was higher than that of TST (81. 2% vs 67. 4%,P 0. 05). The negative prediction value of T-SPOT. TB for non-active pulmonary tuberculosis was 83. 0%, significantly higher than that of TST(47. 8%)and TB-Ab(51. 4%)(P<0. 05). Conclusion Peripheral blood T-SPOT. TB is very sensitive and specific, and the negative prediction value is significantly better than TST and TB-Ab. T-SPOT. TB has a good application value in diagnosis and differenti-al diagnosis of active pulmonary tuberculosis.

  15. Comparison of interferon-gamma release assays and adenosine deaminase of pleural fluid for the diagnosis of pleural tuberculosis



    Objective To compare the diagnostic performance of interferon gamma releasing assays(T-SPOT.TB)and adenosine deaminase(ADA)in pleural tuberculosis,and therefore to evaluate the value of T-SPOT.TB in a high tuberculosis burden country.Methods From June 2011to November 2012,111 patients with pleural fluid in Beijing Chest Hospital,Capital Medical University were

  16. Interferon-Gamma Release Assays versus Tuberculin Skin Testing for the Diagnosis of Latent Tuberculosis Infection: An Overview of the Evidence


    A profusion of articles have been published on the accuracy and uses of interferon-gamma releasing assays. Here we review the clinical applications, advantages, and limitations of the tuberculin skin test and interferon-gamma release assays and provide an overview of the most recent systematic reviews conducted for different indications for the use of these tests. We conclude that both tests are accurate to detect latent tuberculosis, although interferon-gamma release assays have higher speci...

  17. 结核抗原特异性IFN-γ ELISPOT检测技术用于诊断HIV感染者人群中结核潜伏感染的研究%Use of IFN-γ release assay in latent tuberculosis infection diagnosis in general and HTV-infected populations

    王辉; 刘映霞; 周伯平; 陈心春; 卢洪州; 谭艳; 朱秀云; 廖明凤; 张洁云; 刘艳; 刘水腾; 张路坤; 周映


    目的 探讨结核分枝杆菌抗原特异性IFN-γ酶联免疫斑点(ELISPOT)检测技术在HIV感染者中用于诊断结核潜伏感染的应用价值.方法 运用自行研发的结核分枝杆菌抗原特异性IFN-γ ELISPOT检测方法对110例确诊的HIV感染者及205例HIV阴性健康志愿者的外周血结核特异性IFN-γ释放水平进行检测,并同时进行结核菌素皮肤试验(研).结果 ELISPOT在HIV感染和HIV/AIDS正在接受抗病毒治疗人群中的阳性率分别为24.5%和29.8%,明显高于HIV阴性健康人群7.3%(P<0.001);对多种不同部位多肽抗原刺激以P8.10的敏感性最高,但与其他抗原的差异无统计学意义(P>0.05);不同CD4细胞计数水平对ELISPOT检测结果尤影响(P>0.05);PPD试验在HIV感染人群中的阳性率远远低于ELISPOT检测方法,其差异有统计学意义(P<0.0001).结论 结核分枝杆菌抗原特异性IFN-γ ELISPOT检测技术在艾滋病合并结核潜伏感染的早期诊断中有很大的应用价值,推荐用于艾滋病合并结核潜伏感染的辅助诊断.%Objective To assess the validity of a newly developed in-house ELISPOT IFN-γ release assay (IGRA) for the detection of latent tuberculosis infection among HIV infected individuals. Methods In-house ELISPOT assay were performed, together with a tuberculin skin test in 205 health controls and 110 HIV infected individuals , who had no signs of active tuberculosis at time of enrolment . Results Using the ELISPOT assay, positivity rates for the 205 health controls, 110 HIV infected individuals and 47 AIDS patients on highly active antiretrovial therapy (HAART) were 7. 3% , 24.5% , 29. 8% , respectively. These results indicated that the positive rates obtained from HIV infected individuals (include patient on HAART) was significantly higher than health controls( P < 0.001). We found no significant correlation between the CD4 cell count and positivity of ELISPOT assay (P >0.05 ). The proportion of subjects with

  18. Development of a phenotypic assay for characterisation of ethanologenic yeast strain sensitivity to inhibitors released from lignocellulosic feedstocks.

    Greetham, D; Wimalasena, T; Kerruish, D W M; Brindley, S; Ibbett, R N; Linforth, R L; Tucker, G; Phister, T G; Smart, K A


    Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.

  19. Mycobacterium kansasii Infection in a Patient Receiving Biologic Therapy-Not All Reactive Interferon Gamma Release Assays Are Tuberculosis.

    Saleem, Nasir; Saba, Raya; Maddika, Srikanth; Weinstein, Mitchell


    Mycobacterium kansasii, a nontuberculous mycobacterium, can lead to lung disease similar to tuberculosis. Immunotherapeutic biologic agents predispose to infections with mycobacteria, including M kansasii. T-cell-mediated interferon gamma release assays like QuantiFERON-TB Gold Test (QFT) are widely used by clinicians for the diagnosis of infections with Mycobacterium tuberculosis; however, QFT may also show positive result with certain nontuberculous mycobacterial infections. We report a case of M kansasii pulmonary infection, with a positive QFT, in an immunocompromised patient receiving prednisone, leflunomide and tocilizumab, a humanized anti-interleukin-6 receptor monoclonal antibody. This case highlights the risk of mycobacterial infections with the use of various biologic agents and the need for caution when interpreting the results of interferon gamma release assays.

  20. Executive summary of the guidelines for the use of interferon-γ release assays in the diagnosis of tuberculosis infection.

    Santin, Miguel; García-García, José-María; Rigau, David; Altet, Neus; Anibarro, Luis; Casas, Irma; Díez, Nuria; García-Gasalla, Mercedes; Martínez-Lacasa, Xavier; Penas, Antón; Pérez-Escolano, Elvira; Sánchez, Francisca; Domínguez, José


    Interferon-gamma release assays are widely used for the diagnosis of tuberculosis infection in Spain. However, there is no consensus on their application in specific clinical scenarios. To develop a guideline for their use, a panel of experts comprising specialists in infectious diseases, respiratory diseases, microbiology, pediatrics and preventive medicine, together with a methodologist, conducted a systematic literature search, summarized the findings, rated the quality of the evidence, and formulated recommendations following the GRADE (Grading of Recommendations of Assessment Development and Evaluations) methodology. This document provides evidence-based guidance on the use of interferon-gamma release assays for the diagnosis of tuberculosis infection in patients at the risk of tuberculosis or suspected of having active disease. The guidelines will be applicable to specialist and primary care, and public health.

  1. A positive serum basophil histamine release assay is a marker for ciclosporin-responsiveness in patients with chronic spontaneous urticaria

    Iqbal, Kamran; Bhargava, Kapil; Skov, Per Stahl;


    ABSTRACT: The electronic records of 398 patients with chronic spontaneous urticaria (CSU) who had had a serum basophil histamine release assay (BHRA) performed as a marker of functional autoantibodies were audited. The BHRA was positive in 105 patients (26.4%). Fifty eight were treated with ciclo...... with ciclosporin because they were H1 anti-histamine unresponsive. CSU patients with a positive BHRA were more likely to respond clinically (P...

  2. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory


    criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

  3. Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications

    Georgiou, Christos D.; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Kontantinos; Papapostolou, Ioannis; Quinn, Richard C.; McKay, Christopher P.; Sun, Henry J.


    This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2, and their quantification by an O2 electrode based on the stoichiometry of the involved reactions: The intermediate product O2 from the hydrolysis of metal superoxides is converted by cytochrome c to O2, and also by superoxide dismutase (SOD) to 1/2 mol O2 and 1/2 mol H2O2, which is then converted by catalase (CAT) to 1/2 mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to 1/2 mol O2 by CAT. The assay-method was validated in a sealed sample chamber using a liquid-phase Clark-type O2 electrode with known concentrations of O2 and H2O2, and with commercial metal superoxide and peroxide mixed with Mars analogue Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(exp. -3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by gamma-radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, demonstrating the suitability of these enzymes for planetary missions, e.g., in Mars or Europa.

  4. Biosensor cell assay for measuring real-time aldosterone-induced release of histamine from mesenteric arteries

    Dalgaard, Emil G; Andersen, Kenneth; Svenningsen, Per


    as a sensitive biosensor assay for histamine release from isolated mouse mesenteric arteries. Activation of the H1 receptor by histamine was measured as an increased number of intracellular Ca(2+) transient peaks using fluorescence imaging RESULTS: The developed biosensor was sensitive to histamine...... in physiological relevant concentrations and responded to substances released by the artery preparation. Aldosterone treatment of mesenteric arteries from wild type mice for 50 minutes resulted in an increased number of intracellular Ca(2+) transient peaks in the biosensor cells, which was significantly inhibited...... by the histamine H1 blocker pyrilamine. Mesenteric arteries from mast cell deficient SASH mice induced similar pyrilamine-sensitive Ca(2+) transient response in the biosensor cells. Mesenteric arteries from wild type and SASH mice expressed histamine decarboxylase mRNA, indicating that mast cells are not the only...

  5. Identifying Carbohydrate Ligands of a Norovirus P Particle using a Catch and Release Electrospray Ionization Mass Spectrometry Assay

    Han, Ling; Kitova, Elena N.; Tan, Ming; Jiang, Xi; Klassen, John S.


    Noroviruses (NoVs), the major cause of epidemic acute gastroenteritis, recognize human histo-blood group antigens (HBGAs), which are present as free oligosaccharides in bodily fluid or glycolipids and glycoproteins on the surfaces of cells. The subviral P particle formed by the protruding (P) domain of the NoV capsid protein serves as a useful model for the study NoV-HBGA interactions. Here, we demonstrate the application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against the P particle to rapidly identify NoV ligands and potential inhibitors. Carbohydrate libraries of 50 and 146 compounds, which included 18 and 24 analogs of HBGA receptors, respectively, were screened against the P particle of VA387, a member of the predominant GII.4 NoVs. Deprotonated ions corresponding to the P particle bound to carbohydrates were isolated and subjected to collision-induced dissociation to release the ligands in their deprotonated forms. The released ligands were identified by ion mobility separation followed by mass analysis. All 13 and 16 HBGA ligands with intrinsic affinities >500 M-1 were identified in the 50 and the 146 compound libraries, respectively. Furthermore, screening revealed interactions with a series of oligosaccharides with structures found in the cell wall of mycobacteria and human milk. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors, as estimated from the relative abundance of released ligand ions.

  6. Utility value of a T-cell interferon-γ release assay based on recombinant Mycobacterium tuberculosis 11kD protein in the diagnosis of tuberculosis



    Objective To evaluate the diagnostic efficiency of a T-cell interferon-γrelease assay based on recombinant Mycobacterium tuberculosis(MTB)11kD protein for diagnosing tuberculosis.Methods This prospective study enrolled inpatients with suspected tuberculosis at PUMCH to examine the diagnostic sensitivity,specificity,predictive value(PV)and likelihood ratio(LR)of T-cell interferon-γrelease assays based on recombinant MTB-11kD

  7. Quality assessment of an interferon-gamma release assay for tuberculosis infection in a resource-limited setting

    Hang Nguyen TL


    Full Text Available Abstract Background When a test for diagnosis of infectious diseases is introduced in a resource-limited setting, monitoring quality is a major concern. An optimized design of experiment and statistical models are required for this assessment. Methods Interferon-gamma release assay to detect tuberculosis (TB infection from whole blood was tested in Hanoi, Viet Nam. Balanced incomplete block design (BIBD was planned and fixed-effect models with heterogeneous error variance were used for analysis. In the first trial, the whole blood from 12 donors was incubated with nil, TB-specific antigens or mitogen. In 72 measurements, two laboratory members exchanged their roles in harvesting plasma and testing for interferon-gamma release using enzyme linked immunosorbent assay (ELISA technique. After intervention including checkup of all steps and standard operation procedures, the second trial was implemented in a similar manner. Results The lack of precision in the first trial was clearly demonstrated. Large within-individual error was significantly affected by both harvester and ELISA operator, indicating that both of the steps had problems. After the intervention, overall within-individual error was significantly reduced (P Conclusion BIBD and analysis of fixed-effect models with heterogeneous variance are suitable and useful for objective and individualized assessment of proficiency in a multistep diagnostic test for infectious diseases in a resource-constrained laboratory. The action plan based on our findings would be worth considering when monitoring for internal quality control is difficult on site.

  8. Analysis of Ebola Virus and VLP Release Using an Immunocapture Assay


    vaccine efficacy. While other viral pro- teins such as VP40, VP24, VP30, and VP35 expressed by a Venezuelan encephalitis virus (VEE)-based replicon are...VLP release was also achieved by VP30 (Fig. 6). While mouse studies have shown some protective effect by Ebola VP35 expressed in replicons , VP35 is...vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 251 (1), 28–37. uang, Y., Xu, L., Sun, Y., Nabel, G.J

  9. IFN-γ release assay: a diagnostic assistance tool of tuberculin skin test in pediatric tuberculosis in China

    SUN Lin; SHEN A-dong; YAN Hui-min; HU Ying-hui; JIAO Wei-wei; GU Yi; XIAO Jing; LI Hui-min; JIAO An-xia; GUO Ya-jie


    Background Prompt diagnosis of Mycobacterium tuberculosis (MTB) infection is an essential step in tuberculosis control and elimination. However, it is often difficult to accurately diagnose pediatric tuberculosis (TB). The tuberculin test (TST) may have a low specificity because of cross-reactivity with antigens present in Mycobacterium bovis bacillus Calmette-Guerin (BCG) and other mycobacteria, especially in China with a predominantly BCG-vaccinated population.Early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), stand out as suitable antigens that induce an interferon-gamma (IFN-γ) secreting, T-cell-mediated immune response to infection. While,considered the higher costs and complexity of the IFN-γ release assay (TSPOT), we aimed to evaluate the TSPOT and TST test in the clinical diagnosis of pediatric tuberculosis and to establish a diagnostic process suitable for China.Methods The sensitivity and specificity of the assay were evaluated in total seventy four children with active tuberculosis and fifty one nontuberculous children with other disease, and then the results were compared with TST.Logistic regression models were used to identify variables that were associated with positive results for each assay. The independent variables included sex, age, birth place, vaccination history, close contract with an active TB patient.Results The sensitivity of TSPOT was higher than TST in active TB children with or without BCG vaccination, as well as in children with culture-confirmed TB. But the difference was not significant statistically. Combining results of the TSPOT and TST improved the sensitivity to 94.6%. Agreement of the TST and TSPOT was low (77.0%, k=0.203) in active TB patients. The difference in specificity between TSPOT and TST test was statistically significant (94.1% vs.70.6%, P=0.006). Specificity of the two tests in patients without prior BCG vaccination history was similar (80.0% vs.60.0%). The concordance

  10. Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

    Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.


    A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL (mean, 15 +/- 7 (+/- SD) pg/mL; n = 58). Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term (1462 +/- 752 (+/- SD) pg/mL; n = 55), and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA.

  11. Prognostic value of interferon-γ release assays, a population-based study from a TB low-incidence country

    Hermansen, Thomas Stig; Lillebaek, Troels; Langholz Kristensen, Kristina


    BACKGROUND: The ability of interferon-γ release assays to predict the development of TB has been investigated in many studies, but few cases develop TB during follow-up limiting the generalisation of results. METHODS: We assessed QuantiFERON-TB Gold In-Tube test (QFT) results from 15 980 Danish...... individuals and data on all TB cases in Denmark from 2005 to 2012 and determined the predictive value of the QFT for coprevalent TB (0-90 days after testing) and incident TB (>90 days). RESULTS: Coprevalent TB was diagnosed in 10.7% (183/1703) and 0.3% (38/13 463) cases with a positive and negative QFT......, respectively. For the QFT-positive cases, coprevalent TB was more frequent among persons 35  years (19.3% vs 7.2%, pTB, the positive and negative predictive values (PPV...

  12. Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography.

    Harada, Airi; Sasaki, Keiko; Kaneta, Takashi


    Here we describe the application of an in-capillary enzyme assay using micellar electrokinetic chromatography (MEKC) in the determination of enzyme activity in a crude culture medium containing lignin peroxidase released from Phanerochaete chrysosporium (P. chrysosporium). The method consists of a plug-plug reaction between lignin peroxidase and its substrate, veratryl alcohol, the separation of the product, veratraldehyde, from the other components including the enzyme and the culture medium, and the determination of the enzyme activity from the peak area of veratraldehyde produced by the plug-plug reaction. This method is more sensitive than conventional spectrophotometry since the background originates from the enzyme and the culture medium can be removed via MEKC separation. Veratraldehyde was separated at -10kV in a background electrolyte containing 50mM tartrate buffer (pH 2.5) and 50mM sodium dodecyl sulfate (SDS) after a plug-plug reaction in the capillary for 5min. The calibration curve of veratraldehyde was linear up to 4pmol (500μM) with a limit to quantification of 0.026pmol (3.2μM) (SN=10). The activity of lignin peroxidase was directly measured from the peak area of veratraldehyde. The activity of lignin peroxidase released from P. chrysosporium into the medium for 7 days was successfully determined to be 3.40UL(-1).

  13. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

    Thomas, Ajit G; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S


    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  14. Clinical evaluation of thyrotropin-releasing hormone (TRH) test with a sensitive immunoradiometric thyrotropin (TSH) assay kit

    Nakamura, Saeko; Demura, Reiko; Yamanaka, Yukako; Ishiwatari, Naoko; Jibiki, Kazuko; Odagiri, Emi; Demura, Hiroshi


    Thyrotropin-releasing hormone (TRH) test was performed using a commercially available immunoradiometric thyrotropin (TSH) assay kit (RIA-gnost hTSH) in patients with endocrine diseases. The basal serum concentration of TSH ranged from 0.2 to 2.9 in healthy subjects. The values for endocrine diseases, except for Graves' disease, were almost within the normal range. A significant increase in TSH values caused by TRH test was observed in females compared with males (4.4 - 24.7 vs 4.1 - 12.3 In cases of Graves' disease, there was a good correlation between the basal TSH value and the response of TSH to TRH. However, in the other endocrine diseases, including acromegaly, prolactinoma, anorexia nervosa, Cushing syndrome, and hypopituitarism, the response of TSH to TRH did not necessarily correlated with the basal TSH value. TRH test would be of value in elucidating pathophysiologic features, as well as in accurately diagnosing secretion reserve of TSH. (Namekawa, K.).

  15. Measurement Uncertainty of Chromogenic LAL Assays: Reaction Time and Proportion of Endotoxin and LAL Reagent Affect Release of p-Nitroaniline.

    Ostronoff, Celina Silva; Lourenço, Felipe Rebello


    Limulus Amebocyte Lysate (LAL) assays are widely used for detection and quantification of bacterial endotoxins in pharmaceuticals and medical devices. However, there are only a few studies on the measurement uncertainty of LAL assays. The aim of this work was to identify and quantify the main sources of measurement uncertainty for end point and kinetic-chromogenic LAL assays. Response surface methodology was used to study how the release of p-nitroaniline (pNA) is affected by reaction time and proportion of endotoxin and LAL reagent in end point and kinetic-chromogenic LAL assays, respectively. Increased release of pNA was observed when reaction time was increased. In addition, if different volumes of sample (or endotoxin standard) and LAL reagent are used, the pNA release rate will be affected. These results may be due to the increased interaction between the bacterial endotoxin and LAL-activated enzyme. Final measurement uncertainties (95% confidence interval) were 90-120% and 90-127% of bacterial endotoxin content for end point and kinetic-chromogenic assays, respectively. These values are reasonable for the scope of the method and allow the application of these measurement uncertainties in routine analysis of pharmaceuticals and medical devices.

  16. Histamine release-neutralization assay for sera of patients with atopic dermatitis and/or cholinergic urticaria is useful to screen type I hypersensitivity against sweat antigens.

    Shindo, Hajime; Ishii, Kaori; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro


    We previously reported that about 80 % of patients with atopic dermatitis and 60 % with cholinergic urticaria revealed type I allergy against sweat, by means of skin test against autologous sweat and/or histamine-release test for peripheral blood basophils with semi-purified sweat antigen. In this study, we developed an assay for sera to neutralize histamine-releasing activity of semi-purified sweat antigen. The semi-purified sweat antigen was pre-incubated with serially diluted sera for 30 min at 37 °C and was subjected to histamine-release activity. Histamine release-neutralization (HRN) activities were calculated by measuring the amount of histamine release from basophils in the presence or absence of semi-purified sweat antigen. Of 62 subjects, 39 showed positive histamine release (≥5 %) from their basophils in response to semi-purified sweat antigen, and sera of 34 out of 39 subjects (87.2 %) were also positive in HRN activity (≥10 %). The specificity of the HRN assay was 0.522. Moreover, HRN activities in sera were largely correlated with degrees of histamine release from peripheral blood basophils of the same donors in response to sweat antigen. To identify the substance that neutralizes histamine-release activity, we removed IgE and IgG from the sera of HRN (+) subjects by column chromatography. The HRN activities in 30 out of 42 sera were largely reduced by the removal of IgG. On the other hand, sera of four subjects lost HRN activity by the removal of IgE, suggesting that the majority of HRN (+) subjects have serum IgG against the sweat antigen as well as IgE bound to peripheral basophils. Thus, the HRN assay maybe useful for the screening of type I allergy against sweat antigen.

  17. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  18. Response of luteinizing hormone and follicle stimulating hormone to luteinizing hormone-releasing hormone in prepubertal and pubertal chidren, as measured by a highly sensitive immunradiometric assay



    To investigate the age-related changes in the pituitary responsiveness to luteinizing hormone-releasing hormone (LH-RH), the consentrations of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured before and after LH-RH administra-tion using the highly sensitive immunoradiometric assay (IRMA) in 283 normal children (161 males and 77 females) between 4 and 14 years old and in 22 patients (18 males and 4 females) with pituitary dwarfism. Then, the area of response ...

  19. High-sensitive and rapid detection of Mycobacterium tuberculosis infection by IFN-γ release assay among HIV-infected individuals in BCG-vaccinated area

    Jiang Weimin


    Full Text Available Abstract Background An accurate test for Mycobacterium tuberculosis infection is urgently needed in immunosuppressed populations. The aim of this study was to investigate the diagnostic power of enzyme-linked immunospot (ELISPOT-based IFN-γ release assay in detecting active and latent tuberculosis in HIV-infected population in bacillus Calmette-Guerin (BCG-vaccinated area. A total of 100 HIV-infected individuals including 32 active tuberculosis patients were recruited. An ELISPOT-based IFN-γ release assay, T-SPOT.TB, was used to evaluate the M. tuberculosis ESAT-6 and CFP-10 specific IFN-γ response. Tuberculin skin test (TST was performed for all recruited subjects. Results The subjects were divided into group HIV+ATB (HIV-infected individuals with active tuberculosis, n = 32, group HIV+LTB (HIV-infected individuals with positive results of T-SPOT.TB assay, n = 46 and group HIV only (HIV-infected individuals with negative results of T-SPOT.TB assay and without evidence of tuberculosis infection, n = 22. In group HIV+ATB and HIV+LTB, T-SPOT.TB positive rate in subjects with TST P 85% in patients with TB treatment for less than 1 month and CD4+ T cells ≥200/μl, while for patients treated for more than 3 months and CD4+ T cells Conclusion ELISPOT-based IFN-γ release assay is more sensitive and rapid for the diagnosis of TB infection in Chinese HIV-infected individuals with history of BCG vaccination, and could be an effective tool for guiding preventive treatment with isoniazid in latently infected people and for TB control in China.

  20. Diagnosis and follow-up of treatment of latent tuberculosis; the utility of the QuantiFERON-TB Gold In-tube assay in outpatients from a tuberculosis low-endemic country

    Blomberg Bjørn


    Full Text Available Abstract Background Interferon-gamma (IFN-γ Release Assays (IGRA are more specific than the tuberculosis skin test (TST in the diagnosis of latent tuberculosis (TB infection (LTBI. We present the performance of the QuantiFERON®-TB Gold In-tube (QFT-TB assay as diagnostic test and during follow-up of preventive TB therapy in outpatients from a TB low-endemic country. Methods 481 persons with suspected TB infection were tested with QFT-TB. Thoracic X-ray and sputum samples were performed and a questionnaire concerning risk factors for TB was filled. Three months of isoniazid and rifampicin were given to patients with LTBI and QFT-TB tests were performed after three and 15 months. Results The QFT-TB test was positive in 30.8% (148/481 of the total, in 66.9% (111/166 of persons with origin from a TB endemic country, in 71.4% (20/28 previously treated for TB and in 100% (15/15 of those diagnosed with active TB with no inconclusive results. The QFT-TB test was more frequently positive in those with TST ≥ 15 mm (47.5% compared to TST 11-14 mm (21.3% and TST 6-10 mm (10.5%, (p 0.05. Conclusion Only one third of those with suspected TB infection had a positive QFT-TB test. Recent immigration from TB endemic countries and long duration of exposure are risk factors for a positive QFT-TB test and these groups should be targeted through screening. Since most patients remained QFT-TB positive after therapy, the test should not be used to monitor the effect of preventive therapy. Prospective studies are needed in order to determine the usefulness of IGRA tests during therapy.

  1. Dried plasma spots in the diagnosis of tuberculosis

    Aabye, Martine G; Latorre, Irene; Diaz, Jessica


    Interferon-γ release assays (IGRA) are probably the most accurate tests for detection of latent M.tuberculosis infection, but IGRAs are labour intensive and transport of samples over longer distances is difficult. IP-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays...

  2. Use of Interferon-Gamma Release Assays in a Health Care Worker Screening Program: Experience from a Tertiary Care Centre in the United States


    BACKGROUND: Interferon-gamma release assays including the QuantiFERON-TB Gold In-Tube test (QFT-GIT [Cellestis Ltd, Australia]) may be used in place of the tuberculin skin test (TST) in surveillance programs for Mycobacterium tuberculosis infection control. However, data on performance and practicality of the QFT-GIT in such programs for health care workers (HCWs) are limited.OBJECTIVES: To assess the performance, practicality and reversion rate of the QFT-GIT among HCWs at a tertiary health ...

  3. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.


    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  4. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders;


    . However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed...... light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell...... concentration/population varied with whole blood, 10 × 106 cells/ml PBMC and 5 × 106 cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p

  5. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan


    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  6. A survey of monitoring and assay systems for release of metals from radiation controlled areas at LANL.

    Gruetzmacher, K. M. (Kathleen M.); MacArthur, D. W. (Duncan W.)


    At Los Alamos National Laboratory (LANL), a recent effort in waste minimization has focused on scrap metal from radiological controlled areas (RCAs). In particular, scrap metal from RCAs needs to be dispositioned in a reasonable and cost effective manner. Recycling of DOE scrap metals from RCAs is currently under a self-imposed moratorium. Since recycling is not available and reuse is difficult, often metal waste from RCAs, which could otherwise be recycled, is disposed of as low-level waste. Estimates at LANL put the cost of low-level waste disposal at $550 to $4000 per cubic meter, depending on the type of waste and the disposal site. If the waste is mixed, the cost for treatment and disposal can be as high as $50,000 per cubic meter. Disposal of scrap metal as low-level waste uses up valuable space in the low-level waste disposal areas and requires transportation to the disposal site under Department of Transportation (DOT) regulations for low-level waste. In contrast, disposal as non-radioactive waste costs as little as $2 per cubic meter. While recycling is unavailable, disposing of the metal at an industrial waste site could be the best solution for this waste stream. A Green Is Clean (GIC) type verification program needs to be in place to provide the greatest assurance that the waste does not contain DOE added radioactivity. This paper is a review of available and emerging radiation monitoring and assay systems that could be used for scrap metal as part of the LANL GIC program.

  7. From Space to the Patient: A New Cytokine Release Assay to Monitor the Immune Status of HIV Infected Patients and Sepsis Patients

    Kaufmann, I.; Draenert, R.; Gruber, M.; Feuerecker, M.; Crucian, B. E.; Mehta, S. L.; Roider, J.; Pierson, D. L.; Briegel, J. M.; Schelling, G.; Sams, C. F.; Chouker, A.


    Monitoring of humans either in the healthy men under extreme environmental stress like space flight, in human immunodeficiency virus (HIV) infected patients or in sepsis is of critical importance with regard to the timing of adequate therapeutic (counter-)measures. The in vivo skin delayed-type hypersensitivity test (DTH) served for many years as a tool to evaluate cell mediated immunity. However, this standardised in vivo test was removed from the market in 2002 due to the risk of antigen stabilization. To the best of our knowledge an alternative test as monitoring tool to determine cell mediated immunity is not available so far. For this purpose we tested a new alternative assay using elements of the skin DTH which is based on an ex vivo cytokine release from whole blood and asked if it is suitable and applicable to monitor immune changes in HIV infected patients and in patients with septic shock.

  8. Interferon-gamma assay in combination with tuberculin skin test are insufficient for the diagnosis of culture-negative pulmonary tuberculosis.

    Marcin Wlodarczyk

    Full Text Available OBJECTIVE: Early diagnosis of infectious cases and treatment of tuberculosis (TB are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST and interferon (IFN-γ-based assay (IGRA for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb culture. METHODS: In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV, negative predicted value (NPV, and analytic accuracy (Acc of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag - Nil in the cultures of blood from patients with active TB and NMLD patients were also analysed. RESULTS: The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies. CONCLUSIONS: Our results do not show that a combination of IGRA and TST might be a

  9. Interferon-gamma assay in combination with tuberculin skin test are insufficient for the diagnosis of culture-negative pulmonary tuberculosis.

    Wlodarczyk, Marcin; Rudnicka, Wieslawa; Janiszewska-Drobinska, Beata; Kielnierowski, Grzegorz; Kowalewicz-Kulbat, Magdalena; Fol, Marek; Druszczynska, Magdalena


    Early diagnosis of infectious cases and treatment of tuberculosis (TB) are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST) and interferon (IFN)-γ-based assay (IGRA) for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb) culture. In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD) hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and analytic accuracy (Acc) of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag - Nil) in the cultures of blood from patients with active TB and NMLD patients were also analysed. The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies. Our results do not show that a combination of IGRA and TST might be a step forward in the diagnosis of culture-negative TB

  10. Comparison of the tuberculin skin test and interferon gamma release assay for the screening of tuberculosis in adolescents in close contact with tuberculosis TB patients.

    Song, Seung-Eun; Yang, JiYeon; Lee, Kil Soo; Kim, Hyungjun; Kim, Young Mi; Kim, Seonghan; Park, Mi-Sun; Oh, Su Yeon; Lee, Jin Bum; Lee, EunPyo; Park, Sang-Hee; Kim, Hee-Jin


    The tuberculin skin test (TST) frequently yields false positive results among BCG-vaccinated persons thereby limiting its diagnostic value particularly in settings with high BCG vaccination rate. We determined the agreement between IGRA and TST using 2 cutoff values and identified possible relationships between the results of these tests and the development of active tuberculosis. Adolescents aged 11-19 years in close contact with smear-positive tuberculosis cases and with normal chest radiographs were recruited from middle and high schools in South Korea. The TST was conducted by trained nurses, and blood was drawn for the QuantiFERON-TB Gold In-Tube (QFT-GIT). Participants were followed up for 2 years to check for incidence tuberculosis. A total of 2,982 subjects were included in the study, the average age was 15.1 years (SD 1.3), 61% had BCG vaccination scars. The agreement of QFT-GIT and the TST was low (κ = 0.38, 95% CI 0.32 to 0.42) using 10 mm cutoff; however, when the 15 mm cutoff was used, the agreement was intermediate (κ = 0.56, 95% CI 0.50 to 0.61). The odds ratio (OR) for the development of active tuberculosis was 7.9 (95% CI 3.46 to 18.06) for QFT-GIT positive patients, 7.96 (95% CI 3.14-20.22) for TST/QFT-GIT+ and the OR 4.62 (95% CI 2.02 to 10.58) and 16.35 (95% CI 7.09 to 37.71) for TST 10 mm and 15 mm cutoff respectively. The results of this study suggest that the TST cutoff point for patients aged 11-17 years would be 15 mm in other study. The OR of QFT-GIT for the development of active tuberculosis and its intermediate agreement with TST using 15 mm cutoff demonstrates its role as an adjunct diagnostic tool to current clinical practice. Positive responders to both TST and QFT-GIT at the outset may benefit from chemoprophylaxis.

  11. Effect of Pregnancy on Interferon Gamma Release Assay and Tuberculin Skin Test Detection of Latent TB Infection Among HIV-Infected Women in a High Burden Setting.

    LaCourse, Sylvia M; Cranmer, Lisa M; Matemo, Daniel; Kinuthia, John; Richardson, Barbra A; Horne, David J; John-Stewart, Grace


    Peripartum immunologic changes may affect latent tuberculosis infection (LTBI) diagnostic performance among HIV-infected women. HIV-infected women were serially tested with tuberculin skin test (TST) and interferon gamma release assay [QuantiFERON TB Gold In-tube (QFT)] in pregnancy and 6 weeks postpartum in Kenya. Prevalence, sensitivity and agreement, and correlates of QFT/TST positivity were assessed. Quantitative QFT mitogen and Mycobacterium tuberculosis antigen (Mtb-Ag) responses were compared by peripartum stage. Incidence of test conversion at 6 weeks postpartum was evaluated in baseline TST-/QFT- women. Among 100 HIV-infected women, median age was 26 years, median CD4 was 555 cells per cubic millimeter, and 88% were on antiretrovirals. More women were QFT+ than TST+ in both pregnancy (35.4% vs. 13.5%, P = 0.001) and postpartum (29.6% vs. 14.8%, P 2-fold more women with LTBI compared with TST in pregnancy and postpartum. Lower QFT Mtb-Ag and mitogen responses in pregnancy compared with postpartum suggest that pregnancy-associated immunologic changes may influence LTBI test performance.

