LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.

Tumour immunologists strive to develop efficient tumour vaccination and adoptive transfer therapies that enlarge the pool of tumour-specific and -reactive effector T-cells in vivo. To assess the efficiency of the various strategies, ex vivo assays are needed for the longitudinal monitoring of the patient's specific immune responses providing both quantitative and qualitative data. In particular, since tumour cell cytolysis is the end goal of tumour immunotherapy, routine immune monitoring protocols need to include a read-out for the cytolytic efficiency of Ag-specific cells. We propose to combine current immune monitoring techniques in a highly sensitive and reproducible multi-parametric flow cytometry based cytotoxicity assay that has been optimised to require low numbers of Ag-specific T-cells. The possibility of re-analysing those T-cells that have undergone lytic activity is illustrated by the concomitant detection of CD107a upregulation on the surface of degranulated T-cells. To date, the LiveCount Assay provides the only possibility of assessing the ex vivo cytolytic activity of low-frequency Ag-specific cytotoxic T-lymphocytes from patient material.

Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay

Drug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle. The platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library. The unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin.

BACKGROUND: The activity of the renin-angiotensin system is usually evaluated as plasma renin activity (PRA, ngAI/ml per h) but the reproducibility of this enzymatic assay is notoriously scarce. We compared the inter and intralaboratory reproducibilities of PRA with those of a new automated chemiluminescent assay, which allows the direct quantification of immunoreactive renin [chemiluminescent immunoreactive renin (CLIR), microU/ml]. METHODS: Aliquots from six pool plasmas of patients with very low to very high PRA levels were measured in 12 centres with both the enzymatic and the direct assays. The same methods were applied to three control plasma preparations with known renin content. RESULTS: In pool plasmas, mean PRA values ranged from 0.14 +/- 0.08 to 18.9 +/- 4.1 ngAI/ml per h, whereas those of CLIR ranged from 4.2 +/- 1.7 to 436 +/- 47 microU/ml. In control plasmas, mean values of PRA and of CLIR were always within the expected range. Overall, there was a significant correlation between the two methods (r = 0.73, P < 0.01). Similar correlations were found in plasmas subdivided in those with low, intermediate and high PRA. However, the coefficients of variation among laboratories found for PRA were always higher than those of CLIR, ranging from 59.4 to 17.1% for PRA, and from 41.0 to 10.7% for CLIR (P < 0.01). Also, the mean intralaboratory variability was higher for PRA than for CLIR, being respectively, 8.5 and 4.5% (P < 0.01). CONCLUSION: The measurement of renin with the chemiluminescent method is a reliable alternative to PRA, having the advantage of a superior inter and intralaboratory reproducibility.

A highly sensitive in vitro infection assay to explore early stages of mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) infection of adult mice induces a strong response to superantigen (Sag) in their draining lymph nodes, which results from the presentation of Sag by MMTV-infected B cells to Sag-reactive T cells. To date, infection with physiologically relevant doses of MMTV can be detected in vivo only after several days of Sag-mediated T-cell-dependent amplification of infected B cells. Furthermore, no efficient in vitro system of detecting MMTV infection is available. Such a model would allow the dissection of the early phase of infection, the assessment of the contributions of different cell types, and the screening of large panels of molecules for their potential roles in infection and Sag response. For these reasons, we have established an in vitro model for detecting