  12. Assay of 6-gingerol in CO2 supercritical fluid extracts of ginger and evaluation of its sustained release from a transdermal delivery system across rat skin.

    Chen, Yan; Zhang, Cuiping; Zhang, Mei; Fu, Xiaobing


    Ginger has been widely used as healthy food condiment as well as traditional Chinese medicine since antiquity. Multiple potentials of ginger for treatment of various ailments have been revealed. However, the biological half-life of 6-gingerol (a principal pungent ingredient of ginger) is only 7.23 minutes while taken orally. Delivery of ginger compositions by routes other than oral have scarcely been reported. Therefore, we studied a noninvasive transdermal drug delivery system (TDDS) of ginger to bypass hepatic first pass metabolism, avoid gastrointestinal degradation and achieve long persistent release of effective compositions. After establishment of a HPLC analysis method of 6-gingerol, assays of 6-gingerol were performed to compare two kinds of ginger extracts. Then, the characteristics of transdermal delivery of 6-gingerol in TDDS were exhibited. The results showed that the contents of 6-gingerol in two kinds of ginger extracts were significantly different. The maximal delivery percentage of 6-gingerol across rat skin at 20 h was more than 40% in different TDDS formulations. TDDS may provide long-lasting delivery of ginger compounds.

  13. Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

    Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.


    This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

  14. Consistency of Mycobacterium tuberculosis-Specific Interferon-Gamma Responses in HIV-1-Infected Women during Pregnancy and Postpartum

    Sasi R. Jonnalagadda


    Full Text Available Background. We determined the consistency of positive interferon-gamma (IFN-γ release assays (IGRAs to detect latent TB infection (LTBI over one-year postpartum in HIV-1-infected women. Methods. Women with positive IGRAs during pregnancy had four 3-monthly postpartum IGRAs. Postpartum change in magnitude of IFN-γ response was determined using linear mixed models. Results. Among 18 women with positive pregnancy IGRA, 15 (83% had a subsequent positive IGRA; 9 (50% were always positive, 3 (17% were always negative, and 6 (33% fluctuated between positive and negative IGRAs. Women with pregnancy IGRA IFN-γ >8 spot forming cells (SFCs/well were more likely to have consistent postpartum IGRA response (odds ratio: 10.0; 95% confidence interval (CI: 0.9–117.0. Change in IFN-γ response over postpartum was 10.2 SFCs/well (95% CI: −1.5–21.8 SFCs/well. Conclusion. Pregnancy positive IGRAs were often maintained postpartum with increased consistency in women with higher baseline responses. There were modest increases in magnitude of IGRA responses postpartum.

  15. Interferon-gamma release assays and tuberculin skin testing for diagnosing latent Mycobacterium tuberculosis infection in at-risk groups in Poland.

    Kruczak, Katarzyna; Mastalerz, Lucyna; Sładek, Krzysztof


    The diagnostics of latent tuberculosis infection in Poland using the tuberculin skin test is challenging due to the obligatory Bacillus Calmette-Guérin vaccinations. Interferon-gamma release assays are still very rarely used for diagnostics. We compared the tuberculin skin test and the QuantiFERON-TB Gold In-Tube test to evaluate the degree of latent tuberculosis infection in at-risk groups for tuberculosis (homeless, close contacts, periodic contacts, nursing-home attendees) and in healthy individuals. QuantiFERON-TB Gold In-Tube tests were carried out on 785 individuals from the homeless (n=150), close contacts (n=171), periodic contacts (n=163), nursing-home attendees (n=152), and healthy individuals (n=149). The tuberculin skin test was performed on 129, 156, 147, 148, and 121 participants, respectively. We evaluated the (a) correlation between serum concentrations of interferon gamma and the tuberculin-skin-test induration diameter; (b) between the number of QuantiFERON-TB Gold In-Tube-positive results and the tuberculin-skin-test diameter in the studied groups; and (c) agreement between both tests and the kappa coefficient using the tuberculin-skin-test diameters of 5, 10, and 15mm. Larger tuberculin-skin-test induration diameters were associated with elevated serum concentrations of interferon gamma. We found a positive correlation between the number of positive QuantiFERON-TB Gold In-Tube screening results and the tuberculin-skin-test induration diameter. The agreement between QuantiFERON-TB Gold In-Tube and tuberculin-skin-test screening results improved with increasing tuberculin-skin-test induration diameter. Based on measures of tuberculin-skin-test induration diameter alone, it is difficult to diagnose latent tuberculosis infection with certainty. The agreement of the QuantiFERON-TB Gold In-Tube test increases with the tuberculin-skin-test diameter. Tuberculin-skin-test diameters larger than 15mm are more likely to be associated with active infection

  16. Comparison of interferon-γ release assay to two cut-off points of tuberculin skin test to detect latent Mycobacterium tuberculosis infection in primary health care workers.

    de Souza, Fernanda Mattos; do Prado, Thiago Nascimento; Pinheiro, Jair dos Santos; Peres, Renata Lyrio; Lacerda, Thamy Carvalho; Loureiro, Rafaela Borge; Carvalho, Jose Américo; Fregona, Geisa; Dias, Elias Santos; Cosme, Lorrayne Beliqui; Rodrigues, Rodrigo Ribeiro; Riley, Lee Wood; Maciel, Ethel Leonor Noia


    An interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil. A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points. We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST-/QFT- group, risk factors for discordance in the TST+/QFT- group with TST cut-off of ≥5 mm included age between 41-45 years [OR = 2.70; CI 95%: 1.32-5.51] and 46-64 years [OR = 2.04; CI 95%: 1.05-3.93], BCG scar [OR = 2.72; CI 95%: 1.40-5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09-4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03-4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01-2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05-2.62], were significantly associated with the TST+/QFT- group. No statistically significant associations were found among the TST-/QFT+ discordant group with either TST cut-off value. Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed.

  17. High prevalence of latent tuberculosis infection among injection drug users in Tijuana, Mexico

    Garfein, R.S; Lozada, R; Liu, L; Laniado-Laborin, R; Rodwell, T.C; Deiss, R; Alvelais, J; Catanzaro, A; Chiles, P.G; Strathdee, S.A


    ...®-TB Gold In-Tube, a whole-blood interferon-gamma release assay (IGRA). LTBI prevalence was estimated and correlates were identified using RDS-weighted logistic regression.RESULTS: Of 1020 IDUs, 681 (67...

  18. Comparison of a commercial interferon-gamma release assay and tuberculin skin test for the detection of latent tuberculosis infection in Hong Kong arthritis patients who are candidates for biologic agents.

    So, H; Yuen, C Sw; Yip, R Ml


    It is universally agreed that screening for latent tuberculosis infection prior to biologic therapy is necessary, especially in endemic areas such as Hong Kong. There are still, however, controversies regarding how best to accomplish this task. The tuberculin skin test has been the routine screening tool for latent tuberculosis infection in Hong Kong for the past decade although accuracy is far from perfect, especially in patients who have been vaccinated with Bacillus Calmette-Guérin, who are immunocompromised, or who have atypical mycobacterium infection. The new interferon-gamma release assays have been shown to improve specificity and probably sensitivity. This study aimed to evaluate agreement between the interferon-gamma release assay and the tuberculin skin test in the diagnosis of latent tuberculosis infection in patients with arthritic diseases scheduled to receive biologic agents. We reviewed 38 patients with rheumatoid arthritis, psoriatic arthritis, or spondyloarthritis at a local hospital in Hong Kong from August 2013 to April 2014. They were all considered candidates for biologic agents. The patients underwent both the interferon-gamma release assay (ASACIR.TB; A.TB) and the tuberculin skin test simultaneously. Concurrent medications were documented. Patients who tested positive for either test (ie A.TB+ or TST+) were prescribed treatment for latent tuberculosis if they were to be given biologic agents. All patients were followed up regularly for 1 year and the development of active tuberculosis infection was evaluated. Based on an induration of 10 mm in diameter as the cut-off value, 13 (34.2%) of 38 patients had a positive tuberculin skin test. Of the 38 patients, 11 (28.9%) also had a positive interferon-gamma release assay. The agreement between interferon-gamma release assay and tuberculin skin test was 73.7% (kappa=0.39). Six patients were TST+/A.TB- and four were TST-/A.TB+. When positive tuberculin skin test was defined as an induration of 5

  19. γ-干扰素释放分析T-SPOT.TB在结核性疾病中的诊断价值%Diagnostic value of interferon-gamma release assay T-SPOT.TB for tuberculosis

    韦海旭; 席薇莲; 陆利欢; 陈健


    目的 评价γ-干扰素释放分析T-SPOT.TB检测在结核性疾病中的诊断价值.方法 采用T-SPOT.TB试剂盒对疑诊或待排结核患者外周血中释放γ-干扰素的结核分枝杆菌特异性T淋巴细胞进行检测.结果 γ-干扰素释放分析T-SPOT.TB检测在结核性疾病阳性检出率为83.3%(20/24),明显高于结核菌素试验(tuberculin skin test,TST) 的41.7%(10/24)、涂片找抗酸杆菌的26.7%(4/15)、分枝杆菌分离培养的22.2%(2/9),差异有统计学意义(P<0.05).γ-干扰素释放分析T-SPOT.TB检测诊断结核性疾病敏感性、特异性分别为83.3%、96.4%,显著优于结核菌素试验.结论 γ-干扰素释放分析T-SPOT.TB检测是诊断结核的快速敏感方法,在结核性疾病诊断中有重要价值.%Objective To evaluate the diagnostic value of an interferon-gamma release assay T-SPOT. TB in tuberculosis. Methods T-SPOT. TB assay reagent was employed to detect Mycobacterium tuberculosis antigen specific T cells in the peripheral blood samples from the patients with suspected tuberculosis. Results The positive detection rate was 83. 3%( 20/24 ) by interferon-gamma release assay T-SPOT. TB was significantly higher than 41. 7%( 10/24 ) by tuberculin test, 26. 1% (4/15 ) in smear for acid-fast bacillus and 22. 2% ( 2/9 ) in mycobacteria separation culture with statistical differences( P <0. 05 ). The sensitivity and specificity of interferon-gamma release assay T-SPOT. TB were 83. 3% and 96. 4% , which were significantly higher than those of tuberculin test with statistical ditfer-ences( P <0. 05 ). Conclusions Interferon-gamma release assay T-SPOT. TB is rapid and high-sensitive test for diagnosing tuberculosis.

  20. Release patterns of pregnancy-associated plasma protein A in patients with acute coronary syndromes assessed by an optimized monoclonal antibody assay

    Schoos, Mikkel; Iversen, Kasper; Teisner, Ane;


    of ACS. Methods. In 40 patients, PAPP-A concentrations were measured in serially collected samples assessed by a novel ELISA technique. The patients were grouped according to type of ACS. Results. Release patterns for ST elevation myocardial infarction (STEMI) patients who underwent primary percutaneous...

  1. Interferon gamma release test in early diagnosis of pulmonary tuberculosis%γ干扰素释放试验在肺结核早期诊断中的应用价值



    Objective To evaluate the diagnostic efficacy of interferon gamma release test (IGRAs) for early diagnosing pulmonary tuberculosis disease.Methods 120 patients with pulmonary tuberculosis (an experimental group) and 60 healthy controls (a control group) were chosen.Thte results of IGRAs and tuberculin skin test (TST) were compared between the two groups.Results The sensitivity,specificity,positive and negative predictive values,and the Yuden index of IGRAs were 90.83%,90.55%,97.11%,48.73%,and 0.835 and those of TST were 71.67%,62.34%,90.42%,24.25%,and 0.191,respectively,suggesting that IGRAs was more effective than TST for diagnosing tuberculosis (P<0.05).Conclusions IGRAs has a high diagnostic efficacy for pulmonary tuberculosis and should be clinically generalized.%目的 评价γ干扰素释放试验(IGRAs)早期诊断肺结核疾病的效能.方法 取120例肺结核病人(实验组),健康体检者60例(对照组),对比GRAs、结核菌素皮试(TST)检测结果.结果 IGRAs用于肺结核诊断的敏感性、特异性分别为90.83%、90.55%,阳性、阴性预测值分别为97.11%、48.73%,Yuden指数0.835,TST上述指标分别为62.34%、71.67%、90.42%、24.25%、0.191,提示IGRAs诊断结核病的效能优于TST (P<0.05).结论 IGRAs对肺结核具有较高的诊断效能,可予以推广.

  2. The clinical utility of tuberculin skin test and interferon-γ release assay in the diagnosis of active tuberculosis among young adults: a prospective observational study

    Lee Sei Won


    Full Text Available Abstract Background The roles of the tuberculin skin test (TST and QuantiFERON®-TB Gold In-Tube assay (QFT-IT in the diagnosis of active tuberculosis (TB are not clear in young adults. We evaluated the diagnostic accuracy of the TST and QFT-IT in smear-negative TB among young adults with no underlying disease. Methods We prospectively enrolled 166 young participants 20-29 years of age with suspected active TB in a military hospital of South Korea. The TST and QFT-IT were performed for all participants. Results Of the 143 patients included in the analysis, active TB was diagnosed in 100 (69.9%. There were 141 male patients, none of whom had immunosuppressive disease. The sensitivity, specificity, positive predictive value (PPV, and negative predictive value (NPV of TST were 94% (95% CI, 87-98%, 88% (95% CI, 74-96%, 95% (95% CI, 88-98%, and 86% (95% CI, 72-94%, respectively. The sensitivity, specificity, PPV, and NPV of the QFT-IT were 93% (95% CI, 86-97%, 95% (95% CI, 81-99%, 98% (95% CI, 92-99%, and 84% (95% CI, 69-93%, respectively. No significant differences were found between the TST and QFT-IT in any statistic. Conclusions Both the TST and QFT-IT showed high sensitivity and specificity in differentiating active TB from other diseases. The diagnostic accuracy of these two tests did not differ significantly when applied to this clinical population of young, immunocompetent adults in whom neonatal BCG vaccination was common, there was no history of previous TB and in whom suspicion of TB was high. Trial registration NCT00982969

  3. Igra bez pravil v Ukraine zakantshivajetsja


    Välisinvestorite huvist Ukraina kinnisvaraturu vastu. Poliitilise ebastabiilsuse, seaduste ebatäiuslikkuse jm. kõrval pidurdab kinnisvaraturu arengut Ukrainas korruptsioon. 2006. aastal kinnitatud elamute klassifikaatorist

  4. Systematic review of cost and cost-effectiveness of different TB-screening strategies

    Costa José


    Full Text Available Abstract Background Interferon-γ release assays (IGRAs for TB have the potential to replace the tuberculin skin test (TST in screening for latent tuberculosis infection (LTBI. The higher per-test cost of IGRAs may be compensated for by lower post-screening costs (medical attention, chest x-rays and chemoprevention, given the higher specificity of the new tests as compared to that of the conventional TST. We conducted a systematic review of all publications that have addressed the cost or cost-effectiveness of IGRAs. The objective of this report was to undertake a structured review and critical appraisal of the methods used for the model-based cost-effectiveness analysis of TB screening programmes. Methods Using Medline and Embase, 75 publications that contained the terms "IGRA", "tuberculosis" and "cost" were identified. Of these, 13 were original studies on the costs or cost-effectiveness of IGRAs. Results The 13 relevant studies come from five low-to-medium TB-incidence countries. Five studies took only the costs of screening into consideration, while eight studies analysed the cost-effectiveness of different screening strategies. Screening was performed in high-risk groups: close contacts, immigrants from high-incidence countries and healthcare workers. Two studies used the T-SPOT.TB as an IGRA and the other studies used the QuantiFERON-TB Gold and/or Gold In-Tube test. All 13 studies observed a decrease in costs when the IGRAs were used. Six studies compared the use of an IGRA as a test to confirm a positive TST (TST/IGRA strategy to the use of an IGRA-only strategy. In four of these studies, the two-step strategy and in two the IGRA-only strategy was more cost-effective. Assumptions about TST specificity and progression risk after a positive test had the greatest influence on determining which IGRA strategy was more cost-effective. Conclusion The available studies on cost-effectiveness provide strong evidence in support of the use of IGRAs

  5. Clinical application of whole blood interferon-γ release assay on diagnosis of active pulmonary tuberculosis%全血γ干扰素释放试验在活动性肺结核诊断中的应用

    周祖模; 金嘉琳; 王森; 黄河清; 石勇明; 陈林; 张文宏; 翁心华


    Objective To evaluate the application of whole blood interferon-γ release assay QuantiFERON-TB gold in tube (QFT-GIT) on the diagnosis of active pulmonary tuberculosis in China. Methods With the commercial whole blood interferon-γ QFT-GIT kit, interferon-γ released by specific T cells stimulated under early secreted antigenic target 6 kD protein ( ESAT-6) , culture filtrate protein 10 (CFP-10) and TB7. 7 were measured among 48 cases with active pulmonary tuberculosis and 55 BCG (bacillus calmette-guerin ) -vaccinated healthy controls, who had no tuberculosis contact history by questionnaire. Results The QFT-GIT was positive in 93. 8% patients with active pulmonary tuberculosis and 11.1% healthy controls, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of QFT-GIT were 93. 8% , 88. 9% , 88. 2% and 94. 1% , respectively. The antigen-specific IFN-γ release was significantly higher in patients with active pulmonary tuberculosis in comparison with that in the controls (P < 0.0001). Furthermore, after anti-tuberculosis treatment, the antigen-specific IFN-γ decreased significantly (P < 0.05). Conclusions QFT-GIT is a sensitive and specific assay for detecting tuberculosis, which could discriminate the active tuberculosis patients from BCG vaccinated healthy controls and could be a convenient and useful diagnostic tool for the diagnosis of tuberculosis.%目的 评价全血γ干扰素释放试验在活动性肺结核诊断中的应用价值.方法 应用一种全血γ干扰素释放检测试剂盒QuantiFERON-TB gold in tube(QFT-GIT)对48例活动性肺结核患者(活动性结核组)进行检测,同时选取55例健康者作为对照(对照组),均通过调查问卷排除结核病接触史.结果 本研究活动性结核组患者和对照组QFT-GIT阳性率分别为93.8%和11.1%,QFT-GIT诊断活动性肺结核的总体敏感性和特异性分别为93.8%和88.9%,阳性和阴性预测值分别为88.2%和94.1%.活动性

  6. Enzyme assays.

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie


    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  7. Rate of tuberculosis infection in children and adolescents with household contact with adults with active pulmonary tuberculosis as assessed by tuberculin skin test and interferon-gamma release assays.

    Ferrarini, M A G; Spina, F G; Weckx, L Y; Lederman, H M; De Moraes-Pinto, M I


    Tuberculosis (TB) infection was evaluated in Brazilian immunocompetent children and adolescents exposed and unexposed (control group) to adults with active pulmonary TB. Both groups were analysed by clinical and radiological assessment, TST, QFT-IT and T-SPOT.TB. The three tests were repeated after 8 weeks in the TB-exposed group if results were initially negative. Individuals with latent tuberculosis infection (LTBI) were treated and tests were repeated after treatment. Fifty-nine TB-exposed and 42 controls were evaluated. Rate of infection was 69·5% and 9·5% for the exposed and control groups, respectively. The exposed group infection rate was 61% assessed by TST, 57·6% by T-SPOT.TB, and 59·3%, by QFT-IT. No active TB was diagnosed. Agreement between the three tests was 83·1% and 92·8% in the exposed and control groups, respectively. In the exposed group, T-SPOT.TB added four TB diagnoses [16%, 95% confidence interval (CI) 1·6-30·4] and QFT-IT added three TB diagnoses (12%, 95% CI 0-24·7) in 25 individuals with negative tuberculin skin test (TST). Risk factors associated to TB infection were contact with an adult with active TB [0-60 days: odds ratio (OR) 6·9; >60 days: OR 27·0] and sleeping in the same room as an adult with active TB (OR 5·2). In Brazilian immunocompetent children and adolescents, TST had a similar performance to interferon-gamma release assays and detected a high rate of LTBI.

  8. Prevalence of latent tuberculosis infection in BCG-vaccinated healthcare workers by using an interferon-gamma release assay and the tuberculin skin test in an intermediate tuberculosis burden country.

    Hung, Wan-Ting; Lee, Susan Shin-Jung; Sy, Cheng-Len; Wu, Kuang-Sheng; Chen, Jui-Kuang; Tsai, Hung-Chin; Chen, Yao-Shen


    The risk of healthcare workers (HCWs) acquiring tuberculosis (TB) infection is high. We determined the prevalence of latent TB infection (LTBI) in HCWs with a high Bacille Calmette-Guérin (BCG) vaccine coverage in an intermediate TB burden country by using an interferon-gamma release assay [QuantiFERON-TB Gold (QFT-G)] and by using the tuberculin skin test (TST). Risk factors associated with a positive test were determined. This prospective cross-sectional study enrolled HCWs from a medical center in Taiwan. Participants were grouped into workers without exposure (Group 1) and workers who self-reported a history of TB exposure (Group 2). All participants completed a questionnaire to collect demographic information and risk factors for acquiring TB. The QFT-G test and the TST were administered and risk factors for a positive test were analyzed. We recruited 193 HCWs [149 (77.2%) female workers] with a mean age of 35.6 years. All were BCG-vaccinated. The prevalence of LTBI was 88.8% (based on the TST) and 14.5% (based on the QFT-G test). There was no difference between HCWs with and without known exposure to TB. Agreement between the tests was poor (i.e., the kappa value was less than 0.05). Multivariable logistic regression showed that only the QFT-G test was associated with age (35 years or greater) (adjusted OR, 2.53; p = 0.03). By using the QFT-G test or TST, this study found a similar prevalence of LTBI in HCWs with and without known exposure to TB. This suggests that in intermediate TB burden countries exposure to TB may occur within the hospital and within the community. Compared to the TST, the QFT-G test was correlated better with age, which is a known risk factor for latent TB infection. Copyright © 2013. Published by Elsevier B.V.

  9. Evaluation of the usefulness of interferon-gamma release assays and the tuberculin skin test for the detection of latent Mycobacterium tuberculosis infections in Korean rheumatic patients who are candidates for biologic agents.

    Kim, Jae-Hoon; Won, Soyoung; Choi, Chan-Bum; Sung, Yoon-Kyoung; Song, Gwan Gyu; Bae, Sang-Cheol


    The aim of this study was to evaluate the occurrence of active tuberculosis (TB) in patients who received both an interferon-gamma release assay (the QuantiFERON-TB Gold In-Tube test [QFT-GIT]) and tuberculin skin test (TST) in comparison with those who received QFT-GIT or TST alone for the detection of latent TB infection (LTBI). In total, 842 patients who received QFT-GIT or TST and used biologic agents between January 2007 and December 2012 were recruited to determine the usefulness of LTBI screening tests. The incidence of active TB was calculated relative to the LTBI screening method as the number of events per 100 000 person-years exposure. TB occurred in two of the patients who complied with an LTBI prophylaxis strategy. The TB incidence in the group that received both QFT-GIT and TST was 151.05 (95% confidence interval [CI] 150.11-151.98)/100 000 person-years, and the incidence was 169.78 (95% CI 168.73-170.84)/100 000 person-years in the group that received only TST. TB occurred even in some patients who received LTBI prophylaxis in compliance with national guidelines. The incidence of TB in patients who received either the QFT-GIT plus TST prophylaxis strategy or the TST prophylaxis strategy alone was higher than the annual incidence of the general population of the Republic of Korea. It is not possible to conclude which of the LTBI prophylaxis strategies is superior. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  10. Different screening strategies (single or dual for the diagnosis of suspected latent tuberculosis: a cost effectiveness analysis

    Rook Graham


    Full Text Available Abstract Background Previous health economic studies recommend either a dual screening strategy [tuberculin skin test (TST followed by interferon-γ-release assay (IGRA] or a single one [IGRA only] for latent tuberculosis infection (LTBI, the former largely based on claims that it is more cost-effective. We sought to examine that conclusion through the use of a model that accounts for the additional costs of adverse drug reactions and directly compares two commercially available versions of the IGRA: the Quantiferon-TB-Gold-In-Tube (QFT-GIT and T-SPOT.TB. Methods A LTBI screening model directed at screening contacts was used to perform a cost-effectiveness analysis, from a UK healthcare perspective, taking into account the risk of isoniazid-related hepatotoxicity and post-exposure TB (2 years post contact using the TST, QFT-GIT and T-SPOT.TB IGRAs. Results Examining costs alone, the TST/IGRA dual screening strategies (TST/T-SPOT.TB and TST/QFT-GIT; £162,387 and £157,048 per 1000 contacts, respectively cost less than their single strategy counterparts (T-SPOT.TB and QFT-GIT; £203,983 and £202,921 per 1000 contacts which have higher IGRA test costs and greater numbers of persons undergoing LTBI treatment. However, IGRA alone strategies direct healthcare interventions and costs more accurately to those that are truly infected. Subsequently, less contacts need to be treated to prevent an active case of TB (T-SPOT.TB and QFT-GIT; 61.7 and 69.7 contacts in IGRA alone strategies. IGRA single strategies also prevent more cases of post-exposure TB. However, this greater effectiveness does not outweigh the lower incremental costs associated with the dual strategies. Consequently, when these costs are combined with effectiveness, the IGRA dual strategies are more cost-effective than their single strategy counterparts. Comparing between the IGRAs, T-SPOT.TB-based strategies (single and dual; £39,712 and £37,206 per active TB case prevented

  11. Diagnositic value of Xpert mycobacterium tuberculosis/rifampicin assay and γ-interferon release assay for joint tuberculosis%利福平耐药实时荧光定量核酸扩增检测技术与γ-干扰素释放试验在关节结核辅助诊断中的价值

    刘荣梅; 高孟秋; 吴晓光; 张立群; 宋艳华; 马丽萍


    目的:探讨和评价利福平耐药实时荧光定量核酸扩增检测技术(Xpert mycobacterium tuberculosis/rifampicin,Xpert MTB/RIF;简称Xpert检测)与γ-干扰素释放试验在关节结核辅助诊断中的价值。方法选取2015年1~9月首都医科大学附属北京胸科医院住院经手术治疗,病理确诊为关节结核的患者50例,同时行脓液Xpert检测和血酶联免疫斑点技术法(T-SPOT.TB检测)检测结核分枝杆菌感染T细胞。结果 T-SPOT.TB检测在诊断关节结核中的敏感度为88%(44/50);Xpert检测的敏感度为66%(33/50);两种方法联合检测的敏感度为66%(33/50)。T-SPOT.TB检测的敏感度高于Xpert检测及两种方法联合检测(χ2=6.832,P=0.009)。结论 Xpert检测和γ-干扰素释放试验采用T-SPOT.TB检测试剂盒在关节结核快速诊断方面均具有重要的价值,两者结合可提高关节结核诊断的准确度。%ObjectiveTo explore and evaluate the diagnositic value of Xpert mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) andγ-interferon release assays in joint tuberculosis.Method50 patients diagnosed as joint tuberculosis from January 2015 to September 2015 in Capital Medical University, Beijing Chest Hospital were included into this study. Rifampin puss simultaneously real-time quantitative nucleic acid amplification Xpert MTB/RIF and T-SPOT.TB test were tested at the same time.ResultT-SPOT.TB detection sensitivity in the diagnosis of joint tuberculosis was 88% (44/50); Xpert detection sensitivity was 66% (33/50); combined detection sensitivity of the two methods was 66% (33/50). T-SPOT.TB sensitivity is higher than Xpert detection and combined detection of the two methods (χ2=6.832,P = 0.009).ConclusionThe Xpert MTB/RIF andγ-interferon release assay which applied by T-SPOT.TB detection method have significant value in the rapid diagnosis of tuberculosis joints. A combination of these can improve the accuracy of joint

  12. Update on Immunological Test (Quantiferon-TB Gold) Contribution in the Management of Tuberculosis-Related Ocular Inflammation.

    Trad, S; Bodaghi, B; Saadoun, D


    To clarify interferon-gamma release assay (IGRA) contributions to intraocular tuberculosis management, according to related TB endemic prevalence. Despite extensive investigations, including IGRA, the diagnosis of intraocular tuberculosis (TB) is still challenging and remains mostly presumptive. According to the literature, it appears that management of suspected ocular TB differs significantly based on whether patients are from areas of high TB prevalence or from non-TB-endemic countries. The accuracy and final contribution of chest X-rays, tuberculin skin test and IGRA differ significantly according to low or high endemic TB areas. This work aims to review IGRA test outcomes in the light of these clinical and epidemiological settings. We therefore focused our analysis and its subsequent guidelines proposal based on the TB prevalence where the patient lives. We believe that distinct guidelines should be determined for managing patients with suspected ocular TB, first taking into consideration the relative TB prevalence.

  13. Bronchoalveolar lavage enzyme-linked immunospot for diagnosis of smear-negative tuberculosis in HIV-infected patients.

    Adithya Cattamanchi

    Full Text Available Peripheral blood interferon-gamma release assays (IGRAs have sub-optimal sensitivity and specificity for diagnosis of active pulmonary tuberculosis (TB. However, assessment of local immune responses has been reported to improve the accuracy of TB diagnosis.We enrolled HIV-infected adults with cough ≥2 weeks' duration admitted to Mulago Hospital in Kampala, Uganda and referred for bronchoscopy following two negative sputum acid-fast bacillus smears. We performed an ELISPOT-based IGRA (T-SPOT.TB®, Oxford Immunotec, Oxford, UK using peripheral blood and bronchoalveolar lavage (BAL fluid mononuclear cells, and determined the accuracy of IGRAs using mycobacterial culture results as a reference standard.94 HIV-infected patients with paired peripheral blood and BAL IGRA results were included. The study population was young (median age 34 years [IQR 28-40 years] and had advanced HIV/AIDS (median CD4+ T-lymphocyte count 60 cells/µl [IQR 22-200 cells/µl]. The proportion of indeterminate IGRA results was higher in BAL fluid than in peripheral blood specimens (34% vs. 14%, difference 20%, 95% CI 7-33%, p = 0.002. BAL IGRA had moderate sensitivity (73%, 95% CI 50-89% but poor specificity (48%, 95% CI 32-64% for TB diagnosis. Sensitivity was similar (75%, 95% CI 57-89% and specificity was higher (78%, 95% CI 63-88% when IGRA was performed on peripheral blood.BAL IGRA performed poorly for the diagnosis of smear-negative TB in a high HIV/TB burden setting. Further studies are needed to examine reasons for the large proportion of indeterminate results and low specificity of BAL IGRA for active TB in high HIV/TB burden settings.

  14. The corneal pocket assay.

    Ziche, Marina; Morbidelli, Lucia


    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  15. Angiogenesis Assays.

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P


    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  16. γ干扰素释放试验在儿童肺结核诊断中的价值%Performance of interferon γ release assays in diagnosis of childhood pulmonary tuberculosis

    鲍磊; 李涛; 卢水华; 张文宏


    In order to investigate the performance of two kinds of interferon γrelease assays in the diagnosis of childhood pulmonary tuberculosis in China (with a high rate of bacillus Calmette-Guérin vaccination and a high incidence of tuberculosis ) , a total of 114 children with suspected pulmonary tuberculosis were recruited .Among the cases ,45 received QuantiFERON-Gold In-Tube (QFT-GIT ) test while the other 69 were tested with T-SPOT .TB . The clinical information was collected for diagnostic classification . The sensitivity ,specificity ,positive predictive value (PPV) and negative predictive value (NPV) were compared between the two methods .The sensitivity of QFT-GIT in the diagnosis of childhood pulmonary tuberculosis was 86 .4% ,the specificity was 81 .3% ,PPV was 91 .7% ,and NPV was 76 .5% .In contrast ,the sensitivity of T-SPOT .TB was 72 .3% , the specificity was 93 .7% , PPV was 97 .1% , and NPV was 53 .6% . The positive rates of two methods in the cases treated with glucocorticoids decreased significantly compared to those untreated .In conclusion ,both QFT-GIT and T-SPOT .TB have high PPV in the diagnosis of childhood pulmonary tuberculosis since the rate of latent tuberculosis infection was low in children .%为研究2种γ干扰素释放试验(IGRA)试剂盒在结核病高发、卡介苗(BCG)高接种地区用于诊断儿童肺结核的价值,共入组临床怀疑肺结核患儿114例,其中45例行QuantiFERON-Gold In-Tube(QFT-GIT)检测,69例行T-SPOT .TB检测,收集临床资料,比较2种方法的灵敏度、特异度、阳性预测值(PPV)和阴性预测值(NPV )。结果显示,QFT-GIT 在儿童肺结核诊断中的灵敏度为84.6%、特异度为81.3%、PPV 为91.7%、NPV为76.5%。T-SPOT .TB的灵敏度为72.3%、特异度为93.7%、PPV为97.1%、NPV为53.6%。与未治疗患儿相比,激素治疗患儿QFT-GIT和T-SPOT .TB的阳性率显著下降


    贾晨光; 李秀武; 姚黎明; 刘建强; 黄俊; 陈银芳; 张雷


    Objective To compare the sensitivity of interferon gamma release test (IGRA) with suptum smear,sputum culture and tuberculin slcintest (TST ) in the diagnosis of extrapulmonary tuberculosis.To investigate the value of IGRA method in extrapulmonary tuberculosis.Methods Fresh peripheral blood samples were collected from 75 patients of clinically diagnosed extrapulmonary tuberculosis.The result was compared with those of traditional gold standard test methods (sputum smear and sputum culture,TST ),and their clinical application value further were analyzed.Results In 75 cases of extrapulmonary tuberculosis,the positive rate by IGRA reagent test was 84.0%,sputum smear 8.0%,sputum culture 5.3%,TST test 52.0%.The positive rate of IGRA was significantly higher than those of other common methods,and the differences were statistically significant (P<0.01).Conclusion In the diagnosis of extrapulmonary tuberculosis,the sensitivity of IGRA reagent is significantly higher than those of the traditional test methods,IGRA has a good clinical value.%目的:比较干扰素γ释放试验(interferon gamma release assay,IGRA)与结核菌染色涂片、结核菌培养、结核菌素试验(tuberculin skin test,TST)在肺外结核诊断中的敏感性,探讨 IGRA方法在肺外结核中的应用价值。方法采集75例经临床诊断为肺外结核病患者的新鲜外周血,将 IGRA检测结果与传统的检测方法(结核菌染色涂片、结核菌染色涂片培养、TST)的检测结果进行比较。结果75例肺外结核患者中,IGRA检测阳性率为84.0%,结核菌染色涂片(8.0%),结核菌培养(5.3%),TST检测(52.0%),IGRA检测阳性率均明显高于其他临床常用检测方法,差异有统计学意义(P<0.01)。结论 IGRA试剂检测法诊断肺外结核,敏感性明显高于传统的检测方法,有很好的临床应用价值。

  18. 结核菌快速培养法与γ-干扰素释放试验在结核病诊断中的应用价值%The clinical values of TB rapid culture method and γ-interferon release assay in diagnosis of tuberculosis



    目的 探讨结核菌快速培养法与γ-干扰素释放试验在结核病诊断中的应用价值.方法 选择2013年11月至2014年7月在我院进行检查的与结核病密切接触者180例,均进行结核菌快速培养法与γ-干扰素释放试验检测结核杆菌,以结核分枝杆菌培养结果为对照,分析两种检测方法结核杆菌阳性检出率的敏感性及特异性,并分析导致结核菌快速培养法误诊的危险因素.结果 结核菌快速培养法和γ-干扰素释放试验结核菌检出阳性率分别是13.3% 和16.1%,两者比较差异无统计学意义(P>0.05).以结核分枝杆菌培养结果为金标准,结核菌快速培养法和γ-干扰素释放试验的诊断敏感性分别为70.0%和96.7%,特异性分别为98.0%和100.0%.γ-干扰素释放试验的诊断敏感性明显高于结核菌快速培养法,差异有统计学意义(P<0.05 ).logistic多元回归分析表明年龄和既往结核史是导致结核菌快速培养法误诊的主要因素 (P<0.05 ).结论 相对于结核菌快速培养法,γ-干扰素释放试验结核病诊断的敏感性更高.%Objective To investigate the clinical values of TB rapid culture method andγ-interferon release assay in the diagnosis of tuberculosis. Methods A total of 180 close contact cases with tuberculosis from November 2013 to July 2014 in our hospital were selected and given the rapid TB culture and γ-interferon release assay, then the risk factors for misdiagnosis were analysed. Results The detection positive rate of the TB rapid culture method and γ-interferon release assay was respectively 13. 3% and 16. 1%, and the difference had no statistical significance (P>0. 05). With mycobacterium tuberculosis sputum culture results as the gold standard, the sensitivity of the TB rapid culture method and γ-interferon release assay was respectively 70. 0% and 96. 7%, and the specificity was 96. 7% and 100. 0%. The diagnostic sensitivity of the

  19. Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials.

    Martinelli, Elisa; Sarvothaman, Mahesh K; Galli, Giancarlo; Pettitt, Michala E; Callow, Maureen E; Callow, James A; Conlan, Sheelagh L; Clare, Anthony S; Sugiharto, Albert B; Davies, Cait; Williams, David


    Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700™. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.

  20. Optogenetic control of ATP release

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.


    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  1. Mechanism and kinetics of the loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionation-based drug release-/transfer-assay

    Hinna, Askell Hvid; Hupfeld, Stefan; Kuntsche, Judith


    in the vascular bed. A range of in vitro test methods has been suggested over the years for prediction of the release of drug from liposomal carriers. The majority of these fail to give a realistic prediction for poorly water-soluble drugs due to the intrinsic tendency of such compounds to remain associated...... liposome to another was found rate determining as compared to redistribution from the outermost to the inner concentric bilayers, such that the overall process could be adequately described by a single 1st order kinetic model.By varying the donor-to-acceptor lipid mass ratio in the range 1:1 to 1...

  2. Interferon-gamma inducible protein 10 as a biomarker for active tuberculosis and latent tuberculosis infection in children: A case-control study

    Alsleben, Neele; Ruhwald, Morten; Rüssmann, Holger;


    Background: Interferon-gamma (IFN-γ) release assays (IGRAs) are suboptimally sensitive to diagnose tuberculosis (TB) and latent TB infection (LTBI) in young children. In this study we compared Mycobacterium tuberculosis antigen-stimulated IFN-γ inducible protein 10 (IP-10) responses in children w...

  3. Comparative validation of the growth hormone-releasing hormone and arginine test for the diagnosis of adult growth hormone deficiency using a growth hormone assay conforming to recent international recommendations.

    Chanson, Philippe; Cailleux-Bounacer, Anne; Kuhn, Jean-Marc; Weryha, Georges; Chabre, Olivier; Borson-Chazot, Françoise; Dubois, Séverine; Vincent-Dejean, Caroline; Brue, Thierry; Fedou, Christine; Bresson, Jean-Louis; Demolis, Pierre; Souberbielle, Jean-Claude


    The GHRH plus arginine (GHRH+Arg) test is a promising alternative to the insulin tolerance test (ITT) for diagnosis of adult GH deficiency (AGHD). The objectives of the study were to validate the GHRH+Arg test for diagnosis of AGHD, using the ITT as comparator and a GH assay calibrated according to recent international recommendations, and to study the repeatability and tolerance of both tests. This was a multicenter, randomized, open-label, phase III study. The study was conducted at 10 French university hospitals. Sixty-nine subjects (38 and 15 with high and low probability of GH deficiency, respectively, and 16 healthy controls) were randomized: 35 to the GHRH+Arg-GHRH+Arg-ITT test sequence and 34 to the ITT-ITT-GHRH+Arg test sequence. Each subject underwent three tests of GH secretion separated by 24 h or more. The primary variable used for response assessments was serum peak GH response. Test results were compared with the final AGHD diagnosis. Peak GH responses in the two tests were strongly correlated. A cutoff value of 7.89 microg/liter for GHRH+Arg corresponding to 3 microg/liter for ITT was calculated. The cutoff value leading to 95% specificity with the GHRH+Arg test was measured at about 3.67 microg/liter (sensitivity 79.0%). Intermethod agreement and repeatability were high. Both tests were well tolerated. A preference for the GHRH+Arg test was expressed by 74% of subjects. The GHRH+Arg test demonstrated good accuracy and repeatability, was at least as sensitive as the ITT, and was associated with better subject acceptability. The GHRH+Arg test represents a good alternative to the ITT for the diagnosis of AGHD.

  4. Ny test til diagnostik af tuberkulose

    Ravn, Pernille; Rose, Michala Vaaben; Søborg, Bolette;


    The performance of two commercially-available Interferon Gamma Release Assays, QuantiFERON-TB-Gold and T-SPOT-TB, is reviewed. The tests can indirectly detect M. tuberculosis infection based on T-cell recognition of specific proteins which are not present in the BCG vaccine M. avium, or most other...... environmental mycobacteria.The sensitivity (76-88%) of the IGRAs is not optimal for a diagnostic test for active tuberculosis. However, the specificity is high (92-97%), and the IGRAs may play an important role in the investigation for active tuberculosis and screening for latent tuberculosis infection in BCG...... vaccinated populations and immunosuppressed patients. Udgivelsesdato: 2009-Sep-7...

  5. Prevalence and associated risk factors of latent tuberculosis infection among undergraduate and postgraduate dental students: A retrospective study.

    Lamberti, Monica; Muoio, Maria Rosaria; Westermann, Claudia; Nienhaus, Albert; Arnese, Antonio; Ribeiro Sobrinho, Antônio Paulino; Di Giuseppe, Gabriella; Garzillo, Elpidio Maria; Crispino, Vincenzo; Coppola, Nicola; De Rosa, Alfredo


    To estimate the prevalence of latent tuberculosis (TB) infection (LTBI) in Italian dental students exposed to the same occupational risks as dental health care personnel and to evaluate potential risk factors, a cross-sectional study was conducted on undergraduate and postgraduate students. After clinical evaluation, students were given a tuberculin skin test; in those found positive, an interferon-γ release assay (IGRA) was conducted. Of the 281 students enrolled, 10 were only TST positive; 8 were TST or/and IGRA positive. We found that participants testing positive at TST and/or IGRA, a group in which the risk of false LTBI positives is minimal, were older and had been studying longer. Although the prevalence of LTBI among dental students in our study was low, a risk of acquiring a work-related infection exists even in a country with a low incidence of TB. Thus, dental students should be screened to catch LTBI early on.

  6. 胸水γ-干扰素释放试验与老年结核性胸膜炎预后的关系%Relationship of interferon-gamma release assay in pleural effusion and prognostic assessment of tuberculous pleurisy in elderly patients

    张晓光; 刘会; 宋韬; 池跃鹏; 章志华; 张勤凤; 付洪义


    目的 探讨胸水的γ-干扰素释放试验与老年结核性胸膜炎患者预后的关系.方法 选取2012年3月至2014年5月在我院就诊并确诊的结核性胸膜炎患者80例,根据年龄分为2组:A组(年龄<60岁)和B组(年龄≥60岁),每组40例.取胸水采用T-SPOT.TB检测γ-干扰素释放试验.抗结核治疗的同时动态观察患者治疗1个月时的胸水吸收情况和治疗6个月时的胸膜粘连、肥厚情况,综合分析患者预后.结果 A组γ-干扰素释放试验阳性患者37例,阳性率为92.5%;B组γ-干扰素释放试验阳性患者25例,阳性率为62.5%.A组胸水γ-干扰素释放试验阳性率高于B组(x2=10.323,P< 0.05).1个月综合治疗结束后,A组患者胸水吸收比例为85.0%,B组患者胸水吸收比例为70.0%,2组比较差异无统计学意义(x2 =2.581,P>0.05).6个月治疗结束后,A组患者胸膜肥厚比例为27.5%,B组患者胸膜肥厚比例为57.5%,B组发生胸膜肥厚患者比例高于A组(x2=7.366,P<0.05).结论 老年结核性胸膜炎患者早期的胸水γ-干扰素释放试验阳性率相对偏低,可能与老年患者细胞免疫功能低下有关.而老年患者治疗后期胸膜肥厚、粘连较中青年患者更加明显,提示老年胸膜炎预后欠佳.%Objective To explore the relationship of interferon-gamma release assay in pleural effusion and the prognostic assessment of tuberculous pleurisy in elderly patients.Methods 80 patients with tuberculous pleurisy in our hospital from March 2012 to May 2014 were selected.They were divided into group A (<60 years old) and group B (≥60 years old),40 cases in each group.The pleural effusion was taken for interferon-gamma release assay by T-SPOT.TB.The pleural effusion absorption in one month,the pleural adhesion and hypertrophy in six months were observed dynamically when antituberculosis treatment was conducted.The patients' outcomes were analysed comprehensively.Results There were 37 patients in the group

  7. Renin release

    Schweda, Frank; Friis, Ulla; Wagner, Charlotte;


    The aspartyl-protease renin is the key regulator of the renin-angiotensin-aldosterone system, which is critically involved in salt, volume, and blood pressure homeostasis of the body. Renin is mainly produced and released into circulation by the so-called juxtaglomerular epithelioid cells, located......, salt, and volume overload. In contrast, the events controlling the function of renin-secreting cells at the organ and cellular level are markedly less clear and remain mysterious in certain aspects. The unravelling of these mysteries has led to new and interesting insights into the process of renin...

  8. School-based Study to Identify and Treat Adolescent Students at Risk for Tuberculosis Infection.

    Hatzenbuehler, Lindsay A; Starke, Jeffrey R; Graviss, Edward A; Smith, E O'Brian; Cruz, Andrea T


    Screening for and treating tuberculosis (TB) infection in children and adolescents is an effective way of decreasing future TB cases. However, current approaches leave many children at risk for TB unidentified. We recruited adolescent students from 2 public high schools (a magnet and a low-income) in the Houston Independent School District. Compared with the magnet school, the student population at the low-income school was larger, primarily Hispanic and economically disadvantaged. Students were educated about TB, and parents completed a risk factor questionnaire. Students with TB risk factors were tested using 2 interferon gamma release assays (IGRAs). Those with a positive IGRA received a 12-dose regimen of weekly isoniazid/rifapentine (3HP) administered via direct observation at school. Nine hundred twenty-five students received TB education; 73% of their parents submitted the TB questionnaire. Eighty-six percent of students (n = 415) with a TB risk factor identified on the study questionnaire agreed to IGRA testing. Sixteen students had at least one positive IGRA (1% [magnet], 4.1% [low-income]; P = 0.005). Recent student travel to a high-risk country (7) or contact with TB disease (2) were associated with IGRA positivity (P risk for TB.

  9. Microbead agglutination based assays

    Kodzius, Rimantas


    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  10. Colorimetric protein assay techniques.

    Sapan, C V; Lundblad, R L; Price, N C


    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  11. The glucose oxidase-peroxidase assay for glucose

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  12. Absolute nuclear material assay

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA


    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  13. A new fluorescent assay for sialyltransferase.

    Kajihara, Y; Kamitani, T; Sakakibara, T


    A new fluorescent assay for the sialyltransferase reaction was established. After incubation of the sialyltransferase reaction, the sialyloligosaccharide obtained was treated by acid hydrolysis, and then the NeuAc that was released was labeled with 1,2-diamino-4,5-methylenedioxibenzene. The fluorescent-labeled NeuAc could be estimated by HPLC (excitation: 373 nm; emission: 448 nm) and a Lineweaver-Burk plot could be plotted with the data from this analysis.

  14. Cell viability assays: introduction.

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  15. Tube-Forming Assays.

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy


    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  16. Transgenic Animal Mutation Assays

    Tao Chen; Ph.D.D.A.B.T.


    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  17. Assays for thrombopoietin

    McDonald, T.P.


    In summary, thrombopoietin levels have been determined indirectly by measuring thrombocytopoiesis in assay animals (platelet counting, measurement of isotope incorporation into newly formed platelets, changes in platelet sizes, or alterations in number and size of megakaryocytes) and by use of an immunoassay. Although much work remains, it seems clear at the present time that isotopic uptake into platelets of specially prepared assay mice (rebound-thrombocytosis) is superior to the other techniques now available for the measurement of thrombopoietin. However, the ideal assay for TSF which is specific, rapid, and inexpensive is yet to be developed. An immunoassay is in the development stage, but will require additional work before it can be utilized for the routine assay of TSF.

  18. New Rapid Spore Assay

    Kminek, Gerhard; Conley, Catharine


    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  19. News/Press Releases

    Office of Personnel Management — A press release, news release, media release, press statement is written communication directed at members of the news media for the purpose of announcing programs...

  20. Against vaccine assay secrecy.

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors.

  1. Against vaccine assay secrecy

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  2. Rover waste assay system

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)


    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  3. Histamine release from cord blood basophils

    Nielsen, Bent Windelborg; Damsgaard, Tine Engberg; Herlin, Troels


    The histamine release (HR) after challenge with anti-IgE, concanavalin A, N-formyl-met-leu-phe and the calcium ionophore A23187 from 97 cord blood samples was determined by a microfiber-based assay. Maximum HR with anti-IgE showed great inter-individual variation (median: 20.5; range: 1-104 ng...

  4. Histamine release from cord blood basophils

    Nielsen, Bent Windelborg; Damsgaard, Tine Engberg; Herlin, Troels


    The histamine release (HR) after challenge with anti-IgE, concanavalin A, N-formyl-met-leu-phe and the calcium ionophore A23187 from 97 cord blood samples was determined by a microfiber-based assay. Maximum HR with anti-IgE showed great inter-individual variation (median: 20.5; range: 1-104 ng...

  5. CTL ELISPOT assay.

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita


    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  6. Assays for calcitonin receptors

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.


    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  7. Oral suspensions of morphine hydrochloride for controlled release: rheological properties and drug release.

    Morales, M E; López, G; Gallardo, V; Ruiz, M A


    Recent developments in pharmaceutical technology have facilitated the design and production of modified release formulas for drugs whose physical, chemical or biological properties impede release and thus might compromise their efficacy or safety. One such drug is morphine, whose short half-life requires repeated doses at short intervals. The use of biocompatible polymers such as ethylcellulose has made it possible to develop microencapsulated formulations which facilitate liquid, sustained-release pharmaceutical formulas for oral administration. We developed a stable final formulation of morphine with an acceptable release profile by comparing the rheological properties and stability of formulations with different thickeners (xanthan gum, Carbopol, and carboxymethylcellulose with microcrystalline cellulose) at different concentrations from 0.25% to 1.0%. Release assays in a Franz-type cell were done to determine the most suitable release profile for the formulation.

  8. New oligosaccharyltransferase assay method.

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi


    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  9. Hyaluronic Acid Assays

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H


    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  10. Instrument for assaying radiation

    Coleman, Jody Rustyn; Farfan, Eduardo B.


    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  11. Kinetic Tetrazolium Microtiter Assay

    Pierson, Duane L.; Stowe, Raymond; Koenig, David


    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  12. B cell helper assays.

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo


    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  13. Toxics Release Inventory (TRI)

    U.S. Environmental Protection Agency — The Toxics Release Inventory (TRI) is a dataset compiled by the U.S. Environmental Protection Agency (EPA). It contains information on the release and waste...

  14. Growth cone collapse assay.

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J


    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.



    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  16. Radon assay for SNO+

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)


    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  17. Negative effect of smoking on the performance of the QuantiFERON TB gold in tube test

    Aabye Martine G


    Full Text Available Abstract Background False negative and indeterminate Interferon Gamma Release Assay (IGRA results are a well documented problem. Cigarette smoking is known to increase the risk of tuberculosis (TB and to impair Interferon-gamma (IFN-γ responses to antigenic challenge, but the impact of smoking on IGRA performance is not known. The aim of this study was to evaluate the effect of smoking on IGRA performance in TB patients in a low and high TB prevalence setting respectively. Methods Patients with confirmed TB from Denmark (DK, n = 34; 20 smokers and Tanzania (TZ, n = 172; 23 smokers were tested with the QuantiFERON-TB Gold In tube (QFT. Median IFN-γ level in smokers and non smokers were compared and smoking was analysed as a risk factor for false negative and indeterminate QFT results. Results Smokers from both DK and TZ had lower IFN-γ antigen responses (median 0.9 vs. 4.2 IU/ml, p = 0.04 and 0.4 vs. 1.6, p  Conclusions Cigarette smoking was associated with false negative and indeterminate IGRA results in both a high and a low TB endemic setting independent of HIV status.

  18. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    Eriksson, Jonas; Langel, Ülo


    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

  19. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien


    cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...

  20. RAS - Screens & Assays - Drug Discovery

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.


    Michelson, C.


    An electromagnetic release mechanism is offered that may be used, for example, for supporting a safety rod for a nuclear reactor. The release mechanism is designed to have a large excess holding force and a rapid, uniform, and dependable release. The fast release is accomplished by providing the electromagnet with slotttd polts separated by an insulating potting resin, and by constructing the poles with a ferro-nickel alloy. The combination of these two features materially reduces the eddy current power density whenever the magnetic field changes during a release operation. In addition to these features, the design of the armature is such as to provide ready entrance of fluid into any void that might tend to form during release of the armature. This also improves the release time for the mechanism. The large holding force for the mechanism is accomplished by providing a small, selected, uniform air gap between the inner pole piece and the armature.

  2. Bacterial assays for recombinagens.

    Hoffmann, G R


    Two principal strategies have been used for studying recombinagenic effects of chemicals and radiation in bacteria: (1) measurement of homologous recombination involving defined alleles in a partially diploid strain, and (2) measurement of the formation and loss of genetic duplications in the bacterial chromosome. In the former category, most methods involve one allele in the bacterial chromosome and another in a plasmid, but it is also possible to detect recombination between two chromosomal alleles or between two extrachromosomal alleles. This review summarizes methods that use each of these approaches for detecting recombination and tabulates data on agents that have been found to be recombinagenic in bacteria. The assays are discussed with respect to their effectiveness in testing for recombinagens and their potential for elucidating mechanisms underlying recombinagenic effects.

  3. Fluctuating Behavior and Influential Factors in the Performance of the QuantiFERON-TB Gold In-Tube Assay in the Diagnosis of Tuberculosis: e0103763

    Lei Bao; Tao Li; Ni Diao; Yaojie Shen; Lingyun Shao; Ying Zhang; Shuihua Lu; Wenhong Zhang


    ...] release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay's ability to detect TB...

  4. Fluctuating Behavior and Influential Factors in the Performance of the QuantiFERON-TB Gold In-Tube Assay in the Diagnosis of Tuberculosis

    Bao, Lei; Li, Tao; Diao, Ni; Shen, Yaojie; Shao, Lingyun; Zhang, Ying; Lu, Shuihua; Zhang, Wenhong


    ... release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay's ability to detect TB...

  5. Lubiprostone stimulates small intestinal mucin release

    De Lisle Robert C


    Full Text Available Abstract Background Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 (PGE1 used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid secretion. In a mouse model of the genetic disease cystic fibrosis, we previously showed that in vivo administration of lubiprostone resulted in greater mucus accumulation in the small intestine. The aim of this study was to directly test whether lubiprostone stimulates intestinal mucin release. Methods Mucin release was measured by mounting segments (4-5 cm of mouse proximal-mid small intestine in an organ bath, allowing access to the perfusate (luminal and the bath (serosal solutions. Nifedipine (10-6 M and indomethacin (10-5 M were included in all solutions to inhibit smooth muscle activity and endogenous prostaglandin production, respectively. The tissue was equilibrated under flow for 30 min, using the perfusate collected during the final 10 min of the equilibration period to measure unstimulated release rate. Stimulus was then added to either the perfusate or the bath and the perfusate was collected for another 30 min to measure the stimulated mucin release rate. Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using purified pig gastric mucin as a standard. Results When applied luminally at 1 μM lubiprostone was ineffective at stimulating mucin release. When added to the serosal solution, 1 μM lubiprostone stimulated mucin release to ~300% of the unstimulated rate. As a positive control, serosal 1 μM prostaglandin E2 increased mucin release to ~400% of the unstimulated rate. Conclusions These results support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. Stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis. Thus, in




    Full Text Available Oral route gets the highest priority for thedelivery of the drug as well as better patient compliance incase of self delivery dosage formulation. The aim ofpresent investigation was undertaken with the objective offormulating sustain release formulation of Itopridehydrochloride for oral drug delivery. Itopride hydrochlorideis highly water soluble prokinetic drug.Hydroxypropylmethylcellulose K4M (lower viscositygrade and K100M (higher viscosity grade were used as amatrix forming agents to control the release of drug. HPMCK4M and HPMC K100M were used individually as well asin combination with different proportion in the preparationof the Sustained release formulation. 32 factorial designswere applied to the polymer concentration that affects thedrug release profile. Reduced equation for drug release at2hr,6hr,and10hrwere22 1 2 1 Q 37.644 5.41X 3.25X 2.017X ,26 1 2 1 Q 72.367 8.05X 4.4X 3.75X ,and10 1 1 2 90.844 5.8 2.633 2.8 2 Q X X X Xrespectively. Optimized batch F019 shows good tabletproperties like hardness(7-9kg/cm2, thickness(4.48mm,friability(0.024%,assay(99.3% and nearly similar drugrelease profile to the targeted reference drug release profileand it was indicated by similarity factor (f2=86.04.

  7. Release the Body, Release the Mind.

    Stoner, Martha Goff


    A college English teacher describes the anxiety and resentment of students during in-class writing assignments and the successful classroom use of meditation and body movement. Movement seemed to relax the students, change their attitudes, and release their creative impulses to write. Implications related to the body-mind connection are pondered.…

  8. Herbicide resistance screening assay.

    Peterson, Joan M


    Herbicide resistance screening is a method that can be used not only to determine presence of the enzyme, phosphinothricin acetyltransferase, encoded by either the Bar or the Pat gene in transgenic maize, but also to assess the inheritance ratio of those genes in a segregating population. Herbicide screening can also be used to study linkage of a transgene of interest that was cotransformed with the herbicide resistance marker gene. By combining the herbicide screen assay with a PCR-based screen of leaf tissue DNA for the presence of both the Bar or the Pat gene marker and a cotransformed transgene of interest from the same seedling tissue and maintaining that seedling identity, the researcher can identify linkage or the possible breakdown in linkage of the marker gene and the transgene of interest. Further, the occurrence of "DNA silencing" can be evaluated if an individual seedling that was susceptible to the applied herbicide nonetheless gave PCR data that indicated presence of the gene responsible for herbicide resistance. Similarly, "DNA silencing" of the gene of interest may be investigated if the seeds can be screened and scored for that phenotypic trait in a nondestructive manner prior to planting.

  9. Disagreement between Human Papillomavirus Assays

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller


    assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four......% tested positive on all four. Agreement was lower in women aged ≥ 30 years (30%, vs. 49% at samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30...

  10. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    Gibbs, S.; Corsini, E.; Spiekstra, S.W.; Galbiati, V.; Fuchs, H.W.; Degeorge, G.; Troese, M.; Hayden, P.; Deng, W.; Roggen, E.


    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low

  11. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

    Chang, A; Dusing, S


    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  12. Cardiac troponins and high-sensitivity cardiac troponin assays.

    Conrad, Michael J; Jarolim, Petr


    Measurement of circulating cardiac troponins I and T has become integral to the diagnosis of myocardial infarction. This article discusses the structure and function of the troponin complex and the release of cardiac troponin molecules from the injured cardiomyocyte into the circulation. An overview of current cardiac troponin assays and their classification according to sensitivity is presented. The diagnostic criteria, role, and usefulness of cardiac troponin for myocardial infarction are discussed. In addition, several examples are given of the usefulness of high-sensitivity cardiac troponin assays for short-term and long-term prediction of adverse events.

  13. Intracellular drug release nanosystems

    Fenghua Meng


    Full Text Available In order to elicit therapeutic effects, many drugs including small molecule anticancer drugs, proteins, siRNA, and DNA have to be delivered and released into the specific cellular compartments typically the cytoplasm or nucleus of target cells. Intracellular environment-responsive nanosystems that exhibit good extracellular stability while rapidly releasing drugs inside cancer cells have been actively pursued for effective cancer therapy. Here, we highlight novel designs of smart nanosystems that release drugs in response to an intracellular biological signal of cancer cells such as acidic pH in endo/lysosomal compartments, enzymes in lysosomes, and redox potential in cytoplasm and the cell nucleus.

  14. From Antenna to Assay

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.


    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  15. Miniature Release Mechanism Project

    National Aeronautics and Space Administration — The objective is to design, build and functionally test a miniature release mechanism for CubeSats and other small satellites. The WFF 6U satellite structure will be...

  16. Transporter assays and assay ontologies: useful tools for drug discovery.

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P


    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  17. Cryptosporidium cell culture infectivity assay design.

    King, B J; Keegan, A R; Robinson, B S; Monis, P T


    Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

  18. Solid waste transuranic storage and assay facility indoor air sampling

    Pingel, L.A., Westinghouse Hanford


    The purpose of the study is to collect and analyze samples of the indoor air at the Transuranic Storage and Assay Facility (TRUSAF), Westinghouse Hanford. A modified US EPA TO-14 methodology, using gas chromatography/mass spectrography, may be used for the collection and analysis of the samples. The information obtained will be used to estimate the total release of volatile organic compounds from TRUSAF to determine the need for air emmission permits.

  19. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement

    Mack, U; Migliori, G B; Sester, M


    Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected...... with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-gamma release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M....... tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could...

  20. Biomarkers of latent TB infection

    Ruhwald, Morten; Ravn, Pernille


    For the last 100 years, the tuberculin skin test (TST) has been the only diagnostic tool available for latent TB infection (LTBI) and no biomarker per se is available to diagnose the presence of LTBI. With the introduction of M. tuberculosis-specific IFN-gamma release assays (IGRAs), a new area...... of in vitro immunodiagnostic tests for LTBI based on biomarker readout has become a reality. In this review, we discuss existing evidence on the clinical usefulness of IGRAs and the indefinite number of potential new biomarkers that can be used to improve diagnosis of latent TB infection. We also present...... early data suggesting that the monocyte-derived chemokine inducible protein-10 may be useful as a novel biomarker for the immunodiagnosis of latent TB infection....


    Bishnoi Kapil*, , ,; Kataria Mahesh; Singhal Vipin; Gupta Deepika


    Micronutrients added to foods are analyzed using various procedures depending on their nature and properties. The microbiological assays are better than chemical method because any suitable change in vitamin molecule which may not be detected by chemical method will be revealed by change in microbial activity. The microbiological assay of vitamins is based upon the comparison of the stimulation of growth of bacteria by measured concentration of vitamin with that produced by known concentratio...

  2. A fluoride release-adsorption-release system applied to fluoride-releasing restorative materials.

    Suljak, J P; Hatibovic-Kofman, S


    This investigation compared the initial fluoride release and release following refluoridation of three resin-modified glass-ionomer cements (Photac-Fil Applicap, Vitremer, and Fuji II LC) and a new polyacid-modified resin composite material (Dyract). After daily flouride release was measured for 8 days, specimens were refluoridated in 1,000-ppm solutions of fluoride ion for 10 minutes and fluoride release was measured for 5 days. Two further 5-day refluoridation-release periods were carried out. All materials released fluoride initially. Photac released the most; Dyract released the least. Initial release was greatest over the first few days. All materials released significantly more fluoride for 24 to 48 hours after refluoridation. Less fluoride was released with each successive refluoridation for the three glass-ionomer cements. The release from the Dyract compomer remained at a comparatively constant and significantly lower level following each refluoridation.

  3. Calcineurin-dependent galactomannan release in Aspergillus fumigatus

    Mennink-Kersten, M.A.S.H.; Ruegebrink, D.; Verweij, P.E.; Steinbach, W.J.


    The galactomannan assay to diagnose invasive aspergillosis is recommended and clinically utilized, yet the mechanism of galactomannan release from Aspergillus fumigatus is unknown. We used an A. fumigatus strain lacking calcineurin A (cnaA), already shown to be critically important for pathogenicity

  4. RAVEN Beta Release

    Rabiti, Cristian [Idaho National Lab. (INL), Idaho Falls, ID (United States); Alfonsi, Andrea [Idaho National Lab. (INL), Idaho Falls, ID (United States); Cogliati, Joshua Joseph [Idaho National Lab. (INL), Idaho Falls, ID (United States); Mandelli, Diego [Idaho National Lab. (INL), Idaho Falls, ID (United States); Kinoshita, Robert Arthur [Idaho National Lab. (INL), Idaho Falls, ID (United States); Wang, Congjian [Idaho National Lab. (INL), Idaho Falls, ID (United States); Maljovec, Daniel Patrick [Idaho National Lab. (INL), Idaho Falls, ID (United States); Talbot, Paul William [Idaho National Lab. (INL), Idaho Falls, ID (United States)


    This documents the release of the Risk Analysis Virtual Environment (RAVEN) code. A description of the RAVEN code is provided, and discussion of the release process for the M2LW-16IN0704045 milestone. The RAVEN code is a generic software framework to perform parametric and probabilistic analysis based on the response of complex system codes. RAVEN is capable of investigating the system response as well as the input space using Monte Carlo, Grid, or Latin Hyper Cube sampling schemes, but its strength is focused toward system feature discovery, such as limit surfaces, separating regions of the input space leading to system failure, using dynamic supervised learning techniques. RAVEN has now increased in maturity enough for the Beta 1.0 release.

  5. Controlled-release microchips.

    Sharma, Sadhana; Nijdam, A Jasper; Sinha, Piyush M; Walczak, Robbie J; Liu, Xuewu; Cheng, Mark M-C; Ferrari, Mauro


    Efficient drug delivery remains an important challenge in medicine: continuous release of therapeutic agents over extended time periods in accordance with a predetermined temporal profile; local delivery at a constant rate to the tumour microenvironment to overcome much of the systemic toxicity and to improve antitumour efficacy; improved ease of administration, and increasing patient compliance required are some of the unmet needs of the present drug delivery technology. Microfabrication technology has enabled the development of novel controlled-release microchips with capabilities not present in the current treatment modalities. In this review, the current status and future prospects of different types of controlled-release microchips are summarised and analysed with reference to microneedle-based microchips, as well as providing an in-depth focus on microreservoir-based and nanoporous microchips.

  6. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Carina Ladeira


    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  7. Method for quantifying alginate and determining release from a food vehicle in gastrointestinal digesta.

    Houghton, David; Wilcox, Matthew D; Brownlee, Iain A; Chater, Peter; Seal, Chris J; Pearson, Jeffrey P


    To assess the efficacy of alginate as a modifier of enzyme activity, a suitable method to quantify its release must be developed. This paper develops and assesses the ability of the Periodic Acid Schiffs (PAS) assay to quantify alginate, and its release from bread during digestion in a model gut. Control and alginate enriched (4% w/w wet dough) bread were used. A model gut replicating the mouth, stomach and small intestines was used. Standard curves were created for alginate in deionised H2O and model gut solutions using a modified PAS to remove interference. The PAS assay quantified alginate with excellent linearity (R(2)=0.99), and optical density range (0.02-0.5). There was a significant difference in alginate release at 180 min compared to 0 and 60 min. The data indicate the modified PAS assay is a simple method for quantifying alginate release and release rate from alginate enriched products.

  8. Determining drug release rates of hydrophobic compounds from nanocarriers.

    D'Addio, Suzanne M; Bukari, Abdallah A; Dawoud, Mohammed; Bunjes, Heike; Rinaldi, Carlos; Prud'homme, Robert K


    Obtaining meaningful drug release profiles for drug formulations is essential prior to in vivo testing and for ensuring consistent quality. The release kinetics of hydrophobic drugs from nanocarriers (NCs) are not well understood because the standard protocols for maintaining sink conditions and sampling are not valid owing to mass transfer and solubility limitations. In this work, a new in vitroassay protocol based on 'lipid sinks' and magnetic separation produces release conditions that mimic the concentrations of lipid membranes and lipoproteins in vivo, facilitates separation, and thus allows determination of intrinsic release rates of drugs from NCs. The assay protocol is validated by (i) determining the magnetic separation efficiency, (ii) demonstrating that sink condition requirements are met, and (iii) accounting for drug by completing a mass balance. NCs of itraconazole and cyclosporine A (CsA) were prepared and the drug release profiles were determined. This release protocol has been used to compare the drug release from a polymer stabilized NC of CsA to a solid drug NP of CsA alone. These data have led to the finding that stabilizing block copolymer layers have a retarding effect on drug release from NCs, reducing the rate of CsA release fourfold compared with the nanoparticle without a polymer coating.This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

  9. Detecting ATP release by a biosensor method.

    Hayashi, Seiji; Hazama, Akihiro; Dutta, Amal K; Sabirov, Ravshan Z; Okada, Yasunobu


    Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

  10. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. (Univ. of Pittsburgh School of Medicine, PA (USA))


    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

  11. Release of OLe peanut

    OLe is a high oleic Spanish-type peanut that has excellent yield and enhanced Sclerotinia blight and pod rot resistance when compared to other high oleic Spanish cultivars. The purpose for releasing OLe is to provide peanut producers with a true Spanish peanut that is high oleic and has enhanced yi...

  12. Border cell release

    Mravec, Jozef


    Plant border cells are specialised cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localised cell separation which is essential for their release to the environment is little understood. Here I present...

  13. Barcoded microchips for biomolecular assays.

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu


    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  14. Radioreceptor assay method for insulin

    Mori, K.F.; Wood, R.J. (Bureau of Drug Research, Health and Welfare Canada, Ottawa, Ontario. Health Protection Branch)


    A sensitive practical radioreceptor assay method for pharmaceutical insulin products has been developed with partially purified rat liver plasma membranes and the optimal conditions under which the best overall assay performance is obtainable have been defined. Intra- and inter-assay variations of the method averaged 7.3 and 12.2% of the man, respectively, when expressed as the coefficient of variation. Potency estimates of an insulin product obtained with the proposed method correlated well with those determined by the mouse convulsion bioassay method. Liver membranes prepared according to the method could be stored for up to ten weeks at 4/sup 0/C and for 6 months or more at -18/sup 0/C without losing insulin-binding ability.

  15. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    McCourt, M


    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  16. [The diagnosis of latent tuberculosis infection in children in XXI century. Is tuberculin skin test still up to date?].

    Bielecka, Teresa; Komorowska-Piotrowska, Anna; Mazur, Agnieszka; Feleszko, Wojciech


    Tuberculosis morbidity rates in Poland have been gradually decreasing. Nevertheless, there are approximately 8 thousand cases being registered annually, which includes almost 3 thousand massively infectious patients. In the last 3 years, around 100 cases/year have been reported among children below 14 years of age. Infection with Mycobacterium tuberculosis should be considered in all patients who present symptoms suggesting tuberculosis, have had recent contact with a person suffering from lung tuberculosis or are planned to undergo an immunosuppressive treatment. HIV infected patients are also supposed to have screening tests for M. tuberculosis infection performed. For over a 100 years tuberculin skin test (TST) was the only test capable of confirming tuberculous infection. TST is based on the assessment of skin reaction to intracutaneous injection of tuberculin. Due to cross-reaction to the injected tuberculin in BCG vaccinated individuals, the correct interpretation of the test is difficult. Since 13 years new immunological assays have been available. They are based on detecting interferon gamma (Interferon Gamma Release Assay - IGRA) concentration in blood serum, which has previously been incubated with Mycobacterium tuberculosis antigens absent in the BCG strain. In infected individuals interferon gamma is intensively produced by memory cells in reaction to the contact with previously met Mycobacterium antigens. Many trials have proved IGRA's high sensitivity and, higher than TST, specificity. Recent guidelines promote the usage of IGRAs, even in children.

  17. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman


    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  18. A radioactive assay for the degradation of neuropeptide Y

    Ludwig, R.; Lucius, R.; Mentlein, R. [Kiel Univ. (Germany)


    Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian central nervous system. Like other neuropeptides, NPY is inactivated by specialized neuro-peptidases. To trace the degradation of NPY, an assay was established using biotinylated NPY. Biotinyl-NPY was radiolabeled with Na{sup 125}I by the chloramine-T method and bound to a streptavidin-agarose matrix. The amount of radiolabeling was analyzed by reverse-phase HPLC. The assay was carried out with five peptidases and inhibitors to demonstrate different specific activity. Measurable amounts of radioactivity were released by treatment with endopeptidase-24.18, plasmin, and trypsin, whereas dipetidylpeptidase IV (DPPIV) and angiotensin-converting enzyme (ACE) showed no activity in this assay. In the case of DPPIV this is due to a resistance of the assay to aminopeptidase attack. The assay is useful to study the specific degradation of NPY particularly by endopeptidases in all kinds of biological samples. (authors). 31 refs., 6 figs.

  19. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen


    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  20. Assay of mast cell mediators

    Rådinger, Madeleine; Jensen, Bettina M; Swindle, Emily


    Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes...... regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well...

  1. Evolving BioAssay Ontology (BAO): modularization, integration and applications.

    Abeyruwan, Saminda; Vempati, Uma D; Küçük-McGinty, Hande; Visser, Ubbo; Koleti, Amar; Mir, Ahsan; Sakurai, Kunie; Chung, Caty; Bittker, Joshua A; Clemons, Paul A; Brudz, Steve; Siripala, Anosha; Morales, Arturo J; Romacker, Martin; Twomey, David; Bureeva, Svetlana; Lemmon, Vance; Schürer, Stephan C


    The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project ( to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and

  2. Chromosome aberration assays in Allium

    Grant, W.F.


    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  3. Carpal tunnel release

    Larsen, Morten Bo; Sørensen, A I; Crone, K L;


    A single-blind, randomized, controlled trial was done to compare the results of carpal tunnel release using classic incision, short incision, or endoscopic technique. In total, 90 consecutive cases were included. Follow-up was 24 weeks. We found a significantly shorter sick leave in the endoscopi...... incision could be found. There were no serious complications in either group. The results indicate that the endoscopic procedure is safe and has the benefit of faster rehabilitation and return to work....

  4. Cryogenic hydrogen release research.

    LaFleur, Angela Christine [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)


    The objective of this project was to devolop a plan for modifying the Turbulent Combustion Laboratory (TCL) with the necessary infrastructure to produce a cold (near liquid temperature) hydrogen jet. The necessary infrastructure has been specified and laboratory modifications are currently underway. Once complete, experiments from this platform will be used to develop and validate models that inform codes and standards which specify protection criteria for unintended releases from liquid hydrogen storage, transport, and delivery infrastructure.

  5. EIA new releases


    This report was prepared by the Energy Information Administration. It contains news releases on items of interest to the petroleum, coal, nuclear, electric and alternate fuels industries ranging from economic outlooks to environmental concerns. There is also a listing of reports by industry and an energy education resource listing containing sources for free or low-cost energy-related educational materials for educators and primary and secondary students.

  6. Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D.

    Farrell, Christopher-John L; Soldo, Joshua; McWhinney, Brett; Bandodkar, Sushil; Herrmann, Markus


    Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice. 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD₂ recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD₃ cross-reactivity and precision of the automated assays were evaluated. The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD₂ recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD₂ under-recovery, a trend consistent with 3-epi-25-OHD₃ cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD₃ cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations. Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.

  7. Quantitative detection of RT activity by PERT assay: feasibility and limits to a standardized screening assay for human vaccines.

    André, M; Morgeaux, S; Fuchs, F


    The detection of adventitious retroviruses has always been critical for assessing the safety concerns associated with viral vaccines. Assays for the enzymatic activity of reverse transcriptase (RT) are used as general methods for the detection of both known and unknown retroviruses. Several studies using newly-developed ultrasensitive PCR-based RT assays reported RT activity in viral vaccines grown in chicken cells. Here, we have assessed the performances of such a PCR-based RT assay--PERT assay--for the quantitative detection of RT activity in vaccines. Sensitivity, linearity and reproducibility of the method were studied on purified RT and viral vaccines treated to release RT from potentially contaminant retroviruses. The level of RT activity detected in chicken cell-derived vaccines was higher for live attenuated vaccines compared to inactivated ones. Contrary to other studies, RT activity was found in some mammalian cell-derived vaccines. AZT-TP sensitivity of RT activities detected in these vaccines and discrimination between retroviral and RT-like activities was further investigated. Feasibility and limits of PERT assay as a broad-spectrum retroviruses detection method in vaccines are discussed.

  8. Contact: Releasing the news

    Pinotti, Roberto

    The problem of mass behavior after man's future contacts with other intelligences in the universe is not only a challenge for social scientists and political leaders all over the world, but also a cultural time bomb as well. In fact, since the impact of CETI (Contact with Extraterrestrial Intelligence) on human civilization, with its different cultures, might cause a serious socio-anthropological shock, a common and predetermined worldwide strategy is necessary in releasing the news after the contact, in order to keep possible manifestations of fear, panic and hysteria under control. An analysis of past studies in this field and of parallel historical situations as analogs suggests a definite "authority crisis" in the public as a direct consequence of an unexpected release of the news, involving a devastating "chain reaction" process (from both the psychological and sociological viewpoints) of anomie and maybe the collapse of today's society. The only way to prevent all this is to prepare the world's public opinion concerning contact before releasing the news, and to develop a long-term strategy through the combined efforts of scientists, political leaders, intelligence agencies and the mass media, in order to create the cultural conditions in which a confrontation with ETI won't affect mankind in a traumatic way. Definite roles and tasks in this multi-level model are suggested.

  9. Mechanisms of HSP72 release

    Alexzander Asea


    Currently two mechanisms are recognized by which heat shock proteins (HSP) are released from cells; a passive release mechanism, including necrotic cell death, severe blunt trauma, surgery and following infection with lytic viruses, and an active release mechanism which involves the non classical protein release pathway. HSPs are released both as free HSP and within exosomes. This review covers recent findings on the mechanism by which stress induces the release of HSP72 into the circulation and the biological significance of circulating HSP72 to host defense against disease.

  10. Triggered Release from Polymer Capsules

    Esser-Kahn, Aaron P. [Univ. of Illinois, Urbana, IL (United States). Beckman Inst. for Advanced Science and Technology and Dept. of Chemistry; Odom, Susan A. [Univ. of Illinois, Urbana, IL (United States). Beckman Inst. for Advanced Science and Technology and Dept. of Chemistry; Sottos, Nancy R. [Univ. of Illinois, Urbana, IL (United States). Beckman Inst. for Advanced Science and Technology and Dept. of Materials Science and Engineering; White, Scott R. [Univ. of Illinois, Urbana, IL (United States). Beckman Inst. for Advanced Science and Technology and Dept. of Aerospace Engineering; Moore, Jeffrey S. [Univ. of Illinois, Urbana, IL (United States). Beckman Inst. for Advanced Science and Technology and Dept. of Chemistry


    Stimuli-responsive capsules are of interest in drug delivery, fragrance release, food preservation, and self-healing materials. Many methods are used to trigger the release of encapsulated contents. Here we highlight mechanisms for the controlled release of encapsulated cargo that utilize chemical reactions occurring in solid polymeric shell walls. Triggering mechanisms responsible for covalent bond cleavage that result in the release of capsule contents include chemical, biological, light, thermal, magnetic, and electrical stimuli. We present methods for encapsulation and release, triggering methods, and mechanisms and conclude with our opinions on interesting obstacles for chemically induced activation with relevance for controlled release.

  11. Protecting privacy in data release

    Livraga, Giovanni


    This book presents a comprehensive approach to protecting sensitive information when large data collections are released by their owners. It addresses three key requirements of data privacy: the protection of data explicitly released, the protection of information not explicitly released but potentially vulnerable due to a release of other data, and the enforcement of owner-defined access restrictions to the released data. It is also the first book with a complete examination of how to enforce dynamic read and write access authorizations on released data, applicable to the emerging data outsou

  12. The timing of phasic transmitter release is Ca2+-dependent and lacks a direct influence of presynaptic membrane potential

    Felmy, Felix; Neher, Erwin; Schneggenburger, Ralf


    Ca2+ influx through voltage-gated Ca2+ channels and the resulting elevation of intracellular Ca2+ concentration, [Ca2+]i, triggers transmitter release in nerve terminals. However, it is controversial whether in addition to the opening of Ca2+ channels, membrane potential directly affects transmitter release. Here, we assayed the influence of membrane potential on transmitter release at the calyx of Held nerve terminals. Transmitter release was evoked by presynaptic Ca2+ uncaging, or by presyn...

  13. 21 CFR 225.158 - Laboratory assays.


    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  14. Allegheny County Toxics Release Inventory

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — The Toxics Release Inventory (TRI) data provides information about toxic substances released into the environment or managed through recycling, energy recovery, and...

  15. Comet Assay in Cancer Chemoprevention.

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina


    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  16. The skin-blanching assay.

    Smit, P; Neumann, H A M; Thio, H B


    The skin-blanching assay is used for the determination and bioequivalence of dermatologic glucocorticoids (GCs). The exact mechanism of the production of blanching is not fully understood, but it is considered that local vasoconstriction of the skin microvasculature and the consequent blood-flow reduction cause this phenomenon. Several factors influence skin blanching, including drug concentration, duration of application, nature of vehicle, occlusion, posture and location. The intensity of vasoconstriction can be measured in several ways: visual or quantitative methods, such as reflectance spectroscopy, thermography, laser Doppler velocimetry and chromametry. In literature, contradicting results in the correlation of the skin-blanching assay with different tests to determine GC sensitivity have been reported, limiting its clinical usefulness.

  17. Heat release mechanism of energetics

    Kubota, N. [Third Research Center, Technical Research and development Institute (Japan)


    Determination of the heat release mechanism of energetic materials is a major subject of combustion study. In order to elucidate the combustion process of various types of energetic materials a generalized combustion wave structure was proposed and the heat release process was discussed. The heat release process was significantly different between the physical structures of the materials: homogeneous and heterogeneous materials. The thermal structure of an azide polymer was evaluated to demonstrate the heat release mechanism. (author) 6 refs.

  18. Bioluminescence assay for cell viability.

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N


    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  19. Protein binding assay for hyaluronate

    Lacy, B.E.; Underhill, C.B.


    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 or less of hyaluronate, and is unaffected by the presence of proteins.

  20. Radioreceptor assay analysis of tamsulosin and terazosin pharmacokinetics

    Taguchi, Katsunari; Schäfers, Rafael F; Michel, Martin C


    Aims A radioreceptor assay has been developed for α1-adrenoceptor subtypes and applied to a pharmacokinetic analysis of tamsulosin and terazosin. Methods Young, male, healthy volunteers received 0.4 mg tamsulosin (as Omnic® modified release capsules) or 5 mg terazosin (as Flotrin® tablets) in a randomized, cross-over design. Before and after 1, 3, 5, 7, 10 and 23.5 h plasma was analyzed by radioreceptor assay using cloned human α1A-, α1B- and α1D-adrenoceptors and specific h.p.l.c. analysis. Results Following ingestion of tamsulosin median peak plasma levels of 16 ng ml−1 were reached after 5 h and declined to 2 ng ml−1 at 23.5 h. The time course in the radioreceptor assay was similar, and at most time points binding to α1A-adrenoceptors was significantly greater than to α1B- and α1D-adrenoceptors. Following ingestion of terazosin median peak plasma levels of 91 ng ml−1 were reached after 1 h and declined to 11 ng ml−1 at 23.5 h. In the radioreceptor assay binding also peaked at 1 h and declined thereafter, but even after 23.5 h considerable binding activity remained detectable at all three subtypes. At most time points binding to the α1A- and α1D-adrenoceptor was significantly greater than to the α1B-adrenoceptor. Conclusions We conclude that α1-adrenoceptor antagonist pharmacokinetics can be monitored by radioreceptor assays in a subtype-selective manner. Tamsulosin and terazosin exhibit subtype selective receptor binding ex vivo. The discordance between terazosin blood levels as determined by h.p.l.c. and radioreceptor assay at late time points indicates the possible involvement of metabolites in in vivo terazosin effects. PMID:9489594

  1. Is bioexsiccation releasing dioxins?

    Benfenati, E.; Mariani, G.; Lodi, M.; Reitano, G.; Fanelli, R. [' ' Mario Negri' ' Institute for Pharmacological Research, Milan (Italy)


    Bioexsiccation is a relatively new process to treat urban solid wastes. We studied the possible release of dioxins from this process, measuring dioxin concentration in the emissions from a bioexsiccation plant. As a comparison, we measured atmospheric levels nearby the plant. The biofilter treating gaseous emissions was also evaluated to assess its efficiency. Dioxin concentrations in the biofilter effluent were lower than both those before the biofilter and the nearby atmosphere. In the last years the management and treatment of solid urban wastes produced some improved processes, in a general attempt to cope with the problem of the huge amount of wastes produced by the modern society. Bio-exsiccation of waste aims at affording a much more biologically inert and manageable material compared to the original waste. In this process the urban solid waste is kept under an air stream for about two weeks. The waste undergoes biological transformation, due to fermentation, which produces an increase of the temperature up to 60-70 C. At the end of the process the weight waste is typically reduced by one third, due to the loss of water and to the degradation of putrescible compounds. Since this is a relatively new industrial process, we studied the possible release of dioxins in the atmospheric emissions of the bioexsiccation plant.

  2. Investigation on photorespiration with a sensitive C-assay.

    Zelitch, I


    A leaf disk assay for photorespiration has been developed based on the rate of release of recently fixed (14)CO(2) in light in a rapid stream of CO(2)-free air at 30 degrees to 35 degrees . In tobacco leaves (Havana Seed) photorespiration with this assay is 3 to 5 times greater than the (14)CO(2) output in the dark. In maize, photorespiration is only 2% of that in tobacco.The importance of open leaf stomata, rapid flow rates of CO(2)-free air, elevated temperatures, and oxygen in the atmosphere in order to obtain release into the air of a larger portion of the (14)CO(2) evolved within the tissue in the light was established in tobacco. Photorespiration, but not dark respiration, was inhibited by alpha-hydroxy-2-pyridinemethanesulfonic acid, an inhibitor of glycolate oxidase, and by 3-(4-chlorophenyl)-1,1-dimethylurea (CMU), an inhibitor of photosynthetic electron transport, under conditions which did not affect the stomata. These experiments show that the substrates of photorespiration and dark respiration differ and also provide additional support for the role of glycolate as a major substrate of photorespiration. It was also shown that at 35 degrees the quantity of (14)CO(2) released in the assay may represent only 33% of the gross (14)CO(2) evolved in the light, the remainder being recycled within the tissue.It was concluded that maize does not evolve appreciable quantities of CO(2) in the light and that this largely accounts for the greater efficiency of net photosynthesis exhibited by maize. Hence low rates of photorespiration may be expected to be correlated with a high rate of CO(2) uptake at the normal concentrations of CO(2) found in air and at higher light intensities.

  3. Photoresponsive lipid-polymer hybrid nanoparticles for controlled doxorubicin release

    Yao, Cuiping; Wu, Ming; Zhang, Cecheng; Lin, Xinyi; Wei, Zuwu; Zheng, Youshi; Zhang, Da; Zhang, Zhenxi; Liu, Xiaolong


    Currently, photoresponsive nanomaterials are particularly attractive due to their spatial and temporal controlled drug release abilities. In this work, we report a photoresponsive lipid-polymer hybrid nanoparticle for remote controlled delivery of anticancer drugs. This hybrid nanoparticle comprises three distinct functional components: (i) a poly(D,L-lactide-co-glycolide) (PLGA) core to encapsulate doxorubicin; (ii) a soybean lecithin monolayer at the interface of the core and shell to act as a molecular fence to prevent drug leakage; (iii) a photoresponsive polymeric shell with anti-biofouling properties to enhance nanoparticle stability, which could be detached from the nanoparticle to trigger the drug release via a decrease in the nanoparticle’s stability under light irradiation. In vitro results revealed that this core-shell nanoparticle had excellent light-controlled drug release behavior (76% release with light irradiation versus 10% release without light irradiation). The confocal microscopy and flow cytometry results also further demonstrated the light-controlled drug release behavior inside the cancer cells. Furthermore, a CCK8 assay demonstrated that light irradiation could significantly improve the efficiency of killing cancer cells. Meanwhile, whole-animal fluorescence imaging of a tumor-bearing mouse also confirmed that light irradiation could trigger drug release in vivo. Taken together, our data suggested that a hybrid nanoparticle could be a novel light controlled drug delivery system for cancer therapy.

  4. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander


    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Carina Ladeira; Susana Viegas; Manuel C. Gomes


    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  6. An application of the RFQ Linac: Nuclear waste assay characterization

    Lamkin, K.; Schultz, F.; Womble, P.; Humphrey, D.; Vourvopoulos, G.


    A collaboration between Oak Ridge National Laboratory and Western Kentucky University examines the problem of characterization and assay of nuclear waste with high intrinsic neutron and gamma-ray fields. This waste is defined as Remote Handled-Transuranic waste (RH-TRU). A Radiofrequency Quadrupole Linac is used to produce pulses of neutrons, which impinge on the drum that contains the nuclear waste. The neutrons, after being thermalized in the matrix of the drum, are captured by the fissile material (239Pu or 235U), which releases fast neutrons upon fission. Experimental results will be presented to show the versatility of employing the RFQ with the Differential Die-away Technique.

  7. Nonradioactive glycosyltransferase and sulfotransferase assay to study glycosaminoglycan biosynthesis.

    Ethen, Cheryl M; Machacek, Miranda; Prather, Brittany; Tatge, Timothy; Yu, Haixiao; Wu, Zhengliang L


    Glycosaminoglycans (GAGs) are linear polysaccharides with repeating disaccharide units. GAGs include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. All GAGs, except for hyaluronan, are usually sulfated. GAGs are polymerized by mono- or dual-specific glycosyltransferases and sulfated by various sulfotransferases. To further our understanding of GAG chain length regulation and synthesis of specific sulfation motifs on GAG chains, it is imperative to understand the kinetics of GAG synthetic enzymes. Here, nonradioactive colorimetric enzymatic assays are described for these glycosyltransferases and sulfotransferases. In both cases, the leaving nucleotides or nucleosides are hydrolyzed using specific phosphatases, and the released phosphate is subsequently detected using malachite reagents.

  8. Value of immunodiagnostic techniques on diagnosis of pulmonary and extrapulmonary tuberculosis%免疫诊断技术在肺结核及肺外结核诊断中的价值

    程小星; 蒋静


    Serological diagnostic techniques and T-cell interferon gamma release assays (IGRAs)are main immunodiagnostic techniques for tuberculosis. Serological tests are simple and fast, however,they have low specificity and sensitivity in the diagnosis of tuberculosis. Based on cellular immune responses, IGRAs are important breakthrough of immunodiagnostic techniques for tuberculosis. The value of IGRAs in the diagnosis of latent tuberculosis infection has been widely recognized. The diagnostic specificity is 89% in IGRAs, which is significantly higher than that in tuberculin test. However,peripheral blood monouclear cell IGRAs could not be used to differentiate active tuberculosis, latent infection and previous infection, so the poor specificity of diagnosis of active tuberculosis is the main problem. Tuberculosis local immune cell IGRAs has better specificity and sensitivity in the diagnosis of pulmonary and extrapulmonary tuberculosis, so it is worth further study.%目前结核病免疫学诊断技术的研究热点是血清学诊断技术和基于细胞免疫反应的干扰素-γ释放试验.血清学诊断方法具有简单快速的优点,易于推广应用,是研究比较多的结核病诊断方法之一,但现有血清学试验试剂在结核病诊断的特异性和敏感性方面还有待提高.基于细胞免疫反应的干扰素-γ释放试验是结核免疫诊断技术的重要突破,在诊断结核潜伏感染中的价值已经得到广泛的认可,诊断的特异性可达89%,显著高于结核菌素试验.但基于外周血单个核细胞的干扰素-γ释放试验不能区分活动性肺结核、潜伏感染及以往感染,因此诊断活动性结核病的特异性差是其主要问题.基于结核病变局部免疫细胞的干扰素-γ释放试验在肺结核及肺外结核的诊断中具有更好的特异性和敏感性,值得进一步研究.

  9. Attentional priming releases crowding.

    Kristjánsson, Arni; Heimisson, Pétur Rúnar; Róbertsson, Gunnar Freyr; Whitney, David


    Views of natural scenes unfold over time, and objects of interest that were present a moment ago tend to remain present. While visual crowding places a fundamental limit on object recognition in cluttered scenes, most studies of crowding have suffered from the limitation that they typically involved static scenes. The role of temporal continuity in crowding has therefore been unaddressed. We investigated intertrial effects upon crowding in visual scenes, showing that crowding is considerably diminished when objects remain constant on consecutive visual search trials. Repetition of both the target and distractors decreases the critical distance for crowding from flankers. More generally, our results show how object continuity through between-trial priming releases objects that would otherwise be unidentifiable due to crowding. Crowding, although it is a significant bottleneck on object recognition, can be mitigated by statistically likely temporal continuity of the objects. Crowding therefore depends not only on what is momentarily present, but also on what was previously attended.

  10. A Bayesian framework for estimating the incremental value of a diagnostic test in the absence of a gold standard.

    Ling, Daphne I; Pai, Madhukar; Schiller, Ian; Dendukuri, Nandini


    The absence of a gold standard, i.e., a diagnostic reference standard having perfect sensitivity and specificity, is a common problem in clinical practice and in diagnostic research studies. There is a need for methods to estimate the incremental value of a new, imperfect test in this context. We use a Bayesian approach to estimate the probability of the unknown disease status via a latent class model and extend two commonly-used measures of incremental value based on predictive values [difference in the area under the ROC curve (AUC) and integrated discrimination improvement (IDI)] to the context where no gold standard exists. The methods are illustrated using simulated data and applied to the problem of estimating the incremental value of a novel interferon-gamma release assay (IGRA) over the tuberculin skin test (TST) for latent tuberculosis (TB) screening. We also show how to estimate the incremental value of IGRAs when decisions are based on observed test results rather than predictive values. We showed that the incremental value is greatest when both sensitivity and specificity of the new test are better and that conditional dependence between the tests reduces the incremental value. The incremental value of the IGRA depends on the sensitivity and specificity of the TST, as well as the prevalence of latent TB, and may thus vary in different populations. Even in the absence of a gold standard, incremental value statistics may be estimated and can aid decisions about the practical value of a new diagnostic test.

  11. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.


    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-

  12. Analytical validation of the Roche 25-OH Vitamin D Total assay

    Knudsen, Cindy Soendersoe; Nexo, Ebba; Højskov, Carsten Schriver;


    Background: Vitamin D deficiency is considered a major health issue and therefore there is a need for reliable routine tests for measurement of the vitamin in blood samples. Here we present a validation of the recently released Roche 25-OH Vitamin D Total assay (Vitamin D Total). Methods: We anal...

  13. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.


    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-

  14. Cobalt release from inexpensive jewellery

    Thyssen, Jacob Pontoppidan; Jellesen, Morten Stendahl; Menné, Torkil


    Objectives: The aim was to study 354 consumer items using the cobalt spot test. Cobalt release was assessed to obtain a risk estimate of cobalt allergy and dermatitis in consumers who would wear the jewellery. Methods: The cobalt spot test was used to assess cobalt release from all items....... Microstructural characterization was made using scanning electron microscope (SEM) and energy-dispersive spectroscopy (EDS). Results: Cobalt release was found in 4 (1.1%) of 354 items. All these had a dark appearance. SEM/EDS was performed on the four dark appearing items which showed tin-cobalt plating on these....... Conclusions: This study showed that only a minority of inexpensive jewellery purchased in Denmark released cobalt when analysed with the cobalt spot test. As fashion trends fluctuate and we found cobalt release from dark appearing jewellery, cobalt release from consumer items should be monitored in the future...

  15. 21 CFR 864.7525 - Heparin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  16. A colorimetric assay for cytokinin oxidase.

    Libreros-Minotta, C A; Tipton, P A


    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  17. Multicentre comparison of a diagnostic assay

    Waters, Patrick; Reindl, Markus; Saiz, Albert;


    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  18. 21 CFR 866.3210 - Endotoxin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a) Identification. An endotoxin assay is a device that uses serological techniques in whole blood. The device...

  19. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida;


    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...


    S.O. Bader


    The purpose of this design analysis is to specify and document the total and respirable fractions for radioactive materials that are released from an accident event at the Monitored Geologic Repository (MGR) involving commercial spent nuclear fuel (CSNF) in a dry environment. The total and respirable release fractions will be used to support the preclosure licensing basis for the MGR. The total release fraction is defined as the fraction of total CSNF assembly inventory, typically expressed as an activity inventory (e.g., curies), of a given radionuclide that is released to the environment from a waste form. The radionuclides are released from the inside of breached fuel rods (or pins) and from the detachment of radioactive material (crud) from the outside surfaces of fuel rods and other components of fuel assemblies. The total release fraction accounts for several mechanisms that tend to retain, retard, or diminish the amount of radionuclides that are available for transport to dose receptors or otherwise can be shown to reduce exposure of receptors to radiological releases. The total release fraction includes a fraction of airborne material that is respirable and could result in inhalation doses. This subset of the total release fraction is referred to as the respirable release fraction. Potential accidents may involve waste forms that are characterized as either bare (unconfined) fuel assemblies or confined fuel assemblies. The confined CSNF assemblies at the MGR are contained in shipping casks, canisters, or disposal containers (waste packages). In contrast to the bare fuel assemblies, the container that confines the fuel assemblies has the potential of providing an additional barrier for diminishing the total release fraction should the fuel rod cladding breach during an accident. However, this analysis will not take credit for this additional bamer and will establish only the total release fractions for bare unconfined CSNF assemblies, which may however be

  1. Comparative assessment of in vitro release kinetics of calcitonin polypeptide from biodegradable microspheres.

    Prabhu, Sunil; Sullivan, Jennifer L; Betageri, Guru V


    The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.

  2. The impact of BCG vaccination on tuberculin skin test responses in children is age dependent: evidence to be considered when screening children for tuberculosis infection

    Seddon, James A; Paton, James; Nademi, Zohreh; Keane, Denis; Williams, Bhanu; Williams, Amanda; Welch, Steven B; Liebeschutz, Sue; Riddell, Anna; Bernatoniene, Jolanta; Patel, Sanjay; Martinez-Alier, Nuria; McMaster, Paddy; Kampmann, Beate


    Background Following exposure to TB, contacts are screened to target preventive treatment at those at high risk of developing TB. The UK has recently revised its recommendations for screening and now advises a 5 mm tuberculin skin test (TST) cut-off irrespective of age or BCG status. We sought to evaluate the impact of BCG on TST responses in UK children exposed to TB and the performance of different TST cut-offs to predict interferon γ release assay (IGRA) positivity. Methods Children vaccination on TST positivity was evaluated in IGRA-negative children, as was the performance of different TST cut-offs to predict IGRA positivity. Results Of 422 children recruited (median age 69 months; IQR: 32–113 months), 300 (71%) had been vaccinated with BCG. BCG vaccination affected the TST response in IGRA-negative children less than 5 years old but not in older children. A 5 mm TST cut-off demonstrated good sensitivity and specificity in BCG-unvaccinated children, and an excellent negative predictive value but was associated with low specificity (62.7%; 95% CI 56.1% to 69.0%) in BCG-vaccinated children. For BCG-vaccinated children, a 10 mm cut-off provided a high negative predictive value (97.7%; 95% CI 94.2% to 99.4%) with the positive predictive value increasing with increasing age of the child. Discussion BCG vaccination had little impact on TST size in children over 5 years of age. The revised TST cut-off recommended in the recent revision to the UK TB guidelines demonstrates good sensitivity but is associated with impaired specificity in BCG-vaccinated children. PMID:27335104

  3. External Dentin Stimulation Induces ATP Release in Human Teeth.

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T


    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

  4. Steroid assays in paediatric endocrinology.

    Honour, John W


    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  5. Predictive Assay For Cancer Targets

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A


    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  6. The prevalence of latent Mycobacterium tuberculosis infection based on an interferon-γ release assay

    Jensen, Andreas V; Jensen, Lotte; Faurholt-Jepsen, Daniel;


    Introduction:One third of the world's population is estimated to be latently infected with Mycobacterium tuberculosis (LTBI). Surveys of LTBI are rarely performed in resource poor TB high endemic countries like Tanzania although low-income countries harbor the largest burden of the worlds LTBI...

  7. Non-Tuberculous Mycobacteria and the Performance of Interferon Gamma Release Assays in Denmark

    Hermansen, Thomas Stig; Thomsen, Vibeke Østergaard; Lillebaek, Troels


    BACKGROUND: The QuantiFERON-TB-Gold Test (QFT) is more specific than the Mantoux skin-test to discriminate between Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacterial (NTM) infections. Here we study the performance of the QFT in patients with NTM disease. METHODS: From 2005 to 2011...

  8. Species differences in circulating prostaglandin metabolites. Relevance for the assay of prostaglandin release.

    Granström, E; Kindahl, H


    The profiles of circulating metabolites of prostaglandin F2 alpha were investigated in a number of species, viz. rat, rabbit, guinea pig, cattle and sheep. The aim of the study was to identify in each animal major plasma metabolites that outlast the initially formed, short-lived 15-keto-13, 14-dihydroprostaglandin F2 alpha and might thus serve as better parameters for monitoring prostaglandin production in vivo. Tritium-labeled prostaglandin F2 alpha was injected intravenously and frequent blood samples were collected. The metabolic profiles at different stages were visualized using two-dimensional thin-layer chromatography and autoradiography. Identification of circulating products was achieved by comparison with reference compounds using several chromatographic methods, and by gas chromatography-mass spectrometry in cases where larger amounts of the prostaglandin had been administered. In the rabbit a similar study was also done with tritium-labeled prostaglandin E2. Certain species differences were seen in the removal of labeled compounds from the circulation, the elimination being most efficient in the guinea pig. Further differences were seen in the profiles of circulating prostaglandin metabolites. The first appearing major prostaglandin F2 alpha metabolite was always 15-ketodihydroprostaglandin F2 alpha. However, this compound was later replaced in the circulation by a number of more degraded products, the profiles of which were relatively typical for each species. Thus, in cattle, rat and guinea pig, the earliest-formed metabolites, 15-ketodihydroprostaglandin F2 alpha and its tetranor counterpart, 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprostanoic acid, remained comparatively prominent plasma products, whereas highly polar dicarboxylic acids rapidly dominated the metabolite spectrum in the ovine and lapine circulation. These differences were further supported by separate kinetic experiments, using unlabeled prostaglandin F2 alpha and radioimmunological determination of formed products. These latter experiments also demonstrated further pronounced species differences in the rat of elimination of the different prostaglandin metabolites. A considerable interconversion between prostaglandin E and F compounds was also demonstrated in some species. In conclusion, the traditional prostaglandin parameters in plasma, the 15-ketodihydrometabolites, were found not to be the best parameters in all species. It is suggested that species differences in prostaglandin metabolism are taken ito consideration when the optimal analytical protocol is sought for future biological studies. Some alternatives are suggested in the present paper.

  9. Imaging renin content and release in the living kidney.

    Toma, Ildikó; Kang, Jung Julie; Peti-Peterdi, János


    Renin release is the first, and at least initially, the rate-limiting step in the activation of the renin-angiotensin system, which helps to maintain body salt and water balance. Recent advances in our understanding of pathophysiology have generated a renewed interest in the multiple roles of renin and prorenin as a hormone, enzyme, and signaling molecule. The assays available to measure renin content, release and tissue activity are complex, indirect and work with significant internal errors. We developed an imaging approach to directly visualize renin content and study the dynamics of both the release and tissue activity of renin. Our experimental model uses multiphoton fluorescence microscopy, which is ideal for deep optical sectioning of the living renal tissue. Here we review the application of this renin imaging approach to the dissected, in vitro microperfused glomerulus as well as in the intact kidney in vivo.

  10. Activity assay of membrane transport proteins

    Hao Xie


    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  11. Using IGRA in the diagnosis of tuberculosis or latent tuberculosis infection in HIV-positive persons

    Ravn, P.


    Persons with HIV and LTBI are at very high risk of developing active TB. The CD4 count drops in persons during the natural course of HIV/AIDS and at the same time the risk of TB increases. It is clear that there is a TB protective effect when HIV patients are placed on highly active antiretrovira...

  12. [Tuberculosis screening in school health services in Geneva, Switzerland].

    Meynard, Anne; Pfarrwaller, Eva; Lazarevic, Claire-Anne Wyler


    Recent immigrantyouth have multiple health needs that need to be adapted to the context of migration. School health services provide a systematic health check to allyoung immigrants starting school in Geneva, including a tuberculin skin test if coming from a middle or high incidence country. Positive tests are confirmed with Interferon Gamma Release Assay (IGRA) and if indicated, offered treatment of latent tuberculosis even in the absence of clear guidelines. Collective and individual benefits outweigh the difficult logistics: reducing risk of reactivation of latent tuberculosis for populations living in promiscuity, effective collaboration between primary care and school health services to answer the needs of these underserved youth.

  13. Workload Control with Continuous Release

    Phan, B. S. Nguyen; Land, M. J.; Gaalman, G. J. C.


    Workload Control (WLC) is a production planning and control concept which is suitable for the needs of make-to-order job shops. Release decisions based on the workload norms form the core of the concept. This paper develops continuous time WLC release variants and investigates their due date

  14. Press Oil Final Release Survey

    Whicker, Jeffrey Jay [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Ruedig, Elizabeth [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)


    There are forty-eight 55 gallon barrels filled with hydraulic oil that are candidates for release and recycle. This oil needs to be characterized prior to release. Principles of sampling as provided in MARSAME/MARSSIM approaches were used as guidance for sampling.

  15. Affinity-based release of polymer-binding peptides from hydrogels with the target segments of peptides.

    Serizawa, Takeshi; Fukuta, Hiroki; Date, Takaaki; Sawada, Toshiki


    Peptides with affinities for the target segments of polymer hydrogels were identified by biological screening using phage-displayed peptide libraries, and these peptides exhibited an affinity-based release capability from hydrogels. The results from cell culture assays demonstrated the sustained anticancer effects of the drug-conjugated peptides that were released from the hydrogels.



    Objective To study the releases of endothelin-1 and prostacyclin by endothelial cells in culture and to elucidate how these releases were influenced by smoke-treated low density lipoprotein. Methods We exposed endothelial cell cultures to native or oxidized Iow density lipoproteins,low density lipoproteins treated by dimethylsulfoxide-soluble particles from cigarette smoke or dimethylsulfoxide alones. The release of endothelin-1 was assayed by bioassay and the release of prostacyclin was assayed by radioimmunoassay. Results Low density lipoproteins treated by smoke significantly increased the release of endothelin-l (P<0. 025) and decreased the release of prostacyclin (P<0. 02) by endothelial cells in culture, contrast to native or dimethylsulfoxide-treated lipoproteins. Conclusion The main part of vasoconstrictor activity in conditioned medium from bovine aortic EC is endothelin-1.

  17. Sucrose release from polysaccharide gels.

    Nishinari, Katsuyoshi; Fang, Yapeng


    Sucrose release from polysaccharide gels has been studied extensively because it is expected to be useful in understanding flavour release from solid foods and to find a new processing method which produces more palatable and healthier foods. We provide an overview of the release of sucrose and other sugars from gels of agar and related polysaccharides. The addition of sucrose to agar solutions leads to the increase in transparency of the resulting gels and the decrease in syneresis, which is attributed to the decrease in mesh size in gels. The syneresis occurring in the quiescent condition and fluid release induced by compression is discussed. The relationship between the sugar release and the structural, rheological and thermal properties of gels is also discussed. Finally, the future research direction is proposed.

  18. Dry release of suspended nanostructures

    Forsén, Esko Sebastian; Davis, Zachary James; Dong, M.;


    A dry release method for fabrication of suspended nanostructures is presented. The technique has been combined with an anti-stiction treatment for fabrication of nanocantilever based nanoelectromechanical systems (NEMS). The process combines a dry release method, using a supporting layer of photo......A dry release method for fabrication of suspended nanostructures is presented. The technique has been combined with an anti-stiction treatment for fabrication of nanocantilever based nanoelectromechanical systems (NEMS). The process combines a dry release method, using a supporting layer......, the technique enables long time storage and transportation of produced devices without the risk of stiction. By combining the dry release method with a plasma deposited anti-stiction coating both fabrication induced stiction, which is mainly caused by capillary forces originating from the dehydration...

  19. Nitrogen release during coal combustion

    Baxter, L.L.; Mitchell, R.E.; Fletcher, T.H.; Hurt, R.H.


    Experiments in entrained flow reactors at combustion temperatures are performed to resolve the rank dependence of nitrogen release on an elemental basis for a suite of 15 U.S. coals ranging from lignite to low-volatile bituminous. Data were obtained as a function of particle conversion, with overall mass loss up to 99% on a dry, ash-free basis. Nitrogen release rates are presented relative to both carbon loss and overall mass loss. During devolatilization, fractional nitrogen release from low-rank coals is much slower than fractional mass release and noticeably slower than fractional carbon release. As coal rank increases, fractional nitrogen release rate relative to that of carbon and mass increases, with fractional nitrogen release rates exceeding fractional mass and fractional carbon release rates during devolatilization for high-rank (low-volatile bituminous) coals. At the onset of combustion, nitrogen release rates increase significantly. For all coals investigated, cumulative fractional nitrogen loss rates relative to those of mass and carbon passes through a maximum during the earliest stages of oxidation. The mechanism for generating this maximum is postulated to involve nascent thermal rupture of nitrogen-containing compounds and possible preferential oxidation of nitrogen sites. During later stages of oxidation, the cumulative fractional loss of nitrogen approaches that of carbon for all coals. Changes in the relative release rates of nitrogen compared to those of both overall mass and carbon during all stages of combustion are attributed to a combination of the chemical structure of coals, temperature histories during combustion, and char chemistry.

  20. Possible use of psyllium husk as a release retardant

    Desai Angira


    Full Text Available Various hydrophilic polymers from synthetic origin such as methylcellulose, PEGs, HPMC as well as those from natural world such as guar gum, tragacanth, xanthan gum have been used to formulate oral sustained release formulations. Psyllium husk has the ability to swell 10-14 times of its original volume and form a hydrogel. It is biocompatible, inexpensive, inert, non-absorbable, environment friendly and easily available. However, its use as a release retardant has not been fully explored. Owing to large dose, high sensitivity to light, moisture and heat and also very short half-life of 1-2 h; formulation of sustained release dosage form of amoxicillin trihydrate is a challenge. Hence the present study has been undertaken to develop sustained release granules as well as matrix tablets of amoxicillin trihydrate using psyllium husk as a primary release retardant. The drug release of these formulations was compared with those containing HPMC K4M.The results showed insignificant difference in t80% value for drug release as assessed by student′s t-test at 5% level of significance. Selected formulations were kept at controlled conditions of 40o/75% RH and 30o/65% RH for a period of 3 mo. Microbiological assay was used as a stability indicating method of assay. Sustained release granules and tablet formulations containing HPMC alone were found to be more stable than the similar formulations containing husk with percent drug content at the end of 3 mo at 40o/ 75% RH being 92.66%, 93.81%, 86.74%, 88.31% and the rate of degradation being 8.46x10-4 d-1, 7.1x10-4 d-1, 1.58x10-3 d-1 and 1.38x10-3 d-1, respectively. Thus it was concluded that psyllium husk can be effectively used as a hydrogel polymer in sustained release formulations. However, there is a need to keep moisture level under control during and after formulation.

  1. Assay of deoxyhypusine hydroxylase activity.

    Park, Jong Hwan; Wolff, Edith C; Park, Myung Hee


    The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine [N (ε)-(4-amino-2-hydroxybutyl)-lysine]. eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. Deoxyhypusine hydroxylase catalyzes a stereo-specific hydroxylation of the deoxyhypusine residue in the eIF5A intermediate protein, eIF5A(Dhp). The enzyme is totally specific for this protein and does not act on short peptides (hypusine residue in eIF5A. Optimum conditions for the reaction and two detection methods for the product, hypusine-containing eIF5A, are described in this chapter. The first, and most reliable, method is the measurement of the amount of [(3)H]hypusine in the protein hydrolysate after its separation from [(3)H]deoxyhypusine, by ion exchange chromatography. This method does require specialized equipment. The second method is based on counting the total TCA soluble radioactivity after sodium periodate oxidation of the reaction mixture, since the radiolabeled 4-amino-2-hydroxy butyl moiety of the hypusine residue is cleaved and is released from protein as radiolabeled β-propionaldehyde and formaldehyde by periodate oxidation.

  2. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason


    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  3. [Review on characteristics and detecting assay of bacterial endotoxin contamination in water environment].

    Zhang, Can; Liu, Wen-Jun; Zhang, Ming-Lu; Tian, Fang; Yang, Yi; An, Dai-Zhi


    Endotoxins, also known as lipopolysaccharide complexes, are anchored in the outer membrane cell wall of most Gram-negative bacteria and some cyanobacteria. They are continuously released to environment during cell decay. Being common pyrogens and highly immunogenic molecules, endotoxins are related to many human diseases. Due to the tolerances and thermo-stability of endotoxin molecules, they were hard to be removed by common methods. The health risk caused by the endotoxin contamination in drinking water and water environment by various exposure pathways have attracted more and more attention in recent years. In this paper, the physical and chemical properties, biological activities and detection assay of the endotoxin contamination were reviewed, and interfere factors of the main assay, the LAL/TAL (Limulus amebocyte lysate/Tachypleus amebocyte lysate) assay, for detecting endotoxin in water sample were investigated, and the development tendency of the endotoxin detection assay was analyzed.

  4. Development and application of assays for serotonin

    Gow, I.F.


    In this thesis, two assays for serotonin were developed, validated, and used to investigate the relationship between platelet aggregation, serotonin levels and sodium status and serotonin levels and platelet function in patients with cardiovascular disease. A radioimmunoassay (RIA) using an (/sup 125/I)-labelled tracer was developed and validated for the measurement of serotonin in human platelet-rich plasma (PRP) and rat serum. Antisera were raised against N-succinamylserotonin conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. An assay for serotonin using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) was developed, and used to validate the RIA. The RIA can be used to assay up to 100 samples/day compared with 10-20/day by the HPLC-ECD assay.

  5. Commercial SNF Accident Release Fractions

    J. Schulz


    The purpose of this analysis is to specify and document the total and respirable fractions for radioactive materials that could be potentially released from an accident at the repository involving commercial spent nuclear fuel (SNF) in a dry environment. The total and respirable release fractions are used to support the preclosure licensing basis for the repository. The total release fraction is defined as the fraction of total commercial SNF assembly inventory, typically expressed as an activity inventory (e.g., curies), of a given radionuclide that is released to the environment from a waste form. Radionuclides are released from the inside of breached fuel rods (or pins) and from the detachment of radioactive material (crud) from the outside surfaces of fuel rods and other components of fuel assemblies. The total release fraction accounts for several mechanisms that tend to retain, retard, or diminish the amount of radionuclides that are available for transport to dose receptors or otherwise can be shown to reduce exposure of receptors to radiological releases. The total release fraction includes a fraction of airborne material that is respirable and could result in inhalation doses; this subset of the total release fraction is referred to as the respirable release fraction. Accidents may involve waste forms characterized as: (1) bare unconfined intact fuel assemblies, (2) confined intact fuel assemblies, or (3) canistered failed commercial SNF. Confined intact commercial SNF assemblies at the repository are contained in shipping casks, canisters, or waste packages. Four categories of failed commercial SNF are identified: (1) mechanically and cladding-penetration damaged commercial SNF, (2) consolidated/reconstituted assemblies, (3) fuel rods, pieces, and debris, and (4) nonfuel components. It is assumed that failed commercial SNF is placed into waste packages with a mesh screen at each end (CRWMS M&O 1999). In contrast to bare unconfined fuel assemblies, the

  6. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P


    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

  7. Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity

    Dessy Natalia


    Full Text Available Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC is a versatile catalyst for the organic synthesis of chiral α-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the α-hydroxy ketone (R-2,2′-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations.

  8. Highly Efficient Thermoresponsive Nanocomposite for Controlled Release Applications

    Yassine, Omar; Zaher, Amir; Li, Er Qiang; Alfadhel, Ahmed; Perez, Jose E.; Kavaldzhiev, Mincho; Contreras, Maria F.; Thoroddsen, Sigurdur T.; Khashab, Niveen M.; Kosel, Jurgen


    Highly efficient magnetic release from nanocomposite microparticles is shown, which are made of Poly (N-isopropylacrylamide) hydrogel with embedded iron nanowires. A simple microfluidic technique was adopted to fabricate the microparticles with a high control of the nanowire concentration and in a relatively short time compared to chemical synthesis methods. The thermoresponsive microparticles were used for the remotely triggered release of Rhodamine (B). With a magnetic field of only 1 mT and 20 kHz a drug release of 6.5% and 70% was achieved in the continuous and pulsatile modes, respectively. Those release values are similar to the ones commonly obtained using superparamagnetic beads but accomplished with a magnetic field of five orders of magnitude lower power. The high efficiency is a result of the high remanent magnetization of the nanowires, which produce a large torque when exposed to a magnetic field. This causes the nanowires to vibrate, resulting in friction losses and heating. For comparison, microparticles with superparamagnetic beads were also fabricated and tested; while those worked at 73 mT and 600 kHz, no release was observed at the low field conditions. Cytotoxicity assays showed similar and high cell viability for microparticles with nanowires and beads.

  9. Scavenging effect of Trolox released from brushite cements.

    Mestres, Gemma; Santos, Carlos F; Engman, Lars; Persson, Cecilia; Ott, Marjam Karlsson


    In this study a brushite cement was doped with the chain-breaking antioxidant Trolox. The effect of the antioxidant on the physical properties of the cement was evaluated and the release of Trolox was monitored by UV spectroscopy. The ability of the Trolox set free to scavenge reactive oxygen species (ROS) released by macrophages was determined in vitro using a luminol-amplified chemiluminescence assay. Trolox did not modify the crystalline phases of the set cement, which mainly formed crystalline brushite after 7 days in humid conditions. The setting time, compressive strength and morphology of the cement also remained unaltered after the addition of the antioxidant. Trolox was slowly released from the cement following a non-Fickian transport mechanism and nearly 64% of the total amount was released after 3 days. Moreover, the capacity of Trolox to scavenge the ROS released by macrophages increased in a dose-dependent manner. Trolox-loaded cements are expected to reduce some of the first harmful effects of acute inflammation and can thus potentially protect the surrounding tissue during implantation of these as well as other materials used in conjunction.

  10. Radio-synthesized polyacrylamide hydrogels for proteins release

    Ferraz, Caroline C.; Varca, Gustavo H. C.; Lopes, Patricia S.; Mathor, Monica B.; Lugão, Ademar B.


    The use of hydrogels for biomedical purposes has been extensively investigated. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release, using papain and bovine serum albumin as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using γ-radiation under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density and evaluated according to protein release, bioactivity and cytotoxicity. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-5% (w/v) polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 h), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay it was also adequate for biomedical purposes, however this method was not able to generate a matrix with controlled pore sizes.

  11. Sequential-release of anticancer drugs microcapsulated with ethylcellulose

    顾耕华; 黄剑奇; 何虹


    Objective To approach the sequential release of antitumor drugs and promote the effect of chemotherapy.Methods Adriamycin (ADM) and carboplatin (CBP) were respectively microcapsulated with ethylcellulose by organic phase separation. The morphology and sizes of the microcapsules were observed and measured with light microscope and scanning electromicroscope. The contents and the release rates of ADM and CBP in microcapsules were measured with fluorescence spectrophotometer and high-efficiency phantom chromatic (HPC) spectrum respectively. The antitumor sensitivity test in vitro was devised with MTT assay.Results The microcapsules of ADM and CBP were spherical in shape with diameters of 196?4 μm and 214?8 μm respectively. The contents of one-layer and two-layer CBP and ADM microcapsules were 51.4%, 35.7% and 39.8% respectively, with the release rates in vitro of 62.4%/day, 54.8%/day and 48.2% /8h. The results of drug sensitivity test in vitro demonstrated that the current preparation has never affected the stability and antitumor activity of CBP and ADM.Conclusion Microcapsules with different drugs and different thickness of material have different release rate. Combined arterial chemoembolization with different microcapsules could approach the sequential release and promote the effect of chemotherapy.


    Jampani Neeharika


    Full Text Available Candesartan Cilexetil is an esterified prodrug of Candesartan, a non-peptide angiotensin II type-1(AT1 receptor antagonist used in the treatment of hypertension and congestive heart failure. Candesartan meets the requirement of high potency but it is poorly absorbed when administered orally. Therefore, the prodrug Candesartan Cilexetil is developed. It is soluble in methylene chloride, half life is 5.1 to 10.5hrs and bioavailability is 15%. It is marketed as conventional tablets. In this work, it is formulated as immediate release tablets by changing the concentration of ingredients. For many drug substances, conventional immediate-release formulations provide clinically effective therapy while maintaining the required balance of pharmacokinetic and pharmacodynamic profiles with in acceptable level of safety to the patient. The immediate release formulation of Candesartan Cilexetil is prepared by wet granulation method to provide rapid onset of action. In order to optimize the best formulation, ten different trials are developed. The main ingredients used in the formulation are lactose monohydrate, PEG, calcium CMC and MCC. Weight variation, thickness, friability, disintegration time, in-vitro release, pharmaceutical assay are studied as response variables. The formulation containing 38% of MCC is selected as an optimized product in which the different physical properties and in-vitro release profile showed best comparable results with innovator product.

  13. Highly Efficient Thermoresponsive Nanocomposite for Controlled Release Applications

    Yassine, Omar


    Highly efficient magnetic release from nanocomposite microparticles is shown, which are made of Poly (N-isopropylacrylamide) hydrogel with embedded iron nanowires. A simple microfluidic technique was adopted to fabricate the microparticles with a high control of the nanowire concentration and in a relatively short time compared to chemical synthesis methods. The thermoresponsive microparticles were used for the remotely triggered release of Rhodamine (B). With a magnetic field of only 1 mT and 20 kHz a drug release of 6.5% and 70% was achieved in the continuous and pulsatile modes, respectively. Those release values are similar to the ones commonly obtained using superparamagnetic beads but accomplished with a magnetic field of five orders of magnitude lower power. The high efficiency is a result of the high remanent magnetization of the nanowires, which produce a large torque when exposed to a magnetic field. This causes the nanowires to vibrate, resulting in friction losses and heating. For comparison, microparticles with superparamagnetic beads were also fabricated and tested; while those worked at 73 mT and 600 kHz, no release was observed at the low field conditions. Cytotoxicity assays showed similar and high cell viability for microparticles with nanowires and beads.

  14. Birth control - slow release methods

    ... page: // Birth control - slow release methods To use the sharing features on this page, please enable JavaScript. Certain birth control methods contain man-made forms of hormones. These ...

  15. Turbulent lock release gravity current


    The time evolution of a turbulent lock release gravity current, formed by a finite volume ofhomogeneous fluid released instantaneously into another fluid of slightly lower density, was studied byexperimental measurements of the density structure via elaborate digital image processing and by a nu-merical simulation of the flow and mixing using a two-equation turbulence model. The essential fact thatthe gravity current passes through an initial slumping phase in which the current head advances steadilyand a second self-similar phase in which the front velocity decreases like the negative third power of thetime after release is satisfactorily presented by the laboratory observation. An overall entrainment ratioproportional to the distance from the release point is found by the numerical simulation. The renormal-ization group (RNG) k- ε model for Reynolds-stress closure is validated to characterize the gravitycurrent with transitional and localized turbulence.

  16. PCDD/PCDF release inventories

    Fiedler, H. [UNEP Chemicals, Chatelaine (Switzerland)


    The Stockholm Convention on Persistent Organic Pollutants (POPs) entered into force on 17 May 2004 with 50 Parties. In May 2004, 59 countries had ratified or acceded the Convention. The objective of the Convention is ''to protect human health and the environment from persistent organic pollutants''. For intentionally produced POPs, e.g., pesticides and industrial chemicals such as hexachlorobenzene and polychlorinated biphenyls, this will be achieved by stop of production and use. For unintentionally generated POPs, such as polychlorinated dibenzo-pdioxins (PCDD) and polychlorinated dibenzofurans (PCDF), measures have to be taken to ''reduce the total releases derived from anthropogenic sources''; the final goal is ultimate elimination, where feasible. Under the Convention, Parties have to establish and maintain release inventories to prove the continuous release reduction. Since many countries do not have the technical and financial capacity to measure all releases from all potential PCDD/PCDF sources, UNEP Chemicals has developed the ''Standardized Toolkit for the Identification of Quantification of Dioxin and Furan Releases'' (''Toolkit'' for short), a methodology to estimate annual releases from a number of sources. With this methodology, annual releases can be estimated by multiplying process-specific default emission factors provided in the Toolkit with national activity data. At the seventh session of the Intergovernmental Negotiating Committee, the Toolkit was recommended to be used by countries when reporting national release data to the Conference of the Parties. The Toolkit is especially used by developing countries and countries with economies in transition where no measured data are available. Results from Uruguay, Thailand, Jordan, Philippines, and Brunei Darussalam have been published.

  17. Particle-induced artifacts in the MTT and LDH viability assays.

    Holder, Amara L; Goth-Goldstein, Regine; Lucas, Donald; Koshland, Catherine P


    In vitro testing is a common first step in assessing combustion-generated and engineered nanoparticle-related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose-response data. Nanoparticles, well-known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/mL, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/mL and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations.

  18. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Bi, Xiaodong; Liu, Zhen


    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  19. Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

    Llewellyn, David; Miura, Kazutoyo; Fay, Michael P; Williams, Andrew R; Murungi, Linda M; Shi, Jianguo; Hodgson, Susanne H; Douglas, Alexander D; Osier, Faith H; Fairhurst, Rick M; Diakite, Mahamadou; Pleass, Richard J; Long, Carole A; Draper, Simon J


    The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

  20. Sample preparation composite and replicate strategy case studies for assay of solid oral drug products.

    Nickerson, Beverly; Harrington, Brent; Li, Fasheng; Guo, Michele Xuemei


    Drug product assay is one of several tests required for new drug products to ensure the quality of the product at release and throughout the life cycle of the product. Drug product assay testing is typically performed by preparing a composite sample of multiple dosage units to obtain an assay value representative of the batch. In some cases replicate composite samples may be prepared and the reportable assay value is the average value of all the replicates. In previously published work by Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was developed to provide a systematic approach which accounts for variability due to the analytical method and dosage form with a standard error of the potency assay criteria based on compendia and regulatory requirements. In this work, this sample preparation composite and replicate strategy for assay is applied to several case studies to demonstrate the utility of this approach and its application at various stages of pharmaceutical drug product development. Copyright © 2017. Published by Elsevier B.V.

  1. Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays.

    Prather, Brittany; Ethen, Cheryl M; Machacek, Miranda; Wu, Zhengliang L


    Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.

  2. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays.

    Tohidi, Maryam; Arbab, Parvaneh; Ghasemi, Asghar


    Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 μU/mL and -0.14 μU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

  3. Exosomes released from breast cancer carcinomas stimulate cell movement.

    Dinari A Harris

    Full Text Available For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1 exosomes promote cell migration and (2 the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3 exosomes are endocytosed at the same rate regardless of the cell type; (4 exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

  4. Exosomes released from breast cancer carcinomas stimulate cell movement.

    Harris, Dinari A; Patel, Sajni H; Gucek, Marjan; Hendrix, An; Westbroek, Wendy; Taraska, Justin W


    For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

  5. Extracellular adenosine 5'-triphosphate and lipopolysaccharide induce interleukin-1β release in canine blood.

    Spildrejorde, Mari; Curtis, Stephen J; Curtis, Belinda L; Sluyter, Ronald


    Binding of extracellular adenosine 5'-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30 min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs.




    Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand

  7. Assessing sediment contamination using six toxicity assays

    Allen G. BURTON Jr.


    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  8. Radioreceptor assay: theory and applications to pharmacology

    Perret, G. (U.E.R. de Medecine, Sante et Biologie Humaine, 93 - Bobigny (France)); Simon, P. (Faculte de Medecine Pitie-Salpetriere, 75 - Paris (France))

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, ..beta..-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising.

  9. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred


    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  10. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana


    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  11. Assays for Determination of Protein Concentration.

    Olson, Bradley J S C


    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

  12. Immuno-assay techniques for detecting yeasts in foods.

    Middelhoven, W J; Notermans, S


    A brief literature review on immuno-assay of yeast cell wall antigens is given. Special attention is paid to extracellular, thermostable yeast antigens (EPS), which are released to the growth medium by many yeast species. The EPS of Saccharomyces cerevisiae and of Stephanoascus ciferrii (syn. Candida ciferrii) could be specifically and sensitively detected by a sandwich ELISA, using an IgG raised in rabbits immunized with the EPS of these yeasts. The EPS ELISA of three basidiomycetous yeasts tested was not specific, that of Geotrichum candidum was genus-specific but was not sensitive. The EPS of Zygosaccharomyces bailii could be detected in a highly specific competitive ELISA but not in a sandwich ELISA or in a latex agglutination test.

  13. Assessment of gastrointestinal motility using three different assays in vitro.

    Pozzoli, Cristina; Poli, Enzo


    The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug-stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.

  14. The comet assay – how to recognise “good data”

    William Barfield


    Full Text Available Testing of potentially genotoxic materials currently involves use of in vitro assays such as the Ames test (gene mutations, the chromosome aberration assay and in vitro micronucleus assay (predominantly using human peripheral lymphocytes to detect clastogenicity and/or aueuploidy and the mouse lymphoma assay (gene mutations. In addition, one in vivo assay, predominately the in vivo micronucleus assay, is “normally” required to satisfy regulatory testing requirements and on occasion a second in vivo assay is required to assist in interpretation of results. Following release of the OECD 489 guideline “In vivo mammalian alkaline comet assay” in September 2014 the in vivo comet assay is now considered to be the second in vivo genetic toxicology assay of choice, effectively replacing the use of the UDS assay. The comet assay can be used to detect single strand and double strand breaks by measuring the median %tail intensity under alkaline conditions (pH>13. Following the JaCVAM validation trial, subsequent publication of the OECD 489 guideline recommended using the liver (site of metabolism and glandular stomach (site of contact of rats. However, any tissue can be examined if experimental competency with the tissue of interest has been proven. A number of key areas need careful consideration when performing the assay and interpreting the data. Both duration and temperature of tissue preparation have been shown to be critical parameters for obtaining “good data” along with the correct electrophoresis conditions to detect ‘weak’ effects. Study design requires careful thought in terms of animals per group, slides analysed per animal and cells analysed per slide and to facilitate this a special interest group within industry has published recommendations on statistical analysis of the comet assay. The sensitivity of the comet assay within the laboratory can be determined with the use of ‘power curves’ and identifying a high

  15. Improved benzodiazepine radioreceptor assay using the MultiScreen (R) Assay System

    Janssen, MJ; Ensing, K; de Zeeuw, RA


    In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time, The filtration in this method was performed by using the MultiScreen(R) Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom,

  16. Aluminum corrosion product release kinetics

    Edwards, Matt, E-mail:; Semmler, Jaleh; Guzonas, Dave; Chen, Hui Qun; Toor, Arshad; Hoendermis, Seanna


    Highlights: • Release of Al corrosion product was measured in simulated post-LOCA sump solutions. • Increased boron was found to enhance Al release kinetics at similar pH. • Models of Al release as functions of time, temperature, and pH were developed. - Abstract: The kinetics of aluminum corrosion product release was examined in solutions representative of post-LOCA sump water for both pressurized water and pressurized heavy-water reactors. Coupons of AA 6061 T6 were exposed to solutions in the pH 7–11 range at 40, 60, 90 and 130 °C. Solution samples were analyzed by inductively coupled plasma atomic emission spectroscopy, and coupon samples were analyzed by secondary ion mass spectrometry. The results show a distinct “boron effect” on the release kinetics, expected to be caused by an increase in the solubility of the aluminum corrosion products. New models were developed to describe both sets of data as functions of temperature, time, and pH (where applicable)

  17. Controlled Release Formulations of Auxinic Herbicides

    Kowalski, Witold J.; Siłowiecki, Andrzej.; Romanowska, Iwona; Glazek, Mariola; Bajor, Justyna; Cieciwa, Katarzyna; Rychter, Piotr


    Controlled release formulations are applied extensively for the release of active ingredients such as plant protection agents and fertilizers in response to growing concern for ecological problems associated with increased use of plant protection chemicals required for intensive agricultural practices [1]. We synthesized oligomeric mixtures of (R,S)-3-hydroxy butyric acid chemically bonded with 2,4-D, Dicamba and MCPA herbicides (HBA) respectively, and determined their molecular structure and molecular weight dispersion by the size exclusion chromatography, proton magnetic resonance spectrometry and electro-spray ionization mass spectrometry. Further we carried out bioassays of herbicidal effectiveness of the HBA herbicides vs. series of dicotyledonous weeds and crop injury tests [2, 3, 4]. Field bioassays were accomplished according to the EPPO standards [5]. Groups of representative weeds (the development stages in the BCCH scale: 10 - 30) were selected as targets. Statistical variabilities were assessed by the Fisher LSD test for plants treated with the studied herbicides in form of HBA oligomers, the reference herbicides in form of dimethyl ammonium salts (DMA), and untreated plants. No statistically significant differences in the crop injuries caused by the HBA vs. the DMA reference formulation were observed. The effectiveness of the HBA herbicides was lower through the initial period (ca. 2 weeks) relative to the DMA salts, but a significant increase in the effectiveness of the HBA systems followed during the remaining fraction of each assay. After 6 weeks all observed efficiencies approached 100%. The death of weeds treated with the HBA herbicides was delayed when compared with the DMA reference herbicides. The delayed uptake observed for the HBA oligomers relative to the DMA salts was due to controlled release phenomena. In case of the DMA salts the total amount of active ingredients was available at the target site. By contrast, the amount of an active

  18. Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

    Nissen, Dan; Petersen, Lars Jelstrup; Nolte, H;


    OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted...... into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed...... fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual...

  19. Nano-immunosafety: issues in assay validation

    Boraschi, Diana; Italiani, Paola [Institute of Biomedical Technologies, National Research Council, Via G. Moruzzi 1, 56124 Pisa (Italy); Oostingh, Gertie J; Duschl, Albert [Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Casals, Eudald; Puntes, Victor F [Institut Catala de Nanotecnologia, Campus de la UAB - Facultat de Ciencies, Edifici CM7, 08193 Bellaterra (Spain); Nelissen, Inge, E-mail: [VITO NV, Boeretang 200, BE-2400 Mol (Belgium)


    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  20. Mobile non-destructive assay system

    Colarusso, A.P.; Audas, J.H.; Bieri, J.M.; Herrera, G.C.; Hastings, R.D.; Horton, W.S.; Kuckertz, T.H.; Kunz, W.E.; Medvick, P.A.; Vogel, P.A.


    A mobile system for non-destructive assay (NDA), developed at the Los Alamos National Laboratory, provides accurate and sensitive measurements for transuranic (TRU) isotopes contained in 208-iota drums of miscellaneous nuclear wastes. The NDA unit consists of four major subsystems: an assay chamber, counting and digital electronics, data acquisition, and a neutron generator. It performs both active and passive neutron waste measurements. The former determines the amount of fissile isotopes at a sensitivity level of 1 mg plutonium. The latter determines spontaneous fission and ..cap alpha..,n) isotopes at a comparable level. A complete assay consists of sequential active and passive measurements. The assay measurement and other supporting data are incorporated in a commercial spreadsheet program (Lotus 1,2,3) for further analysis, which includes various matrix corrections and a determination of whether or not the drum exceeds the 100-nCi/g threshold for TRU wastes. Field tests have been performed on three separate occasions, accomplishing more than 1800 waste drum assays. These waste drum assays are discussed, especially those comparing passive and active neutron measurements with independent segmented gamma scan assays. Results obtained with a set of 15 drums containing plutonium prepared from standards and actual hot waste matrices are also reviewed.

  1. Analytical characterization of the APTIMA HPV Assay.

    Dockter, Janel; Schroder, Astrid; Eaton, Barbara; Wang, Ann; Sikhamsay, Nathan; Morales, Liezel; Giachetti, Cristina


    Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. To determine the analytical performance characteristics of the APTIMA HPV Assay. Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems. The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or low-risk HPV types. Spermicides, anti-fungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-lot, and inter-run signal variability were 99% of the data. Intra-run variability was HPV Assay showed excellent performance and robustness.

  2. A high-throughput assay of NK cell activity in whole blood and its clinical application

    Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)


    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  3. Triggered Drug Release from Superhydrophobic Meshes using High-Intensity Focused Ultrasound

    Yohe, Stefan T.; Kopechek, Jonathan A.; Porter, Tyrone M; Colson, Yolonda L.; Grinstaff, Mark W.


    Application of high-intensity focused ultrasound to drug-loaded superhydrophobic meshes affords triggered drug release by displacing an entrapped air layer. The air layer within the superhydrophobic meshes is characterized using direct visualization and B-mode imaging. Drug-loaded superhydrophobic meshes are cytotoxic in an in vitro assay after ultrasound treatment.

  4. Ammonium tetrathiomolybdate as a water-soluble and slow-release hydrogen sulfide donor.

    Xu, Shi; Yang, Chun-Tao; Meng, Fu-Hui; Pacheco, Armando; Chen, Li; Xian, Ming


    Ammonium tetrathiomolybdate (TTM) was found to be a slow hydrogen sulfide (H2S) releasing agent. Its H2S generation capability in aqueous solutions was confirmed by UV-vis and fluorescence assays. TTM also showed H2S-like cytoprotective effects in hydrogen peroxide (H2O2)-induced oxidative damage in HaCaT cells.

  5. Controlled release from recombinant polymers.

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza


    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed.

  6. Nuclear energy release from fragmentation

    Li, Cheng; Tsang, M B; Zhang, Feng-Shou


    Nuclear energy released by splitting Uranium and Thorium isotopes into two, three, four, up to eight fragments with nearly equal size are studied. We found that the energy released come from equally splitting the $^{235,238}$U and $^{230,232}$Th nuclei into to three fragments is largest. The statistical multifragmentation model is employed to calculate the probability of different breakup channels for the excited nuclei. Weighing the the probability distributions of fragments multiplicity at different excitation energies for the $^{238}$U nucleus, we found that an excitation energy between 1.2 and 2 MeV/u is optimal for the $^{235}$U, $^{238}$U, $^{230}$Th and $^{232}$Th nuclei to release nuclear energy of about 0.7-0.75 MeV/u.

  7. Training Materials for Release 3

    Wake, Jo Dugstad; Hansen, Cecilie; Debus, Kolja;

    This document, D7.4 – training materials for release 3, provides an overview of the training material for version 3 of the NEXT-TELL tools and methods. Previous documents submitted as part of work package 7, which is about teacher training, are D7.1 – Training Concept, D7.2 – Training Materials...... for Release 1 and D7.3 – Training Materials for Release 2. D7.4 builds on D7.1 and D7.2 and D7.3. D7.4 contains further development of previous work within WP7, essentially a revised theoretical approach to the teacher training, and expansion of the notion of tool training. The media in use have been expanded...

  8. Lipase assay in soils by copper soap colorimetry.

    Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R


    A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

  9. Polymerase chain reaction assay for avian polyomavirus.

    Phalen, D.N.; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By us...

  10. Optical control of insulin release using a photoswitchable sulfonylurea.

    Broichhagen, Johannes; Schönberger, Matthias; Cork, Simon C; Frank, James A; Marchetti, Piero; Bugliani, Marco; Shapiro, A M James; Trapp, Stefan; Rutter, Guy A; Hodson, David J; Trauner, Dirk


    Sulfonylureas are widely prescribed for the treatment of type 2 diabetes mellitus (T2DM). Through their actions on ATP-sensitive potassium (KATP) channels, sulfonylureas boost insulin release from the pancreatic beta cell mass to restore glucose homeostasis. A limitation of these compounds is the elevated risk of developing hypoglycemia and cardiovascular disease, both potentially fatal complications. Here, we describe the design and development of a photoswitchable sulfonylurea, JB253, which reversibly and repeatedly blocks KATP channel activity following exposure to violet-blue light. Using in situ imaging and hormone assays, we further show that JB253 bestows light sensitivity upon rodent and human pancreatic beta cell function. Thus, JB253 enables the optical control of insulin release and may offer a valuable research tool for the interrogation of KATP channel function in health and T2DM.

  11. Modified drug release using atmospheric pressure plasma deposited siloxane coatings

    Dowling, D. P.; Maher, S.; Law, V. J.; Ardhaoui, M.; Stallard, C.; Keenan, A.


    This pilot study evaluates the potential of atmospheric plasma polymerised coatings to modify the rate of drug release from polymeric substrates. The antibiotic rifampicin was deposited in a prototype multi-layer drug delivery system, consisting of a nebulized layer of active drug between a base layer of TEOS deposited on a plastic substrate (polystyrene) and an overlying layer of plasma polymerised PDMS. The polymerised TEOS and PDMS layers were deposited using a helium atmospheric plasma jet system. Elution of rifampicin was measured using UV-VIS spectroscopy, in addition to a antimicrobial well diffusion assay with an established indicator organism. The multi-layered plasma deposited coatings significantly extended the duration of release of the rifampicin from 24 h for the uncoated polymer to 144 h for the coated polymer.

  12. Gum Ghatti--a pharmaceutical excipient: development, evaluation and optimization of sustained release mucoadhesive matrix tablets of domperidone.

    Gurpreetarora; Malik, Karan; Rana, Vikas; Singh, Inderbir


    The objective of this study was to extend the GI residence time of the dosage form and to control the release of domperidone using directly compressible sustained release mucoadhesive matrix (SRMM) tablets. A 2-factor centre composite design (CCD) was employed to study the influence of independent variables like gum ghatti (GG) (X1) and hydroxylpropylmethyl cellulose K 15M (HPMC K 15M) (X2) on dependent variable like mucoadhesive strength, tensile strength, release exponent (n), t50 (time for 50% drug release), rel(10 h) (release after 10 h) and rel(18 h) (release after 18 h). Tablets were prepared by direct compression technology and evaluated for tablet parametric test (drug assay, diameter, thickness, hardness and tensile strength), mucoadhesive strength (using texture analyzer) and in vitro drug release studies. The tensile strength and mucoadhesive strength were found to be increased from 0.665 +/- 0.1 to 1.591 +/- 0.1 MN/cm2 (Z1 to Z9) and 10.789 +/- 0.985 to 50.924 +/- 1.150 N (Z1 to Z9), respectively. The release kinetics follows first order and Hixson Crowell equation indicating drug release following combination of diffusion and erosion. The n varies between 0.834 and 1.273, indicating release mechanism shifts from non fickian (anomalous release) to super case II, which depict that drug follows multiple drug release mechanism. The t50 time was found to increase from 5 +/- 0.12 to 11.4 +/- 0.14 h (Z1 to Z9) and release after 10 and 18 h decreases with increasing concentration of both polymers concluding with release controlling potential of polymers. The accelerated stability studies were performed on optimized formulation as per ICH guideline and the result showed that there was no significant change in tensile strength, mucoadhesive strength and drug assay.

  13. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    Buch Karl


    Full Text Available Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98 were included in the experiment to study its principal and general applicability.

  14. Herpes Murine Model as a Biological Assay to Test Dialyzable Leukocyte Extracts Activity

    Nohemí Salinas-Jazmín; Sergio Estrada-Parra; Miguel Angel Becerril-García; Alberto Yairh Limón-Flores; Said Vázquez-Leyva; Emilio Medina-Rivero; Lenin Pavón; Marco Antonio Velasco-Velázquez; Sonia Mayra Pérez-Tapia


    Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Tra...

  15. Release of Growth Factors into Root Canal by Irrigations in Regenerative Endodontics.

    Zeng, Qian; Nguyen, Sean; Zhang, Hongming; Chebrolu, Hari Priya; Alzebdeh, Dalia; Badi, Mustafa A; Kim, Jong Ryul; Ling, Junqi; Yang, Maobin


    The aim of this study was to investigate the release of growth factors into root canal space after the irrigation procedure of regenerative endodontic procedure. Sixty standardized root segments were prepared from extracted single-root teeth. Nail varnish was applied to all surfaces except the root canal surface. Root segments were irrigated with 1.5% NaOCl + 17% EDTA, 2.5% NaOCl + 17% EDTA, 17% EDTA, or deionized water. The profile of growth factors that were released after irrigation was studied by growth factor array. Enzyme-linked immunosorbent assay was used to validate the release of transforming growth factor (TGF)-β1 and basic fibroblast growth factor (bFGF) at 4 hours, 1 day, and 3 days after irrigation. The final concentrations were calculated on the basis of the root canal volume measured by cone-beam computed tomography. Dental pulp stem cell migration on growth factors released from root segments was measured by using Transwell assay. Total of 11 of 41 growth factors were detected by growth factors array. Enzyme-linked immunosorbent assay showed that TGF-β1 was released in all irrigation groups. Compared with the group with 17% EDTA (6.92 ± 4.49 ng/mL), the groups with 1.5% NaOCl + 17% EDTA and 2.5% NaOCl + 17% EDTA had significantly higher release of TGF-β1 (69.04 ± 30.41 ng/mL and 59.26 ± 3.37 ng/mL, respectively), with a peak release at day 1. The release of bFGF was detected at a low level in all groups (0 ng/mL to 0.43 ± 0.22 ng/mL). Migration assay showed the growth factors released from root segments induced dental pulp stem cell migration. The root segment model in present study simulated clinical scenario and indicated that the current irrigation protocol released a significant amount of TGF-β1 but not bFGF. The growth factors released into root canal space induced dental pulp stem cell migration. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Triggered release of therapeutic antibodies from nanodiamond complexes.

    Smith, Adrienne H; Robinson, Erik M; Zhang, Xue-Qing; Chow, Edward K; Lin, Yang; Osawa, Eiji; Xi, Jianzhong; Ho, Dean


    Recent reports have revealed that detonation nanodiamonds (NDs) can serve as efficient, biocompatible, and versatile drug delivery platforms. Consequently, further investigations exploring additional therapeutic applications are warranted. Current limitations associated with the non-specific nature of intravenous drugs limit the potential of certain pharmacological agents. One such treatment that could benefit from a stable delivery platform is antibody (Ab) therapy. Determination of Ab adsorption and desorption to a ND surface was subsequently examined using the transforming growth factor β (TGF-β) antibody as a model therapeutic. ND-Ab complexes were found to be stable in water through enzyme-linked immunosorbent assays (ELISAs), UV-vis spectroscopy and TEM, with no Ab released after ten days. Released Abs were detected in extreme pH solutions (3.5), DMEM (+) serum with pH levels ranging from 4 to 10.5, and inorganic saline solutions. Preserved activity of Abs released in DMEM (+) serum was confirmed using an ELISA. These results suggest ND-Ab complexes are synthesized and stabilized in water and are triggered to release active Abs upon exposure to physiological conditions.

  17. Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles

    Yuana Yuana


    Full Text Available Microbubbles-assisted ultrasound (USMB has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs. USMB was performed on a monolayer of head-and-neck cancer cells (FaDu with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm. Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

  18. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters.

    O Buß

    Full Text Available β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD, the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

  19. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters

    Dold, S. -M.; Zimmermann, S.; Hamacher, K.; Schmitz, K.; Rudat, J.


    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical Z’-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  20. Bradykinin release avoids high molecular weight kininogen endocytosis.

    Igor Z Damasceno

    Full Text Available Human H-kininogen (120 kDa plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type and CHO-745 (mutant deficient in proteoglycans biosynthesis cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular

  1. Isolation, production, purification, assay and characterization of ...

    Isolation, production, purification, assay and characterization of fibrinolytic ... are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample. ... recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp.

  2. 21 CFR 864.7250 - Erythropoietin assay.


    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass)...

  3. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    Schultz, R.M.; Nanda, S.; Altom, M.G.


    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  4. Organic Phosphorus Characterisation in Agricultural Soils by Enzyme Addition Assays

    Jarosch, Klaus; Frossard, Emmanuel; Bünemann, Else K.


    Phosphorus (P) is a non-renewable resource and it is a building block of many molecules indispensable for life. Up to 80 per cent of total soil P can be in organic form. Hydrolysability and thereby availability to plants and microorganisms differ strongly among the multitude of chemical forms of soil organic P. A recent approach to characterise organic P classes is the addition of specific enzymes which hydrolyse organic P to inorganic orthophosphate, making it detectable by colorimetry. Based on the substrate specificity of the added enzymes, conclusions about the hydrolysed forms of organic P can then be made. The aim of this study was to determine the applicability of enzyme addition assays for the characterisation of organic P species in soil:water suspensions of soils with differing properties. To this end, ten different soil samples originating from four continents, with variable pH (in water) values (4.2-8.0), land management (grassland or cropped land) and P fertilization intensity were analysed. Three different enzymes were used (acid phosphatase, nuclease and phytase). Acid phosphatase alone or in combination with nuclease was applied to determine the content of P in simple monoesters (monoester-like P) and P in DNA (DNA-like P), while P hydrolysed from myo-inositol hexakisphosphate (Ins6P-like P) was calculated from P release after incubation with phytase minus P release by acid phosphatase. To reduce sorption of inorganic P on soil particles of the suspension, especially in highly weathered soils, soil specific EDTA additions were determined in extensive pre-tests. The results of these pre-tests showed that recoveries of at least 30 per cent could be achieved in all soils. Thus, detection of even small organic P pools, such as DNA-like P, was possible in all soils if a suitable EDTA concentration was chosen. The enzyme addition assays provided information about the hydrolysable quantities of the different classes of soil organic P compounds as affected

  5. The comet assay: a heavenly method!

    Collins, Andrew R


    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey.

  6. Electrochemical Assay of Gold-Plating Solutions

    Chiodo, R.


    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  7. Variables Affecting Two Electron Transport System Assays

    Burton, G. Allen; Lanza, Guy R.


    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methan...

  8. Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016).

    Berns, Eric J; Cabezas, Maria D; Mrksich, Milan


    On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

  9. Digital Drug Dosing: Dosing in Drug Assays by Light-Defined Volumes of Hydrogels with Embedded Drug-Loaded Nanoparticles

    Faralli, Adele; Melander, Fredrik; Larsen, Esben Kjær Unmack;


    Polyethylene glycol (PEG)-based hydrogels are widely used for biomedical applications, including matrices for controlled drug release. We present a method for defining drug dosing in screening assays by light-activated cross-linking of PEG-diacrylate hydrogels with embedded drug-loaded liposome...

  10. The impact of BCG vaccination on tuberculin skin test responses in children is age dependent: evidence to be considered when screening children for tuberculosis infection.

    Seddon, James A; Paton, James; Nademi, Zohreh; Keane, Denis; Williams, Bhanu; Williams, Amanda; Welch, Steven B; Liebeschutz, Sue; Riddell, Anna; Bernatoniene, Jolanta; Patel, Sanjay; Martinez-Alier, Nuria; McMaster, Paddy; Kampmann, Beate


    Following exposure to TB, contacts are screened to target preventive treatment at those at high risk of developing TB. The UK has recently revised its recommendations for screening and now advises a 5 mm tuberculin skin test (TST) cut-off irrespective of age or BCG status. We sought to evaluate the impact of BCG on TST responses in UK children exposed to TB and the performance of different TST cut-offs to predict interferon γ release assay (IGRA) positivity. Children <15 years old were recruited from 11 sites in the UK between January 2011 and December 2014 if exposed in their home to a source case with sputum smear or culture positive TB. Demographic details were collected and TST and IGRA undertaken. The impact of BCG vaccination on TST positivity was evaluated in IGRA-negative children, as was the performance of different TST cut-offs to predict IGRA positivity. Of 422 children recruited (median age 69 months; IQR: 32-113 months), 300 (71%) had been vaccinated with BCG. BCG vaccination affected the TST response in IGRA-negative children less than 5 years old but not in older children. A 5 mm TST cut-off demonstrated good sensitivity and specificity in BCG-unvaccinated children, and an excellent negative predictive value but was associated with low specificity (62.7%; 95% CI 56.1% to 69.0%) in BCG-vaccinated children. For BCG-vaccinated children, a 10 mm cut-off provided a high negative predictive value (97.7%; 95% CI 94.2% to 99.4%) with the positive predictive value increasing with increasing age of the child. BCG vaccination had little impact on TST size in children over 5 years of age. The revised TST cut-off recommended in the recent revision to the UK TB guidelines demonstrates good sensitivity but is associated with impaired specificity in BCG-vaccinated children. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to

  11. 7 CFR 550.29 - Press releases.


    ... 7 Agriculture 6 2010-01-01 2010-01-01 false Press releases. 550.29 Section 550.29 Agriculture... Program Management § 550.29 Press releases. Press releases or other forms of public notification will be... opportunity to review, in advance, all written press releases and any other written information to be...

  12. 28 CFR 2.83 - Release planning.


    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Release planning. 2.83 Section 2.83... Release planning. (a) All grants of parole shall be conditioned on the development of a suitable release... parole date for purposes of release planning for up to 120 days without a hearing. If efforts to...

  13. Lignin based controlled release coatings

    Mulder, W.J.; Gosselink, R.J.A.; Vingerhoeds, M.H.; Harmsen, P.F.H.; Eastham, D.


    Urea is a commonly used fertilizer. Due to its high water-solubility, misuse easily leads to excess nitrogen levels in the soil. The aim of this research was to develop an economically feasible and biodegradable slow-release coating for urea. For this purpose, lignin was selected as coating material

  14. Heparin release from thermosensitive hydrogels

    Gutowska, Anna; Bae, You Han; Feijen, Jan; Kim, Sung Wan


    Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic) co

  15. Flavor Release from French Fries

    Loon, W.A.M.; Linssen, J.P.H.; Boelrijk, A.E.M.; Burgering, M.J.M.; Voragen, A.G.J.


    Flavor release from French fries was measured with the MS-NOSE using both panelists and a mouth-model system. The identity of several volatiles measured with the MS-NOSE was verified with MS-MS. The effect of frying time and the effect of adding salt on I-max (maximum intensity of compounds) and on

  16. Spatial release from informational masking

    Rakerd, Brad; Aaronson, Neil L.


    A new method for investigating spatial release from informational masking was developed and employed in two experiments. The new method is computer controlled and efficient. It employs the versatile coordinate response measure speech stimulus set [Bolia et al., J. Acoust. Soc. Am. 107, 1065 (2000)]. The experiments were conducted in an anechoic room, with a primary loudspeaker in front of the listener and a secondary loudspeaker at 60 deg to the right. Target messages were presented from the primary speaker only. For a standard, distractor messages, simultaneous with the target, were also presented from the primary speaker only. Spatial release was measured by presenting the distractors from both primary and secondary speakers with a temporal offset. Experiment 1 fixed the offset (secondary leading, +4 ms) and varied the number of distractors (1 to 3) and the target-to-distractor ratio (-12 to +4 dB). Masking release, sometimes as large as 10 dB, was found for all combinations of these variables. Experiment 2 varied the offset over a wide range of values. Substantial release from masking was found for both positive and negative offsets, but only in the range in which speech echoes are suppressed (<50 ms). [Work supported by NIDCD grant DC 00181.

  17. Lignin based controlled release coatings

    Mulder, W.J.; Gosselink, R.J.A.; Vingerhoeds, M.H.; Harmsen, P.F.H.; Eastham, D.


    Urea is a commonly used fertilizer. Due to its high water-solubility, misuse easily leads to excess nitrogen levels in the soil. The aim of this research was to develop an economically feasible and biodegradable slow-release coating for urea. For this purpose, lignin was selected as coating

  18. Lignin based controlled release coatings

    Mulder, W.J.; Gosselink, R.J.A.; Vingerhoeds, M.H.; Harmsen, P.F.H.; Eastham, D.


    Urea is a commonly used fertilizer. Due to its high water-solubility, misuse easily leads to excess nitrogen levels in the soil. The aim of this research was to develop an economically feasible and biodegradable slow-release coating for urea. For this purpose, lignin was selected as coating material

  19. Kisspeptin regulates gonadotropin-releasing hormone secretion in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats

    Haogang Xue; Chunying Yang; Xiaodong Ge; Weiqi Sun; Chun Li; Mingyu Qi


    Kisspeptin is essential for activation of the hypothalamo-pituitary-gonadal axis. In this study, we established gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. Rats were injected with 1, 10, or 100 pM kisspeptin-10, a peptide derived from full-length kisspeptin, into the arcuate nucleus and medial preoptic area, and with the kisspeptin antagonist peptide 234 into the lateral cerebral ventricle. The results of immunohistochemical staining revealed that pulsatile luteinizing hormone secretion was suppressed after injection of antagonist peptide 234 into the lateral cerebral ventricle, and a significant increase in luteinizing hormone level was observed after kisspeptin-10 injection into the arcuate nucleus and medial preoptic area. The results of an enzyme-linked immunosorbent assay showed that luteinizing hormone levels during the first hour of kisspeptin-10 infusion into the arcuate nucleus were significantly greater in the 100 pM kisspeptin-10 group than in the 10 pM kisspeptin-10 group. These findings indicate that kisspeptin directly promotes gonadotropin-releasing hormone secretion and luteinizing hormone release in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. The arcuate nucleus is a key component of the kisspeptin-G protein-coupled receptor 54 signaling pathway underlying regulating luteinizing hormone pulse secretion.

  20. Modelling and simulations of controlled release fertilizer

    Irfan, Sayed Ameenuddin; Razali, Radzuan; Shaari, Ku Zilati Ku; Mansor, Nurlidia


    The recent advancement in controlled release fertilizer has provided an alternative solution to the conventional urea, controlled release fertilizer has a good plant nutrient uptake they are environment friendly. To have an optimum plant intake of nutrients from controlled release fertilizer it is very essential to understand the release characteristics. A mathematical model is developed to predict the release characteristics from polymer coated granule. Numerical simulations are performed by varying the parameters radius of granule, soil water content and soil porosity to study their effect on fertilizer release. Understanding these parameters helps in the better design and improve the efficiency of controlled release fertilizer.

  1. Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

    Maeda, Akihisa; Takahashi, Kei; Tsuchiyama, Hiromi; Oshida, Keiyu


    The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products.

    Randlev, Britta; Huang, Li-chun; Watatsu, Mitsuko; Marcus, Matthew; Lin, Andy; Shih, Shian-Jiun


    GVAX immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

  3. Controlling variation in the comet assay

    Andrew Richard Collins


    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  4. Cell Culture Assay for Human Noroviruses [response

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.


    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  5. A perifusion method for examining arginine vasopressin (AVP release from hypothalamo-neurohypophyseal system.



    Full Text Available A perifusion method has been developed using rat hypothalamo-neurohypophyseal system (HNS or neural lobe to investigate the control mechanism of arginine vasopressin (AVP release. A specific radioimmunoassay (RIA for AVP was developed to measure AVP in perifusion medium employing anti-AVP serum which was obtained by immunizing rabbits. At a final dilution of 1/12,000, the antiserum showed less than 0.66 and 0.01% cross reactivity with lysine-vasopressin and oxytocin, respectively. But it did not cross reacted with other peptide hormones. The lowest detectable level of vasopressin was 0.5 pg/tube. The intra-assay coefficient of variation averaged 10.4%. The dilution curve of perifused medium was well paralled to the standard curve of AVP assay. AVP release from HNS or neural lobe gradually declined to the stable level in 90-120 min after the initiation of perifusion. Good repeatability of the AVP release from neural lobe was recognized by repeated stimulation with 10 min perifusion of 60 mM KCl at every 60 min. HNS released AVP in dose related manner to the osmotic challenge of sodium or glucose, and AVP release was stimulated from HNS by prostaglandin E2, but not by dopamine. These results show that the perifusion methods using AVP-RIA is a useful method to examine the AVP release from HNS or neural lobe.

  6. Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

    Gnavi, S; di Blasio, L; Tonda-Turo, C; Mancardi, A; Primo, L; Ciardelli, G; Gambarotta, G; Geuna, S; Perroteau, I


    Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  7. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Bas-jan Van Der Leede


    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  8. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    Gibbs, Susan, E-mail: [Department of Dermatology, VU University Medical Centre, Dept of Oral Cell Biology, ACTA, Amsterdam (Netherlands); Corsini, Emanuela [Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano (Italy); Spiekstra, Sander W. [Department of Dermatology, VU University Medical Centre, Dept of Oral Cell Biology, ACTA, Amsterdam (Netherlands); Galbiati, Valentina [Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano (Italy); Fuchs, Horst W. [CellSystems GmbH, Troisdorf (Germany); DeGeorge, George; Troese, Matthew [MB Research Labs, Spinnerstown, PA (United States); Hayden, Patrick; Deng, Wei [MatTek Corporation, Ashland, MA (United States); Roggen, Erwin [3Rs Management and Consultancy (Denmark)


    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24 h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC{sub 50}) with human and animal data showed a superior correlation with human DSA{sub 05} (μg/cm{sup 2}) data (Spearman r = 0.8500; P value (two-tailed) = 0.0061) compared to LLNA data (Spearman r = 0.5968; P value (two-tailed) = 0.0542). DSA{sub 05} = induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA{sub 05} data (Spearman r = 0.8333; P value (two-tailed) = 0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model. - Highlights: • A potential epidermal equivalent assay to label and classify sensitizers • Il-18 release distinguishes sensitizers from non sensitizers • IL-18 release can rank sensitizer potency

  9. Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.

    Chung, Shan; Nguyen, Van; Lin, Yuwen Linda; Kamen, Lynn; Song, An


    In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., (51)Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)


    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  11. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J; Meyer, Matthew R.; Mesri, Mehdi; Abbatiello, Susan E; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J. C.; Fenyö, David; Hiltke, Tara; Ketchum, Karen A.; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C.; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D.; Thomas, Stefani; Townsend, R. Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G


    Summary The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

  12. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.


    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  13. 14 CFR 125.373 - Original flight release or amendment of flight release.


    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Original flight release or amendment of flight release. 125.373 Section 125.373 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT... Flight Release Rules § 125.373 Original flight release or amendment of flight release. (a) A...

  14. Microwave Activation of Drug Release

    Jónasson, Sævar Þór

    Due to current limitations in control of pharmaceutical drug release in the body along with increasing medicine use, methods of externally-controlled drug release are of high interest. In this thesis, the use of microwaves is proposed as a technique with the purpose of externally activating...... setup, called the microwave activation system has been developed and tested on a body phantom that emulates the human torso. The system presented in this thesis, operates unobtrusively, i.e. without physically interfering with the target (patient). The torso phantom is a simple dual-layered cylindrical...... the phantom is of interest for disclosing essential information about the limitations of the concept, the phantom and the system. For these purposes, a twofold operation of the microwave activation system was performed, which are reciprocal of each other. In the first operation phase, named mapping...

  15. Zegerid--immediate-release omeprazole.


    The FDA has approved marketing of Zegerid powder for oral suspension (Santarus), an immediate-release formulation of the proton-pump inhibitor (PPI) omeprazole (Prilosec, and others). All other oral PPIs are delayed-release, enteric-coated formulations designed to prevent degradation of the drug by gastric acid. Each 20- or 40-mg packet of Zegerid contains 1680 mg sodium bicarbonate, which protects the drug from gastric acid degradation. A dose of Zegerid contains 460 mg of sodium, which may be excessive for some patients. Zegerid is the first oral PPI to be approved by the FDA for reduction of risk of upper GI bleeding in critically ill patients. The drug may be useful for patients who are unable to swallow and have nasogastric (NG) tubes in place. Zegerid cost $70.00 for 14 days' treatment, compared to less than $10 for 14 tablets of Prilosec OTC.

  16. Neutron-absorber release device

    VAN Erp, Jan B.; Kimont, Edward L.


    A resettable device is provided for supporting an object, sensing when an environment reaches a critical temperature and releasing the object when the critical temperature is reached. It includes a flexible container having a material inside with a melting point at the critical temperature. The object's weight is supported by the solid material which gives rigidity to the container until the critical temperature is reached at which point the material in the container melts. The flexible container with the now fluid material inside has insufficient strength to support the object which is thereby released. Biasing means forces the container back to its original shape so that when the temperature falls below the melting temperature the material again solidifies, and the object may again be supported by the device.

  17. Comet assay on mice testicular cells

    Anoop Kumar Sharma


    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  18. Research highlights: digital assays on chip.

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino


    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  19. China's First Luxury Brands Released

    Richard Zhu


    @@ China now has its own luxury brands.The four brands Feitian (Moutai spirit), Yong-Qi Zitan (furniture), Rongbaozhai (works of art) and Dongding Oolong (tea) were released as the first Chinese luxury brands by the China Luxury Institute on Nov.11.Based on the Institute's investigation of over 500 enterprises and brands within over 30 kinds of industries like garments, tea, wine, china and jade, in combination with market questionnaire, the first four brands have made their debut.

  20. Real-time imaging of renin release in vitro.

    Peti-Peterdi, János; Fintha, Attila; Fuson, Amanda L; Tousson, Albert; Chow, Robert H


    Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 microM isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release.

  1. Cortisol release in response to UVB exposure in Xiphophorus fish.

    Contreras, Adam J; Boswell, Mikki; Downs, Kevin P; Pasquali, Amanda; Walter, Ronald B


    Xiphophorus fishes are comprised of 26 known species. Interspecies hybridization between select species has been utilized to produce experimental models to study melanoma development. Xiphophorus melanoma induction protocols utilize ultraviolet light (UVB) to induce DNA damage and associated downstream tumorigenesis. However, the impact of induced stress caused by the UVB treatment of the experimental animals undergoing tumor induction protocols has not been assessed. Stress is an adaptive physiological response to excessive or unpredictable environmental stimuli. The stress response in fishes may be measured by an assay of cortisol released into the water. Here, we present results from investigations of stress response during an experimental treatment and UVB exposure in Xiphophorus maculatus Jp 163 B, Xiphophorus couchianus, and F1 interspecies hybrids produced from the mating X. maculatus Jp 163 B×X. couchianus. Overall, cortisol release rates for males and females after UVB exposure showed no statistical differences. At lower UVB doses (8 and 16kJ/m(2)), X. couchianus exhibited 2 fold higher levels of DNA damage then either X. maculatus or the F1 hybrid. However, based on the cortisol release rates, none of the fish types tested induced a primary stress response at the UVB lower doses (8 and 16kJ/m(2)). In contrast, at a very high UVB dose (32kJ/m(2)) both X. maculatus and the F1 hybrid showed a 5 fold increase in the cortisol release rate. To determine the effect of pigmentation on UVB induced stress, wild type and albino Xiphophorus hellerii were exposed to UVB (32kJ/m(2)). Albino X. hellerii exhibited 3.7 fold increase in the cortisol release while wild type X. hellerii did not exhibit a significant cortisol response to UVB. Overall, the data suggest the rather low UVB doses often employed in tumor induction protocols do not induce a primary stress response in Xiphophorus fishes.

  2. Hypoxic enhancement of exosome release by breast cancer cells

    King Hamish W


    Full Text Available Abstract Background Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. Methods Breast cancer cell lines were cultured under either moderate (1% O2 or severe (0.1% O2 hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of  Results Exposure of three different breast cancer cell lines to moderate (1% O2 and severe (0.1% O2 hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. Conclusions These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic

  3. Matters of life and death. How neutrophils die or survive along NET release and is "NETosis" = necroptosis?

    Desai, Jyaysi; Mulay, Shrikant R; Nakazawa, Daigo; Anders, Hans-Joachim


    Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders.

  4. Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies.

    Wang, Lan; Xu, Gang-Ling; Gao, Kai; Wilkinson, Jennifer; Zhang, Feng; Yu, Lei; Liu, Chun-Yu; Yu, Chuan-Fei; Wang, Wen-Bo; Li, Meng; Chen, Wei; Fan, Frank; Cong, Mei; Wang, Jun-Zhi


    Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics. Copyright © 2016. Published by Elsevier B.V.

  5. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.


    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  6. EDTA interference in electrochemiluminescence ACTH assay.

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer


    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  7. Comet assay on tetraploid yeast cells

    Rank, Jette; Syberg, Kristian; Jensen, Klara


    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  8. Fungicide resistance assays for fungal plant pathogens.

    Secor, Gary A; Rivera, Viviana V


    Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

  9. Detection of Eperythrozoon wenyoni by PCR assay

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO


    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  10. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    Dozier, Jonathan K; Distefano, Mark D


    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  11. Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

    陈宝玉; 郭勤; 郭智; 王希成


    This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid-reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N-phospho-L-arginine (PArg) formed in the forward catalysis reaction.The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized.For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L)-1 · cm-1 for the phosphate.Standard curves for phosphate show a good linearity of 0.999.Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies.This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

  12. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page:

  13. A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.

    Kraaij, Tineke; Tengström, Fredrik C; Kamerling, Sylvia W A; Pusey, Charles D; Scherer, H Ulrich; Toes, Rene E M; Rabelink, Ton J; van Kooten, Cees; Teng, Y K Onno


    A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.

  14. Hydrolytic conversion of amorphous calcium phosphate into apatite accompanied by sustained calcium and orthophosphate ions release.

    Niu, Xufeng; Chen, Siqian; Tian, Feng; Wang, Lizhen; Feng, Qingling; Fan, Yubo


    The aim of this study is to investigate the calcium and orthophosphate ions release during the transformation of amorphous calcium phosphate (ACP) to hydroxyapatite (HA) in aqueous solution. The ACP is prepared by a wet chemical method and further immersed in the distilled water for various time points till 14d. The release of calcium and orthophosphate ions is measured with calcium and phosphate colorimetric assay kits, respectively. The transition of ACP towards HA is detected by x-ray diffraction (XRD), transmission electron microscopy (TEM), and fourier transform infrared spectroscopy (FTIR). The results indicate that the morphological conversion of ACP to HA occurs within the first 9h, whereas the calcium and orthophosphate ions releases last for over 7d. Such sustained calcium and orthophosphate ions release is very useful for ACP as a candidate material for hard tissue regeneration.

  15. The Effect of Ethanol on the Release of Opioids from Oral Prolonged-Release Preparations

    Walden, Malcolm; Nicholls, Fiona A.; Smith, Kevin J.; Tucker, Geoffrey T


    Recent experience has prompted the US FDA to consider whether ethanol ingestion may modify the release characteristics of prolonged-release formulations, where dose dumping may be an issue for patient safety. The influence of ethanol on the in vitro release of opioid drugs from some prolonged-release formulations utilizing different release technologies was examined. Results indicated that the prolonged-release mechanisms remained intact under the testing conditions, although one product show...

  16. Toxic Release Inventory Chemicals by Groupings

    U.S. Environmental Protection Agency — The Toxics Release Inventory (TRI) makes available information for more than 600 toxic chemicals that are being used, manufactured, treated, transported, or released...

  17. Section 9: Ground Water - Likelihood of Release

    HRS training. the ground water pathway likelihood of release factor category reflects the likelihood that there has been, or will be, a release of hazardous substances in any of the aquifers underlying the site.

  18. Drugs and brain death: drug assay perspectives.

    Morris, R G


    The ability to make any meaningful interpretation of a drug assay result is very dependent upon a knowledge of the limitations of the method(s) used (sensitivity, specificity etc.), and the concentration that may be measured in plasma and its relationship to CNS effects. We need more information about 'critical' concentrations for each drug and sedation in the setting of the brain-injured patient before meaningful interpretation can be applied to such data. While the above discussion is critical of screen-type assays, the alternative specific assays are not easily provided for, as obviously the resourcing of laboratories to be able to deliver such specialized services for a range of therapeutic drugs, in addition to 'social' drugs or other toxins (e.g. glues, pesticides, solvents, environmental substances etc), becomes an increasingly complex issue in the current economic climate. Hence, the analytical laboratory can offer valuable support to the clinical team however, the interpretation of such results must be assessed in the light of many limitations of such assay methods and not seen as the 'gold standard' for assessment of brain function.

  19. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W


    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  20. Endoproteolytic activity assay in malting barley

    Blanca Gómez Guerrero


    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  1. Comet assay on mice testicular cells

    Sharma, Anoop Kumar


    for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity...

  2. In vitro solubility assays in drug discovery.

    Kerns, Edward H; Di, Li; Carter, Guy T


    The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.

  3. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Fletcher, James T.; Boriraj, Grit


    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  4. Mars Express releases Beagle 2


    At 9:31 CET, the crucial sequence started to separate the Beagle 2 lander from Mars Express. As data from Mars Express confirm, the pyrotechnic device was fired to slowly release a loaded spring, which gently pushed Beagle 2 away from the mother spacecraft. An image from the onboard visual monitoring camera (VMC) showing the lander drifting away is expected to be available later today. Since the Beagle 2 lander has no propulsion system of its own, it had to be put on the correct course for its descent before it was released. For this reason, on 16 December the trajectory of the whole Mars Express spacecraft had to be adjusted to ensure that Beagle 2 would be on course to enter the atmosphere of Mars. This manoeuvre, called "retargeting'' was critical: if the entry angle is too steep, the lander could overheat and burn up in the atmosphere; if the angle is too shallow, the lander might skim like a pebble on the surface of a lake and miss its target. This fine targeting and today's release were crucial manoeuvres for which ESA's Ground Control Team at ESOC (European Space Operations Centre) had trained over the past several months. The next major milestone for Mars Express will be the manoeuvre to enter into orbit around Mars. This will happen at 3:52 CET on Christmas morning, when Beagle 2 is expected to land on the surface of Mars. "Good teamwork by everybody - ESA, industry and the Beagle 2 team - has got one more critical step accomplished. Mars, here comes Europe!" said David Southwood, ESA Director of Science.

  5. DVD With High Gapacity Released


    After two years of research and development, the Center for International Cultural Exchange under the Ministry of Culture of China has recently released a new type of DVD which is double-faced, double-system and bears ten languages. Teststatistics show that all its indicators come up withthose of its foreign counterparts. Now it has beenput into mass production.This new type of DVD features the following performances. It is a high-clarity one, with the horizontal clarity more than 500. It has a superb sound effect, capable of recording various stereo signals such as DOLBY DIGITAL and DTS. It can record dubbing in eight languages and subtitles

  6. Production and assay of forskolin antibodies

    Ho, L.T.; Ho, R.J.


    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  7. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina


    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  8. Individualized optimal release angles in discus throwing.

    Leigh, Steve; Liu, Hui; Hubbard, Mont; Yu, Bing


    The purpose of this study was to determine individualized optimal release angles for elite discus throwers. Three-dimensional coordinate data were obtained for at least 10 competitive trials for each subject. Regression relationships between release speed and release angle, and between aerodynamic distance and release angle were determined for each subject. These relationships were linear with subject-specific characteristics. The subject-specific relationships between release speed and release angle may be due to subjects' technical and physical characteristics. The subject-specific relationships between aerodynamic distance and release angle may be due to interactions between the release angle, the angle of attack, and the aerodynamic distance. Optimal release angles were estimated for each subject using the regression relationships and equations of projectile motion. The estimated optimal release angle was different for different subjects, and ranged from 35 degrees to 44 degrees . The results of this study demonstrate that the optimal release angle for discus throwing is thrower-specific. The release angles used by elite discus throwers in competition are not necessarily optimal for all discus throwers, or even themselves. The results of this study provide significant information for understanding the biomechanics of discus throwing techniques.

  9. 28 CFR 2.33 - Release plans.


    ... Administration DEPARTMENT OF JUSTICE PAROLE, RELEASE, SUPERVISION AND RECOMMITMENT OF PRISONERS, YOUTH OFFENDERS, AND JUVENILE DELINQUENTS United States Code Prisoners and Parolees § 2.33 Release plans. (a) A grant of parole is conditioned upon the approval of release plans by the Regional Commissioner. In general...

  10. Prisoners' Perspectives on Strategies for Release.

    Muhammad, Mika'il A.


    Examines questions regarding the perceptions of New York State prisoners (N=263), their chances of an early release, and their strategies to expedite the release process. Findings suggest that inmates view all activities thought to expedite release as important. Many prisoners wished to eliminate the "game playing" thought associated…

  11. Data release of the LAMOST pilot survey

    A-Li Luo; Jian-Nan Zhang; Shi-Hai Yang; Zheng-Qiu Yao; Yong Yu; Hui Yuan; Chao Zhai; En-Peng Zhang; Jing Zhang; Li-Ping Zhang; Wei Zhang; Yong Zhang; Zhong-Rui Bai; Zhen-Chao Zhang; Ming Zhao; Fang Zhou; Yong-Tian Zhu; Jie Zhu; Si-Cheng Zou; Xiao-Yan Chen; Feng-Fei Wang; Yan-Xin Guo; Jian-Jun Chen; Bing Du; Xiao Kong; Ya-Juan Lei; Yin-Bi Li; Hao-Tong Zhang; Yi-Han Song; Yue Wu; Yan-Xia Zhang; Xin-Lin Zhou; Fang Zuo; Peng Du; Lin He; Wen Hou; Yi-Qiao Dong; Jian Li; Yong-Heng Zhao; Guang-Wei Li; Shuang Li; Jing Song; Yuan Tian; Meng-Xin Wang; Ke-Fei Wu; Hui-Qin Yang; Hai-Long Yuan; Shu-Yun Cao; Hai-Yuan Chen; Gang Zhao; Kun-Xin Chen; Ying Chen; Jia-Ru Chu; Lei Feng; Xue-Fei Gong; Bo-Zhong Gu; Yong-Hui Hou; Zhi-Ying Huo; Hong-Zhuan Hu; Ning-Sheng Hu; Xiang-Qun Cui; Zhong-Wen Hu; Lei Jia; Fang-Hua Jiang; Xiang Jiang; Zi-Bo Jiang; Ge Jin; Ai-Hua Li; Qi Li; Xin-Nan Li; Yan Li; Guo-Ping Li; Ye-Ping Li; Gen-Rong Liu; Guan-Qun Liu; Zhi-Gang Liu; Qi-Shuai Lu; Wen-Zhi Lu; Yu Luo; Yin-Dun Mao; Li Men; Ji-Jun Ni; Yao-Quan Chu; Yong-Jun Qi; Zhao-Xiang Qi; Huo-Ming Shi; Ding-Qiang Su; Shi-Wei Sun; Hong-Jun Su; Zheng-Hong Tang; Qing-Sheng Tao; Liang-Ping Tu; Da-Qing Wang; Jian-Rong Shi; Dan Wang; Guo-Min Wang; Hai Wang; Jia-Ning Wang; Jian Wang; Jian-Ling Wang; Jian-Ping Wang; Lei Wang; Shou-Guan Wang; Shu-Qing Wang; Gang Wang; Ya-Nan Wang; You Wang; Yue-Fei Wang; Ming-Zhi Wei; Xiang-Xiang Xue; Xiao-Zheng Xing; Ling-Zhe Xu; Xin-Qi Xu; Yan Xu; De-Hua Yang


    This paper describes the data release of the LAMOST pilot survey,which includes data reduction,calibration,spectral analysis,data products and data access.The accuracy of the released data and the information about the FITS headers of spectra are also introduced.The released data set includes 319 000 spectra and a catalog of these objects.

  12. Nuclear energy release from fragmentation

    Li, Cheng [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Souza, S.R. [Instituto de Física, Universidade Federal do Rio de Janeiro Cidade Universitária, Caixa Postal 68528, 21945-970 Rio de Janeiro (Brazil); Tsang, M.B. [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); National Superconducting Cyclotron Laboratory and Physics and Astronomy Department, Michigan State University, East Lansing, MI 48824 (United States); Zhang, Feng-Shou, E-mail: [The Key Laboratory of Beam Technology and Material Modification of Ministry of Education, College of Nuclear Science and Technology, Beijing Normal University, Beijing 100875 (China); Beijing Radiation Center, Beijing 100875 (China); Center of Theoretical Nuclear Physics, National Laboratory of Heavy Ion Accelerator of Lanzhou, Lanzhou 730000 (China)


    It is well known that binary fission occurs with positive energy gain. In this article we examine the energetics of splitting uranium and thorium isotopes into various numbers of fragments (from two to eight) with nearly equal size. We find that the energy released by splitting {sup 230,232}Th and {sup 235,238}U into three equal size fragments is largest. The statistical multifragmentation model (SMM) is applied to calculate the probability of different breakup channels for excited nuclei. By weighing the probability distributions of fragment multiplicity at different excitation energies, we find the peaks of energy release for {sup 230,232}Th and {sup 235,238}U are around 0.7–0.75 MeV/u at excitation energy between 1.2 and 2 MeV/u in the primary breakup process. Taking into account the secondary de-excitation processes of primary fragments with the GEMINI code, these energy peaks fall to about 0.45 MeV/u.

  13. Nuclear energy release from fragmentation

    Li, Cheng; Souza, S. R.; Tsang, M. B.; Zhang, Feng-Shou


    It is well known that binary fission occurs with positive energy gain. In this article we examine the energetics of splitting uranium and thorium isotopes into various numbers of fragments (from two to eight) with nearly equal size. We find that the energy released by splitting 230,232Th and 235,238U into three equal size fragments is largest. The statistical multifragmentation model (SMM) is applied to calculate the probability of different breakup channels for excited nuclei. By weighing the probability distributions of fragment multiplicity at different excitation energies, we find the peaks of energy release for 230,232Th and 235,238U are around 0.7-0.75 MeV/u at excitation energy between 1.2 and 2 MeV/u in the primary breakup process. Taking into account the secondary de-excitation processes of primary fragments with the GEMINI code, these energy peaks fall to about 0.45 MeV/u.

  14. Nanoparticle release from dental composites.

    Van Landuyt, K L; Hellack, B; Van Meerbeek, B; Peumans, M; Hoet, P; Wiemann, M; Kuhlbusch, T A J; Asbach, C


    Dental composites typically contain high amounts (up to 60 vol.%) of nanosized filler particles. There is a current concern that dental personnel (and patients) may inhale nanosized dust particles (composite dust was analyzed in real work conditions. Exposure measurements of dust in a dental clinic revealed high peak concentrations of nanoparticles in the breathing zone of both dentist and patient, especially during aesthetic treatments or treatments of worn teeth with composite build-ups. Further laboratory assessment confirmed that all tested composites released very high concentrations of airborne particles in the nanorange (>10(6)cm(-3)). The median diameter of airborne composite dust varied between 38 and 70 nm. Electron microscopic and energy dispersive X-ray analysis confirmed that the airborne particles originated from the composite, and revealed that the dust particles consisted of filler particles or resin or both. Though composite dust exhibited no significant oxidative reactivity, more toxicological research is needed. To conclude, on manipulation with the bur, dental composites release high concentrations of nanoparticles that may enter deeply into the lungs. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  16. Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

    Bausinger, Julia; Schütz, Petra; Piberger, Ann Liza; Speit, Günter


    The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the

  17. Quantification of cerivastatin toxicity supports organismal performance assays as an effective tool during pharmaceutical safety assessment.

    Gaukler, Shannon M; Ruff, James S; Galland, Tessa; Underwood, Tristan K; Kandaris, Kirstie A; Liu, Nicole M; Morrison, Linda C; Veranth, John M; Potts, Wayne K


    A major problem in pharmaceutical development is that adverse effects remain undetected during preclinical and clinical trials, but are later revealed after market release when prescribed to many patients. We have developed a fitness assay known as the organismal performance assay (OPA), which evaluates individual performance by utilizing outbred wild mice (Mus musculus) that are assigned to an exposed or control group, which compete against each other for resources within semi-natural enclosures. Performance measurements included reproductive success, survival, and male competitive ability. Our aim was to utilize cerivastatin (Baycol(®), Bayer), a pharmaceutical with known adverse effects, as a positive control to assess OPAs as a potential tool for evaluating the safety of compounds during preclinical trials. Mice were exposed to cerivastatin (~4.5 mg/kg/day) into early adulthood. Exposure ceased and animals were released into semi-natural enclosures. Within enclosures, cerivastatin-exposed females had 25% fewer offspring and cerivastatin-exposed males had 10% less body mass, occupied 63% fewer territories, sired 41% fewer offspring, and experienced a threefold increase in mortality when compared to controls. OPAs detected several cerivastatin-induced adverse effects indicating that fitness assays, commonly used in ecology and evolutionary biology, could be useful as an additional tool in safety testing during pharmaceutical development.

  18. Analytical and assay issues for use of cardiac troponin testing for risk stratification in primary care.

    Wu, Alan H B; Christenson, Robert H


    Cardiac troponin is the standard marker for diagnosis of acute myocardial infarction and risk stratification of patients who present to an emergency department with signs and symptoms of acute cardiac ischemia. Over the past few years, the analytical sensitivity of assays for cardiac troponin has improved significantly to the point where a detectable amount of troponin can be measured in essentially all healthy subjects. Recent studies have shown that use of a highly sensitive troponin assays may provide value to traditional markers of primary disease risk for patients, i.e., for those who have no history of heart disease. There are barriers to the adoption of cardiac troponin for screening high risk cohorts such as the elderly, diabetics and perhaps even the asymptomatic population. Strategies used for the assignment of cutoff concentrations in acute care, i.e., the 99 th percentile, may not be appropriate for primary care as changes over baseline levels may provide more accurate information of risk than cross-sectional results. A review of biological variation has shown that cardiac troponin as a biomarker has low index of individuality, indicating that reference values are of little utility. Whether or not cardiac troponin can be released in reversible injury is a debate that could have significance for detecting minor myocardial injury. A major hurdle for use of troponin in primary care is the lack of assay standardization and nomenclature for the different generations of troponin assays. Standardization requires knowledge of what is released after cardiac injury and what the various cardiac troponin assays are measuring. Currently it is not clear if the cardiac troponin release after ischemic injury is identical to that in circulation of healthy individuals. This may affect the design of future assays and standardization approaches. There is potential that a marker of myocardial injury such as troponin can add to the value of existing indicators and biomarkers

  19. Formulation and evaluation of ranolazine extended release tablets: Influence of polymers

    TEGK Murthy


    Full Text Available An extended release tablet provides prolonged periods of drug in plasma levels thereby reduce dosing frequency, improve patient compliance and reduce the dose-related side effects. Ranolazine is indicated for the chronic treatment of angina in patients who have not achieved an adequate response with other anti-anginal agents. The present investigation was undertaken to design extended release tablets of Ranolazine employing hypromellose phthalate grade HP-55, ethocel standard 7FP premium ethyl cellulose, Surelease E-7-19040, Klucel HF pharm and Natrosol Type 250 HHX as matrix forming agents using wet granulation method. Formulated tablets were evaluated for uniformity of weight, assay, water content, in vitro drug release studies and stability studies. The drug release followed first order kinetics with both erosion and diffusion as the release mechanism. It is concluded that the desired drug release pattern can be obtained by using natrosol type 250 HHX compared to other polymers. The similarity factor (f2 was calculated to select best formulation by comparing in vitro dissolution data of the commercial formulation Ranexa® . The formulated tablets fulfilled the compendia requirements. The formulated Ranolazine Extended release tablets were found to be stable.

  20. Arg-Phe-amide-related peptides influence gonadotropin-releasing hormone neurons

    Haluk Kelestimur; Emine Kacar; Aysegul Uzun; Mete Ozcan; Selim Kutlu


    The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1–7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels (at 120 minutes of exposure) and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.

  1. Liquid crystalline systems for transdermal delivery of celecoxib: in vitro drug release and skin permeation studies.

    Estracanholli, Eder André; Praça, Fabíola Silva Garcia; Cintra, Ana Beatriz; Pierre, Maria Bernadete Riemma; Lara, Marilisa Guimarães


    Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.

  2. Hydrophobic Drug-Loaded PEGylated Magnetic Liposomes for Drug-Controlled Release

    Hardiansyah, Andri; Yang, Ming-Chien; Liu, Ting-Yu; Kuo, Chih-Yu; Huang, Li-Ying; Chan, Tzu-Yi


    Less targeted and limited solubility of hydrophobic-based drug are one of the serious obstacles in drug delivery system. Thus, new strategies to enhance the solubility of hydrophobic drug and controlled release behaviors would be developed. Herein, curcumin, a model of hydrophobic drug, has been loaded into PEGylated magnetic liposomes as a drug carrier platform for drug controlled release system. Inductive magnetic heating (hyperthermia)-stimulated drug release, in vitro cellular cytotoxicity assay of curcumin-loaded PEGylated magnetic liposomes and cellular internalization-induced by magnetic guidance would be investigated. The resultant of drug carriers could disperse homogeneously in aqueous solution, showing a superparamagnetic characteristic and could inductive magnetic heating with external high-frequency magnetic field (HFMF). In vitro curcumin release studies confirmed that the drug carriers exhibited no significant release at 37 °C, whereas exhibited rapid releasing at 45 °C. However, it would display enormous (three times higher) curcumin releasing under the HFMF exposure, compared with that without HFMF exposure at 45 °C. In vitro cytotoxicity test shows that curcumin-loaded PEGylated magnetic liposomes could efficiently kill MCF-7 cells in parallel with increasing curcumin concentration. Fluorescence microscopy observed that these drug carriers could internalize efficiently into the cellular compartment of MCF-7 cells. Thus, it would be anticipated that the novel hydrophobic drug-loaded PEGylated magnetic liposomes in combination with inductive magnetic heating are promising to apply in the combination of chemotherapy and thermotherapy for cancer therapy.

  3. Release of segregated nuclides from spent fuel

    Johnson, L.H.; Tait, J.C. [Atomic Energy Canada Ltd., Pinawa, MB (Canada). Whiteshell Laboratories


    The potential release of fission and activation products from spent nuclear fuel into groundwater after container failure in the Swedish deep repository is discussed. Data from studies of fission gas release from representative Swedish BWR fuel are used to estimate the average fission gas release for the spent fuel population. Information from a variety of leaching studies on LWR and CANDU fuel are then reviewed as a basis for estimating the fraction of the inventory of key radionuclides that could be released preferentially (the Instant Release Fraction of IRF) upon failure of the fuel cladding. The uncertainties associated with these estimates are discussed. 33 refs, 6 figs, 3 tabs.

  4. Flouride release from various restorative materials.

    Bala, O; Uçtaşli, M; Can, H; Türköz, E; Can, M


    Fluoride release from six light-activated restorative materials, including two resinmodified glass-ionomers, two composites, and two compomers, was evaluated and compared with one conventional acid-based glass-ionomer cement. The amount and rate of release varied among the tested materials. Both resin-modified glass-ionomers and the conventional acid-base glass-ionomer cements released more fluoride then the composites and compomers (p < 0.05). Additionally, composite materials released less fluoride than compomer materials (p < 0.05). Release of fluoride by the tested materials showed a significant decrease after all the tested time intervals.

  5. Control of noradrenaline release from hippocampal synaptosomes

    West, D.P.; Fillenz, M.


    Potassium-evoked tritiated noradrenaline (NA) release from hippocampal synaptosomes was measured with a superfusion method. A single 2-min high-K+ pulse released 39% of the vesicular NA by a Ca2+-dependent mechanism: the Ca2+-independent release was negligible. After changing the vesicular NA store size by pretreating rats with either alpha-methyl-para-tyrosine, 500 mg/kg, or tranylcypromine, 10 mg/kg, a single K+ pulse released a constant percentage of the vesicular NA. With two K+ pulses, however, there was a reduction in the percentage of vesicular NA released in response to the second pulse.

  6. Beta-tricalcium phosphate release from brushite cement surface.

    Alkhraisat, M Hamdan; Mariño, F Tamimi; Retama, J Rubio; Jerez, L Blanco; López-Cabarcos, E


    Different in vivo studies demonstrated that brushite cements are biocompatible, bioresorbable, and osteoconductive. However, the decay of brushite cements has been scarcely studied even though it may be of great concern for clinical applications in highly blood-perfused regions. This work was elaborated to elucidate factors that determine brushite cement surface disintegration. For that, brushite cements were modified using in their preparation different aqueous solutions of phosphoric, glycolic, tartaric, and citric acids in concentrations that were reported to improve the cement properties. Two-viscosity enhancing polysaccharides, chondroitin-4 sulfate and hyaluronic acid, were also assayed. Thereafter, pre- and set cement samples were immersed in distilled water for 24 h. The cement-solid weight loss, microstructure, liquid phase viscosity, mean size of the released particles, and zeta potential were analyzed using X-ray diffraction, FTIR spectroscopy, light scattering, scanning electron microscopy and optical microscopy. It was found that the particles released from the cement surface were beta-TCP, and their amount depends on the carboxylic acid used in the preparation of the cement. The addition of hyaluronic acid and chondroitin-4 sulfate decreased the amount of released particles from the surface of the set brushite cement made with citric acid. Furthermore, the hyaluronic acid increased significantly the viscosity of the citric acid solution and the cement paste prepared with this liquid phase showed a pronounced step down in particle release. In this study, we showed that the water solubility of calcium carboxylate and the viscosity of mixing liquid may dictate the superficial disintegration of brushite cements.

  7. Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes.

    Hamid, Omar; Pensch, Manuela; Agis, Hermann


    Collagen barrier membranes are used in guided tissue regeneration to support healing. This strategy, however, relies on the healing capacity of the tissue. Pharmacological inhibitors of prolyl hydroxylases can support regeneration by enhancing angiogenesis and are therefore a promising tool for periodontology. Here we evaluate the release kinetics of the prolyl hydroxylase inhibitors dimethyloxalylglycine and L-mimosine from collagen barrier membranes. Dimethyloxalylglycine and L-mimosine were lyophilized onto the collagen barrier membranes. The morphology of the collagen barrier membranes was analysed using scanning electron microscopy. The release of prolyl hydroxylase inhibitors was assessed by colorimetric and spectroscopic methods. Their ability to induce a cellular response was assessed in bioassays with gingival and periodontal ligament fibroblasts based on vascular endothelial growth factor production, proliferation, and metabolic activity of the cells. We found that loading of collagen barrier membranes with prolyl hydroxylase inhibitors did not change the overall membrane morphology. Assessment of the release kinetics by direct measurements and based on vascular endothelial growth factor production showed that supernatants obtained from the collagen barrier membranes in the first 6 hours had a sufficient level of prolyl hydroxylase inhibitors to induce vascular endothelial growth factor production. A similar kinetic was found when cell proliferation was assessed. Changes in metabolic activity did not reach the level of significance in the MTT assay. In conclusion, collagen barrier membranes can release prolyl hydroxylase inhibitors thereby increasing the pro-angiogenic capacity of periodontal cells in vitro. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontology and oral surgery.

  8. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader


    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  9. Lump corrections for radioactive waste assay.

    Miller, T J


    Previous studies have shown that automated radioactive waste assay techniques, such as segmented gamma scanner (SGS) and automated qualitative and quantitative (AQ2), have severely underestimated fissile material due to either the malfunction or absence of appropriate lump correction routines. This paper examines the application of manual techniques, such as Monte Carlo N particle (MCNP) and spectral non-destructive assay platform (SNAP) software, to lump corrections in plutonium (Pu), enriched uranium (EU) and depleted uranium (DU) waste streams. Excellent results have been obtained when comparing MCNP with SNAP and applying the SNAP lump correction routine to a range of simulated and typical wastes containing various Pu and EU lump sizes. It has been concluded that the need for lump corrections was relatively rare and usually apparent from abnormal gamma ray peak area ratios, since most AWE waste streams are only lightly shielded.

  10. Transient expression assays in tobacco protoplasts.

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain


    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  11. Identification of irradiated pepper with comet assay

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail:;; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)


    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  12. Posttranslational Modification Assays on Functional Protein Microarrays.

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng


    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  13. Comparative assay of Vipera ammodytes antivenom potency.

    Capitanescu, Cristian; Macovei Oprescu, Anca Monica; Supeanu, Alexandru; Coculescu, Bogdan Ioan; Strambu, Victor; Macovei, Radu Alexandru; Manole, Gheorghe


    The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

  14. Electrochemical impedance spectroscopy based-on interferon-gamma detection

    Li, Guan-Wei; Kuo, Yi-Ching; Tsai, Pei-I.; Lee, Chih-Kung


    Tuberculosis (TB) is an ancient disease constituted a long-term menace to public health. According to World Health Organization (WHO), mycobacterium tuberculosis (MTB) infected nearly a third of people of the world. There is about one new TB occurrence every second. Interferon-gamma (IFN-γ) is associated with susceptibility to TB, and interferongamma release assays (IGRA) is considered to be the best alternative of tuberculin skin test (TST) for diagnosis of latent tuberculosis infection (LTBI). Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, adopting this assay is still labor-intensive and time-consuming. To alleviate these drawbacks, we used IFN-γ antibody to facilitate the detection of IFN-γ. An experimental verification on the performance of IGRA was done in this research. We developed two biosensor configurations, both of which possess high sensitivity, specificity, and rapid IFN-γ diagnoses. The first is the electrochemical method. The second is a circular polarization interferometry configuration, which incorporates two light beams with p-polarization and s-polarization states individually along a common path, a four photo-detector quadrature configuration to arrive at a phase modulated ellipsometer. With these two methods, interaction between IFN-γ antibody and IFN-γ were explored and presented in detail.

  15. Kinetic viability assays using DRAQ7 probe.

    Wlodkowic, Donald; Akagi, Jin; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew


    Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy-to-use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death-inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single-color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.

  16. Delivery of High-Quality Biomarker Assays

    Brian N. Swanson


    Full Text Available Biomarker measurements now support key decisions throughout the drug development process, from lead optimization to regulatory approvals. They are essential for documenting exposure-response relationships, specificity and potency toward the molecular target, untoward effects, and therapeutic applications. In a broader sense, biomarkers constitute the basis of clinical pathology and laboratory medicine. The utility of biomarkers is limited by their specificity and sensitivity toward the drug or disease process and by their overall variability. Understanding and controlling sources of variability is not only imperative for delivering high-quality assay results, but ultimately for controlling the size and expense of research studies. Variability in biomarker measurements is affected by: biological and environmental factors (e.g., gender, age, posture, diet and biorhythms, sample collection factors (e.g., preservatives, transport and storage conditions, and collection technique, and analytical factors (e.g., purity of reference material, pipetting precision, and antibody specificity. The quality standards for biomarker assays used in support of nonclinical safety studies fall under GLP (FDA regulations, whereas, those assays used to support human diagnostics and healthcare are established by CLIA (CMS regulations and accrediting organizations such as the College of American Pathologists. While most research applications of biomarkers are not regulated, biomarker laboratories in all settings are adopting similar laboratory practices in order to deliver high-quality data. Because of the escalation in demand for biomarker measurements, the highly-parallel (multi-plexed assay platforms that have fueled the rise of genomics will likely evolve into the analytical engines that drive the biomarker laboratories of tomorrow.

  17. Methods and devices for protein assays

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee


    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  18. Immunoreagents and competitive assays to fludioxonil

    Abad Fuentes, Antonio; Agulló, Consuelo; Esteve Turrillas, Francesc Albert; Abad Somovilla, Antonio; Mercader Badia, Josep Vicent


    Fludioxonil is a new-generation fungicide widely used for postharvest fruit protection. The aim of this study was to produce hitherto unreported immunoreagents for Fludioxonil analysis by immunoassay. Derivatives of this agrochemical were synthesized with different linker tethering sites. Those functionalized haptens were activated, and the purified active esters were efficiently conjugated to different carrier proteins for immunogen and assay antigen preparation. Antibodies to Fludioxonil we...

  19. Cell based assay for hypoglycemic drugs screening

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  20. Cobalt (II)-EDTA complex as a new reductant for phosphomolybdic acid used for the assay of trazodone

    A V S S Prasad; C S P Sastry


    A new spectrophotometric method for the assay of trazodone (TZ) has been described. TZ forms a complex in stoichiometric proportions with phosphomolybdic acid. The released phosphomolybdic acid from the complex with acetone is reduced with a new reductant (cobalt nitrate-ethylenediamine tetra acetic acid) to molybdenum blue, which has maximum absorption at 840 nm. Beer's law limits, precision and accuracy of the methods are checked by the UV reference method. This method is found to be suitable for the assay of TZ in the presence of other ingredients that are usually present in tablets. Recoveries are almost quantitative.

  1. High-sensitivity cardiac troponin assays: From improved analytical performance to enhanced risk stratification.

    Kozinski, Marek; Krintus, Magdalena; Kubica, Jacek; Sypniewska, Grazyna


    Implementation of cardiac troponin (cTn) assays has revolutionized the diagnosis, risk stratification, triage and management of patients with suspected myocardial infarction (MI). The Universal Definition of MI brought about a shift in the diagnostics of MI, from an approach primarily based on electrocardiography (ECG) to one primarily based on biomarkers. Currently, detection of a rise and/or fall in concentration or activity of myocardial necrosis biomarkers, preferentially cTns, with at least one value above the 99th percentile upper reference limit (URL), is the essential component for the diagnosis of MI. High-sensitivity cardiac troponin (hs-cTn) assays with their superior analytical performance were designed to further facilitate clinical decision making. The ability of hs-cTn assays to detect measurable cTn concentrations in at least 50% of healthy individuals, along with their improved precision (expressed as coefficient of variation ≤10% at the 99th percentile URL) associated with increased recognition of changing values, leads to enhanced risk stratification of patients with suspected MI, and also enables them to be used as prognostic tools potentially useful in other patient subsets. In this comprehensive review, we aim to integrate updated laboratory and clinical knowledge regarding hs-cTn assays in order to promote their optimal use in daily practice. We primarily focus on the role of hs-cTn assays in patients with suspected MI, discussing recommended diagnostic algorithms and result interpretation. Emphasis is also placed on the release of cTns following myocardial injury, the characteristics of antibodies used in available cTn immunoassays, and analytical performance of hs-cTn assays. In this paper, we also review potential challenges related to the selection of a healthy reference population in determining 99th percentile values, biological variation of hs-cTns, inequality between hs-cTn assays, and outline the current status of c

  2. The 2013 Release of Cloudy

    Ferland, G J; van Hoof, P A M; Williams, R J R; Abel, N P; Lykins, M L; Shaw, Gargi; Henney, W J; Stancil, P C


    This is a summary of the 2013 release of the plasma simulation code Cloudy. Cloudy models the ionization, chemical, and thermal state of material that may be exposed to an external radiation field or other source of heating, and predicts observables such as emission and absorption spectra. It works in terms of elementary processes, so is not limited to any particular temperature or density regime. This paper summarizes advances made since the last major review in 1998. Much of the recent development has emphasized dusty molecular environments, improvements to the ionization / chemistry solvers, and how atomic and molecular data are used. We present two types of simulations to demonstrate the capability of the code. We consider a molecular cloud irradiated by an X-ray source such as an Active Nucleus and show how treating EUV recombination lines and the full SED affects the observed spectrum. A second example illustrates the very wide range of particle and radiation density that can be considered.

  3. Release of RANKERN 16A

    Bird Adam


    Full Text Available RANKERN 16 is the latest version of the point-kernel gamma radiation transport Monte Carlo code from AMEC Foster Wheeler’s ANSWERS Software Service. RANKERN is well established in the UK shielding community for radiation shielding and dosimetry assessments. Many important developments have been made available to users in this latest release of RANKERN. The existing general 3D geometry capability has been extended to include import of CAD files in the IGES format providing efficient full CAD modelling capability without geometric approximation. Import of tetrahedral mesh and polygon surface formats has also been provided. An efficient voxel geometry type has been added suitable for representing CT data. There have been numerous input syntax enhancements and an extended actinide gamma source library. This paper describes some of the new features and compares the performance of the new geometry capabilities.

  4. Biomediated continuous release phosphate fertilizer

    Goldstein, Alan H.; Rogers, Robert D.


    A composition is disclosed for providing phosphate fertilizer to the root zone of plants. The composition comprises a microorganism capable of producing and secreting a solubilization agent, a carbon source for providing raw material for the microorganism to convert into the solubilization agent, and rock phosphate ore for providing a source of insoluble phosphate that is solubilized by the solubilization agent and released as soluble phosphate. The composition is provided in a physical form, such as a granule, that retains the microorganism, carbon source, and rock phosphate ore, but permits water and soluble phosphate to diffuse into the soil. A method of using the composition for providing phosphate fertilizer to plants is also disclosed.

  5. Release of RANKERN 16A

    Bird, Adam; Murphy, Christophe; Dobson, Geoff


    RANKERN 16 is the latest version of the point-kernel gamma radiation transport Monte Carlo code from AMEC Foster Wheeler's ANSWERS Software Service. RANKERN is well established in the UK shielding community for radiation shielding and dosimetry assessments. Many important developments have been made available to users in this latest release of RANKERN. The existing general 3D geometry capability has been extended to include import of CAD files in the IGES format providing efficient full CAD modelling capability without geometric approximation. Import of tetrahedral mesh and polygon surface formats has also been provided. An efficient voxel geometry type has been added suitable for representing CT data. There have been numerous input syntax enhancements and an extended actinide gamma source library. This paper describes some of the new features and compares the performance of the new geometry capabilities.

  6. Colloid Release from Soil Aggregates

    Vendelboe, Anders Lindblad; Møldrup, Per; Schjønning, Per;


    The content of water-dispersible colloids (WDC) has a major impact on soil functions and structural stability. In addition, the presence of mobile colloids may increase the risk of colloid-facilitated transport of strongly sorbing environmental contaminants. The WDC content was measured in 39 soils......, using laser diffraction, by agitating the samples using a wet-dispersion unit. This approach eliminated the need for long sedimentation times required by the more classical end-over-end shaking approach and provided information about the time-dependent release of WDC. The total clay content of the soils...... ranged from 0.1 to 0.44 kg kg−1. The WDC content was measured on air-dry and moist 1- to 2-mm aggregates. The WDC content at a reference time was highly correlated to the total clay content (r > 0.91, P soils. Only for two sites was the WDC content correlated to the content of clay...

  7. Diagnosis of latent tuberculosis: Can we do better?

    Al-Orainey Ibrahim


    Full Text Available Latent tuberculosis infection (LTBI is often diagnosed by the tuberculin skin test (TST. The latter has several limitations with regard to its sensitivity and specificity. It may be positive in people with prior bacille Calmette-Guιrin (BCG vaccination or exposure to nontuberculous mycobacteria. False negative TST results frequently occur in patients with impaired T-cell function. Therefore TST results have to be interpreted taking into consideration the pretest risk of TB infection or reactivation. Recently, interferon gamma release assays (IGRA were introduced for the diagnosis of LTBI. These include the T-SPOT-TB and the QuantiFERON ® -TB Gold tests .These tests measure interferon gamma released in response to T-cell stimulation by specific Mycobacterium tuberculosis antigens. These tests have been shown to be more specific than the TST as they are not affected by BCG vaccination. Their sensitivity was similar to that of the TST and in some studies they correlated better with the degree of exposure. In immune-compromised patients their sensitivity was better than that of the TST. IGRA tests were shown to have better predictive value for the development of active disease among individuals with LTBI. These tests are expensive. Their most cost-effective utilization is as confirmatory tests in patients with positive TST results, particularly in areas with high rates of BCG vaccination.

  8. Polymerase chain reaction assay for avian polyomavirus.

    Phalen, D N; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  9. Diagnostic Certified Assay: Neuromuscular and Cardiac Assessments

    Rea Valaperta


    Full Text Available The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%. Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results.

  10. Assessment of plaque assay methods for alphaviruses.

    Juarez, Diana; Long, Kanya C; Aguilar, Patricia; Kochel, Tadeusz J; Halsey, Eric S


    Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a "semisolid-variant method" using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.

  11. Hyperpolarized NMR Probes for Biological Assays

    Sebastian Meier


    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  12. A single step protein assay that is both detergent and reducer compatible: The cydex blue assay.

    Rabilloud, Thierry


    Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single-step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay.

  13. Grafting polymer brushes on biomimetic structural surfaces for anti-algae fouling and foul release.

    Wan, Fei; Pei, Xiaowei; Yu, Bo; Ye, Qian; Zhou, Feng; Xue, Qunji


    Sylgard-184 silicone elastomer negative replica and resorcinol-formaldehyde (RF) positive replica were made by biomimicking the patterns of natural Trifolium and three other kinds of leaves using the micromolding lithography. An effective antifouling (AF) polymer, poly(3-sulfopropyl methacrylate) (PSPMA), was then grafted on these replica surfaces via the surface-initiated atom transfer radical polymerization (SI-ATRP). The AF property of the modified biomimetic surfaces was tested via the settlement assay with two microalgae in different sizes, and their fouling-release (FR) property was evaluated by the removal assay. The results indicate that the structure of microspines on Trifolium leaf can inhibit settlement of microalgae and facilitate the cell release. The AF property was improved by modification with PSPMA brushes.

  14. HDT-1, a new synthetic compound, inhibits glutamate release in rat cerebral cortex nerve terminals (synaptosomes)

    Su-jane WANG; Shiu-huey CHOU; Yuh-chi KUO; Shang-shing Peter CHOU; Woan-fang TZENG; Jxh-yih LEU; Rwei-fen S HUANG; Yih-fong LIEW


    Aim: Excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. In this study, we investigated the effect of HDT-1 (3, 4, 4a, 5, 8, 8a-hexahydro-6,7-dimethyl-4a-(phenyisulfonyl)-2-tosylisoquinolin-1(2H)-one), a novel synthetic compound, on glutamate release in rat cerebrocortical nerve terminals and explored the possible mechanism. Methods: The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) or the high external [K+] and measured by one-line enzyme-coupled fluorometric assay. We also determined the loci at which HDT-1 impinges on cerebrocortical nerve terminals by using membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and Ca2+ indicator Fura-2 to monitor Ca2+ influx. Results: HDT-1 inhibited the release of glutamate evoked by 4-AP and KCI in a concentration-dependent manner. HDT-1 did not alter the resting synaptosomal membrane potential or 4-AP-evoked depolarization. Examination of the effect of HDT-1 on cytosolic [Ca2+] revealed that the diminution of glutamate release could be attributed to reduction in voltage-dependent Ca2+ influx. Consistent with this, the HDT-1-mediated inhibition of glutamate release was significantly prevented in synaptosomes pretreated with the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC. Conclusion: In rat cerebrocortical nerve terminals, HDT-1 inhibits glutamate release through a reduction of voltage-dependent Ca2+ channel activity and subsequent decrease of Ca2+ influx into nerve terminals, rather than any upstream effect on nerve terminal excitability.

  15. Immunotoxicological Assays Using the Japanese Medaka


    Medical Research and Development Command, Fort Detrick Frederick , Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for public release; distribution...34Sc. ADDRESS (City. State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS Fort Detrick PROGRAM PROJECT TASK WORK UNIT Frederick , Maryland 21702-5012...toxicity, as manifested by decreased condition index (Roesijadi and Klerks 1989) and reduced shell growth (Shuster and Pringle 1969), starts to become

  16. 3H-D-aspartate release from cerebellar granule neurons is differentially regulated by glutamate- and K(+)-stimulation

    Belhage, B; Rehder, V; Hansen, Gert Helge


    in neurites as well as cell bodies employing the fluorescent Ca2+ indicator fura-2. Transmitter release was assayed using 3H-D-aspartate to label the exogenously accessible glutamate pools, which in these neurons is believed to also include the transmitter pool. In an attempt to distinguish whether...

  17. Norepinephrine release in the rat pineal gland : The input from the biological clock measured by in vivo microdialysis

    Drijfhout, WJ; vanderLinde, AG; Kooi, SE; Grol, CJ; Westerink, BHC


    The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on-line in vivo microdialysis, NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable t

  18. Fluoride release from newly marketed fluoride varnishes.

    Jablonowski, Beth L; Bartoloni, Joseph A; Hensley, Donna M; Vandewalle, Kraig S


    New fluoride varnishes have been marketed that reportedly release more fluoride (Enamel Pro) or release fluoride more slowly (Vanish XT). The purpose of this study was to compare the amount and rate of fluoride release of new fluoride varnishes with other traditional fluoride varnishes. Extracted molars were cut into block sections. The enamel surfaces of the sections were painted with Enamel Pro, Duraphat, Vanish, or Vanish XT fluoride varnishes. One group was not treated and served as a negative control. The tooth sections were immersed in artificial saliva. The concentration of fluoride in parts per million was measured after the first 30 minutes, daily for the first week, and weekly until the level was below the limit of detection. Fluoride release was plotted over time. Cumulative fluoride release and rate of release (slope) were analyzed using one-way ANOVA/Tukey (α = .05). Enamel Pro had the greatest cumulative fluoride release. There was no significant difference between Duraphat and Vanish. Vanish XT had the lowest cumulative fluoride release. The rate of fluoride release from 1 week to limit of detection was Enamel Pro > Vanish > Duraphat > Vanish XT. The two newly marketed fluoride varnishes (Enamel Pro and Vanish XT) had significantly different fluoride release from the two conventional fluoride varnishes (Duraphat and Vanish).

  19. Evaluation of glibenclamide microspheres for sustained release

    Kambham Venkateswarlu


    Full Text Available Context: Sustained release drug delivery systems are more preferred than the conventional drug delivery systems due to its enhanced bioavailability and patient compliance. Earlier studies reported on glibenclamide (GBCM were not clear and hence, the step has been taken to explore the sustained release drug delivery system of GBCM. Aims: To evaluate the sustained release microspheres obtained of GBCM. Methods: Microspheres were prepared by ionic gelation method using the polymers like Eudragit RS 100 and xanthan gum. Polymers can sustain the drug release from microspheres. The prepared microspheres were subjected to micromeritic studies like Carr’s index, Hausner’s ratio and angle of repose. Results: Micromeritic studies confirmed that the microspheres possessing acceptable flow properties. It was observed from the in vitro release studies, formulations F8 and F9 showed sustained drug release for desired time of 12 h and when compared to F9, formulation F8 showed maximum drug release for 12 h. Conclusions: Results confirmed the formulation F8 consist of the polymers such as Eudragit RS 100 about 150 mg and xanthan gum about 100 mg showed desired sustained release of 12 h with 96.07% and kinetic studies confirmed that the release from microspheres followed non-Fickian diffusion mechanism. Due to its sustained release property, it could enhance the bioavailability of drug thereby improves the patient compliance and expect better treatment than conventional dosage forms.

  20. Release Data Package for Hanford Site Assessments

    Riley, Robert G.; Lopresti, Charles A.; Engel, David W.


    Beginning in fiscal year (FY) 2003, the U.S. Department of Energy (DOE) Richland Operations Office initiated activities, including the development of data packages, to support a Hanford assessment. This report describes the data compiled in FY 2003 through 2005 to support the Release Module of the System Assessment Capability (SAC) for the updated composite analysis. This work was completed as part of the Characterization of Systems Project, part of the Remediation and Closure Science Project, the Hanford Assessments Project, and the Characterization of Systems Project managed by Pacific Northwest National Laboratory. Related characterization activities and data packages for the vadose zone and groundwater are being developed under the remediation Decision Support Task of the Groundwater Remediation Project managed by Fluor Hanford, Inc. The Release Module applies release models to waste inventory data from the Inventory Module and accounts for site remediation activities as a function of time. The resulting releases to the vadose zone, expressed as time profiles of annual rates, become source terms for the Vadose Zone Module. Radioactive decay is accounted for in all inputs and outputs of the Release Module. The Release Module is implemented as the VADER (Vadose zone Environmental Release) computer code. Key components of the Release Module are numerical models (i.e., liquid, soil-debris, cement, saltcake, and reactor block) that simulate contaminant release from the different waste source types found at the Hanford Site. The Release Module also handles remediation transfers to onsite and offsite repositories.

  1. A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.

    Yu, Haixiang; Xu, Xiaowen; Liang, Pingping; Loh, Kang Yong; Guntupalli, Bhargav; Roncancio, Daniel; Xiao, Yi


    DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

  2. Stabilizing Alginate Confinement and Polymer Coating of CO-Releasing Molecules Supported on Iron Oxide Nanoparticles To Trigger the CO Release by Magnetic Heating.

    Meyer, Hajo; Winkler, Felix; Kunz, Peter; Schmidt, Annette M; Hamacher, Alexandra; Kassack, Matthias U; Janiak, Christoph


    Maghemite (Fe2O3) iron oxide nanoparticles (IONPs) were synthesized, modified with covalent surface-bound CO-releasing molecules of a tri(carbonyl)-chlorido-phenylalaninato-ruthenium(II) complex (CORM), and coated with a dextran polymer. The time- and temperature-dependent CO release from this CORM-3 analogue was followed by a myoglobin assay. A new measurement method for the myoglobin assay was developed, based on confining "water-soluble" polymer-coated Dextran500k@CORM@IONP particles in hollow spheres of nontoxic and easily prepared calcium alginate. Dropping a mixture of Dextran500k@CORM@IONP and sodium alginate into a CaCl2 solution leads to stable hollow spheres of Ca(2+) cross-linked alginate which contain the Dextran500k@CORM@IONP particles. This "alginate-method" (i) protects CORM-3 analogues from rapid CO-displacement reactions with a protein, (ii) enables a spatial separation of the CORM from its surrounding myoglobin assay with the alginate acting as a CO-permeable membrane, and (iii) allows the use of substances with high absorptivity (such as iron oxide nanoparticles) in the myoglobin assay without interference in the optical path of the UV cell. Embedding the CORM@IONP nanoparticles in the alginate vessel represents a compartmentation of the reactive component and allows for close contact with, yet facile separation from, the surrounding myoglobin assay. The half-life of the CO release from Dextran500k@CORM@IONP particles surrounded by alginate was determined to be 890 ± 70 min at 20 °C. An acceleration of the CO release occurs at higher temperature with a half-life of 172 ± 27 min at 37 °C and 45 ± 7 min at 50 °C. The CO release can be triggered in an alternating current magnetic field (31.7 kA m(-1), 247 kHz, 39.9 mT) through local magnetic heating of the susceptible iron oxide nanoparticles. With magnetic heating at 20 °C in the bulk solution, the half-life of CO release from Dextran500k@CORM@IONP particles decreased to 155 ± 18 min

  3. Simultaneous measurement of hormone release and secretagogue binding by individual pituitary cells

    Smith, P.F.; Neill, J.D.


    The quantitative relationship between receptor binding and hormone secretion at the single-cell level was investigated in the present study by combining a reverse hemolytic plaque assay for measurement of luteinizing hormone (LH) secretion from individual pituitary cells with an autoradiographic assay of /sup 125/I-labeled gonadontropin-releasing hormone (GnRH) agonist binding to the same cells. In the plaque assay, LH secretion induces complement-mediated lysis of the LH-antibody-coated erythrocytes around the gonadotropes, resulting in areas of lysis (plaques). LH release from individual gonadotropes was quantified by comparing radioimmunoassayable LH release to hemolytic area in similarly treated cohort groups of cells; plaque area was linearly related to the amount of LH secreted. Receptor autoradiography was performed using /sup 125/I-labeled GnRH-A (a superagonist analog of GnRH) both as the ligand and as the stimulant for LH release in the plaque assay. The grains appeared to represent specific and high-affinity receptors for GnRH because (i) no pituitary cells other than gonadotropes bound the labeled ligand and (ii) grain development was progressively inhibited by coincubation with increasing doses of unlabeled GnRH-A. The authors conclude that GnRH receptor number for any individual gonadotrope is a weak determinant of the amount of LH it can secrete; nevertheless, full occupancy of all its GnRH receptors is required for any gonadotrope to reach its full LH-secretory capacity. Apparently the levels of other factors comprising the steps along the secretory pathway determine the secretory capacity of an individual cell.

  4. Inhibition of inflammatory mediator release from microglia can treat ischemic/hypoxic brain injury★

    Wang, Huaibo; Guo,Weitao; Liu, Hongliang; Zeng, Rong; Lu, Mingnan; Chen, Ziqiu; Xiao, Qixian


    Interleukin-1α and interleukin-1β aggravate neuronal injury by mediating the inflammatory reaction following ischemic/hypoxic brain injury. It remains unclear whether interleukin-1α and interleukin-1β are released by microglia or astrocytes. This study prepared hippocampal slices that were subsequently subjected to oxygen and glucose deprivation. Hematoxylin-eosin staining verified that neurons exhibited hypoxic changes. Results of enzyme-linked immunosorbent assay found that interleukin-1α a...

  5. Inhibition of inflammatory mediator release from microglia can treat ischemic/hypoxic brain injury★

    Wang, Huaibo; Guo, Weitao; Liu, Hongliang; Zeng, Rong; Lu, Mingnan; Chen, Ziqiu; Xiao, Qixian


    Interleukin-1α and interleukin-1β aggravate neuronal injury by mediating the inflammatory reaction following ischemic/hypoxic brain injury. It remains unclear whether interleukin-1α and interleukin-1β are released by microglia or astrocytes. This study prepared hippocampal slices that were subsequently subjected to oxygen and glucose deprivation. Hematoxylin-eosin staining verified that neurons exhibited hypoxic changes. Results of enzyme-linked immunosorbent assay found that interleukin-1α a...

  6. Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

    Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph


    This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  7. Fabrication, in vitro Degradation and Cytotoxic Assay of Different Cystalline Phases Calcium Polyphosphate


    Fabrication, in vitro degradation and cytotoxic assay of different crystalline phases calcium polyphosphate (CPP) were reported. The CPP ceramics were fabricated by crystallizing the amorphous frits , and sintered at 550 ℃ ,875 ℃ ,1000 ℃ for 1 h to obtain the γ-CPP, β-CPP anda-CPP respectively. The effects of the different crystalline phases on their weight loss and released PO4 3- were investigated during the degradation.And the surface change was observed by the SEM. The osteoblastic ROS17/2.8 cell line was used to estimate the cytotoxicity of CPP. The effects of CPP on cells' proliferation were evaluated by using MTT assay. The experimental results showed that γ-CPP, β- CPP and α-CPP did not exert cytotoxic effect on the cells. In addition, the proliferation of the growth of ROS17/2.8 cells on β-CPP was optimal.

  8. Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

    Li, Ru; Huang, Jiqing; Kast, Juergen


    Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

  9. Detection of airborne bacteria with disposable bio-precipitator and NanoGene assay.

    Lee, Eun-Hee; Chua, Beelee; Son, Ahjeong


    We demonstrated the detection of airborne bacteria by a disposable bio-precipitator and NanoGene assay combination. The bio-precipitator employed micro corona discharge at 1960V and at less than 35µA to simultaneously charge, capture and lyse the airborne bacteria. This was enabled by the use of a 15μL liquid anode. Using a custom exposure setup, the target bacterium Bacillus subtilis in the atomization solution was rendered airborne. After exposure, the liquid anode in the bio-precipitator was subsequently measured for DNA concentration and analyzed with the NanoGene assay. As the bacterial concentration increased from 0.0104 to 42.6 g-DCW/L the released DNA concentration in the liquid anode increased from 2.10±1.57 to 75.00±7.15ng/μL. More importantly, the NanoGene assay showed an increase in normalized fluorescence (gene quantification) from 18.03±1.18 to 49.71±1.82 as the bacterial concentrations increased from 0.0104 to 42.6 g-DCW/L. the electrical power consumption of the bio-precipitator was shown to be amenable for portable use. In addition, the detection limit of bio-precipitator and NanoGene assay combination in the context of environmentally relevant levels of airborne bacteria was also discussed.

  10. Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs.

    Caviglia, Claudia; Zór, Kinga; Montini, Lucia; Tilli, Valeria; Canepa, Silvia; Melander, Fredrik; Muhammad, Haseena B; Carminati, Marco; Ferrari, Giorgio; Raiteri, Roberto; Heiskanen, Arto; Andresen, Thomas L; Emnéus, Jenny


    In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.

  11. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio


    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

  12. Phage anti-immunocomplex assay for clomazone: two-site recognition increasing assay specificity and facilitating adaptation into an on-site format.

    Rossotti, M A; Carlomagno, M; González-Techera, A; Hammock, B D; Last, J; González-Sapienza, G


    The impact of the use of herbicides in agriculture can be minimized by compliance with good management practices that reduce the amount used and their release into the environment. Simple tests that provide real time on-site information about these chemicals are a major aid for these programs. In this work, we show that phage anti-immunocomplex assay (PHAIA), a method that uses phage-borne peptides to detect the formation of antibody-analyte immunocomplexes, is an advantageous technology to produce such field tests. A monoclonal antibody to the herbicide clomazone was raised and used in the development of conventional competitive and noncompetitive PHAIA immunoassays. The sensitivity attained with the PHAIA format was over 10 times higher than that of the competitive format. The cross-reactivity of the two methods was also compared using structurally related compounds, and we observed that the two-site binding of PHAIA "double-checks" the recognition of the analyte, thereby increasing the assay specificity. The positive readout of the noncompetitive PHAIA method allowed adaptation of the assay into a rapid and simple format where as little as 0.4 ng/mL clomazone (more than 10-fold lower than the proposed standard) in water samples from a rice field could be easily detected by simple visual inspection.

  13. Comparison of Acute Toxicity of Algal Metabolites Using Bioluminescence Inhibition Assay

    Hansa Jeswani


    Full Text Available Microalgae are reported to degrade hazardous compounds. However, algae, especially cyanobacteria are known to produce secondary metabolites which may be toxic to flora, fauna and human beings. The aim of this study was selection of an appropriate algal culture for biological treatment of biomass gasification wastewater based on acute toxicity considerations. The three algae that were selected were Spirulina sp., Scenedesmus abundans and a fresh water algal consortium. Acute toxicity of the metabolites produced by these algal cultures was tested at the end of log phase using the standard bioluminescence inhibition assay based on Vibrio fischeri NRRLB 11174. Scenedesmus abundans and a fresh water algal consortium dominated by cyanobacteria such as Phormidium, Chroococcus and Oscillatoria did not release much toxic metabolites at the end of log phase and caused only about 20% inhibition in bioluminescence. In comparison, Spirulina sp. released toxic metabolites and caused 50% bioluminescence inhibition at 3/5 times dilution of the culture supernatant (EC50.

  14. Development of a radioimmunoassay for measuring gonadotrophin releasing hormone in patients receiving treatment

    Mobsby, V.A.; Knapp, M.L.; Mayne, P.D. (Charing Cross and Westminster Medical School, Westminster, London (UK)); Fink, R.S. (West Middlesex Univ. Hospital, Isleworth (UK)); Osgood, V.M. (Royal Free Hospital, London (UK))


    This gonadotrophin releasing hormone (GnRH) assay showed good precision, recovery, and parallelism over a wide range of GnRH concentrations with a sensitivity of 15 pg/ml. The assay was compared with a commercially available kit (Buhlmann Laboratories). Although the Buhlmann kit showed acceptable precision, recovery, sensitivity, and correlation with the developed GnRH assay for plasma samples, lack of parallelism of serially diluted plasma and urine samples was consistently observed, together with poor correlation with the developed GnRH assay for urine, suggesting a matrix effect with the Buhlmann kit. The developed assay is suitable for measuring GnRH in samples obtained from patients receiving pulsatile infusions of GnRH. In contrast, the commercially available Buhlmann kit was unsuitable for measuring plasma GnRH as the kit had a top standard of only 160 pg/ml, well below peak plasma concentration. It would not be possible to dilute samples for analysis because of lack of parallelism of diluted samples compared with standards obtained with the Buhlmann assay. (author).

  15. A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays.

    Hsu, Denise C; Zaunders, John J; Plit, Marshall; Leeman, Craig; Ip, Susanna; Iampornsin, Thatri; Pett, Sarah L; Bailey, Michelle; Amin, Janaki; Ubolyam, Sasiwimol; Avihingsanon, Anchalee; Ananworanich, Jintanat; Ruxrungtham, Kiat; Cooper, David A; Kelleher, Anthony D


    A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.

  16. Formulation and evaluation of controlled release matrix mucoadhesive tablets of domperidone using Salvia plebeian gum

    Gurpreet Arora


    Full Text Available The aim of study was to prepare controlled release matrix mucoadhesive tablets of domperidone using Salvia plebeian gum as natural polymer. Tablets were formulated by direct compression technology employing the natural polymer in different concentrations (5, 10, 15 and 20% w/w. The prepared batches were evaluated for drug assay, diameter, thickness, hardness and tensile strength, swelling index, mucoadhesive strength (using texture analyzer and subjected to in vitro drug release studies. Real-time stability studies were also conducted on prepared batches. In vitro drug release data were fitted in various release kinetic models for studying the mechanism of drug release. Tensile strength was found to increase from 0.808 ± 0.098 to 1.527 ± 0.10 mN/cm 2 and mucoadhesive strength increased from 13.673 ± 1.542 to 40.378 ± 2.345 N, with an increase in the polymer concentration from 5 to 20% (A1 to A4. Swelling index was reported to increase with both increase in the concentration of gum and the time duration. The in vitro drug release decreased from 97.76 to 83.4% (A1 to A4 with the increase in polymer concentration. The drug release from the matrix tablets was found to follow zero-order and Higuchi models, indicating the matrix-forming potential of natural polymer. The value of n was found to be between 0.5221 and 0.8992, indicating the involvement of more than one drug release mechanism from the formulation and possibly the combination of both diffusion and erosion. These research findings clearly indicate the potential of S. plebeian gum to be used as binder, release retardant and mucoadhesive natural material in tablet formulations.

  17. Prevalence of latent and active tuberculosis among dairy farm workers exposed to cattle infected by Mycobacterium bovis.

    Pedro Torres-Gonzalez

    Full Text Available BACKGROUND: Human tuberculosis caused by M. bovis is a zoonosis presently considered sporadic in developed countries, but remains a poorly studied problem in low and middle resource countries. The disease in humans is mainly attributed to unpasteurized dairy products consumption. However, transmission due to exposure of humans to infected animals has been also recognized. The prevalence of tuberculosis infection and associated risk factors have been insufficiently characterized among dairy farm workers (DFW exposed in settings with poor control of bovine tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Tuberculin skin test (TST and Interferon-gamma release assay (IGRA were administered to 311 dairy farm and abattoir workers and their household contacts linked to a dairy production and livestock facility in Mexico. Sputa of individuals with respiratory symptoms and samples from routine cattle necropsies were cultured for M. bovis and resulting spoligotypes were compared. The overall prevalence of latent tuberculosis infection (LTBI was 76.2% (95% CI, 71.4-80.9% by TST and 58.5% (95% CI, 53.0-64.0% by IGRA. Occupational exposure was associated to TST (OR 2.72; 95% CI, 1.31-5.64 and IGRA (OR 2.38; 95% CI, 1.31-4.30 adjusting for relevant variables. Two subjects were diagnosed with pulmonary tuberculosis, both caused by M. bovis. In one case, the spoligotype was identical to a strain isolated from bovines. CONCLUSIONS: We documented a high prevalence of latent and pulmonary TB among workers exposed to cattle infected with M. bovis, and increased risk among those occupationally exposed in non-ventilated spaces. Interspecies transmission is frequent and represents an occupational hazard in this setting.

  18. Assessment of the influence of direct tobacco smoke on infection and active TB management.

    Altet, Neus; Latorre, Irene; Jiménez-Fuentes, María Ángeles; Maldonado, José; Molina, Israel; González-Díaz, Yoel; Milà, Celia; García-García, Esther; Muriel, Beatriz; Villar-Hernández, Raquel; Laabei, Maisem; Gómez, Andromeda-Celeste; Godoy, Pere; de Souza-Galvão, Maria Luiza; Solano, Segismundo; Jiménez-Ruiz, Carlos A; Domínguez, Jose


    Smoking is a risk factor for tuberculosis (TB) infection and disease progression. Tobacco smoking increases susceptibility to TB in a variety of ways, one of which is due to a reduction of the IFN-γ response. Consequently, an impaired immune response could affect performance of IFN-γ Release Assays (IGRAs). In the present study, we assess the impact of direct tobacco smoking on radiological manifestations, sputum conversion and immune response to Mycobacterium tuberculosis, analyzing IFN-γ secretion by IGRAs. A total of 525 participants were studied: (i) 175 active pulmonary TB patients and (ii) 350 individuals coming from contact tracing studies, 41 of whom were secondary TB cases. Clinical, radiological and microbiological data were collected. T-SPOT.TB and QFN-G-IT were processed according manufacturer's instructions. In smoking patients with active TB, QFN-G-IT (34.4%) and T-SPOT.TB (19.5%) had high frequencies of negative results. In addition, by means of an unconditional logistic regression, smoking was a main factor associated with IGRAs' false-negative results (aOR: 3.35; 95%CI:1.47-7.61; pTB presented a high probability of having cavitary lesions (aOR: 1.88; 95%CI:1.02-3.46;pTB infection (LTBI) was favored in smoking contacts, being a risk factor associated with infection (aOR: 11.57; 95%CI:5.97-22.41; pTB due to a weakened IFN-γ response caused by direct tobacco smoke.

  19. Oral Vaccination with Heat-Inactivated Mycobacterium bovis Does Not Interfere with the Antemortem Diagnostic Techniques for Tuberculosis in Goats

    Alvaro Roy


    Full Text Available Vaccination against tuberculosis (TB is prohibited in cattle or other species subjected to specific TB eradication campaigns, due to the interference that it may cause with the official diagnostic tests. However, immunization with a heat-inactivated (HI Mycobacterium bovis vaccine via the oral route has been suggested to overcome this issue. In this study, the main goal was to assess the interference of the HI vaccine by different routes of administration using a previous vaccination and re-vaccination (boosting protocol. TB-free kid goats were divided into three groups: oral (n = 16, intramuscular (IM; n = 16, and control (n = 16. Results showed that there was a significant difference in the percentage of animals positive to the single intradermal test (SIT and blood based interferon-gamma release assay (IGRA caused by vaccination when performed in the IM group compared to the oral group (p < 0.001. Nevertheless, no positivity to the SIT or IGRA test was observed in orally vaccinated goats regardless of the different interpretation criteria applied. None of the groups presented positive antibody titers using an in-house ELISA and samples collected 2 months after the boost. These results suggest the potential usefulness of the HI vaccine by the oral route in goats to minimize the interference on diagnostic tests (skin and IGRA tests and reducing the necessity of defined antigens to replace the traditional purified protein derivatives for diagnosis. Finally, the results pave the way to future efficacy studies in goats using different routes of HI vaccination.

  20. Mycobacterium Tuberculosis Infection, Immigration Status, and Diagnostic Discordance: A Comparison of Tuberculin Skin Test and QuantiFERON-TB Gold In-Tube Test Among Immigrants to the U.S.

    Wilson, Fernando A; Miller, Thaddeus L; Stimpson, Jim P


    We used a recent source of nationally representative population data on tuberculosis (TB) infection to characterize concordance between the tuberculin skin test (TST) and the QuantiFERON-TB Gold In-Tube (QFT-GIT) blood test for immigrants in the United States. We used TB screening data from the 2011-2012 National Health and Nutrition Examination Survey to examine concordance between the TST and QFT-GIT--an interferon-gamma release assay (IGRA) blood test--for 7,097 U.S. natives, naturalized citizens, and noncitizens. Consistent with prior findings, one in five immigrants in the survey was identified with latent TB infection (LTBI), a rate 14 times higher than for U.S. natives. We also found higher rates of discordant TST/IGRA results among immigrants than among U.S. natives. Unadjusted discordance between TST and IGRA was 3% among U.S. natives (weighted N=5,684,274 of 191,179,213) but ranged up to 19% for noncitizens (weighted N=3,722,960 of 19,377,147). Adjusting for age, sex, and race/ethnicity, noncitizens had more than nine times the odds of having a positive TST result but negative QFT-GIT result compared with U.S. natives. Our findings suggest that whether and how either of these tests should be deployed is highly context sensitive. Significant discordance in test results when used among immigrants raises the possibility of missed opportunities for harm reduction in this already at-risk population. However, we found little distinction between the tests in terms of diagnostic outcome when used in a U.S. native population, suggesting little benefit to the adoption and use of the QFT-GIT test in place of TST on the basis of test performance alone for this population.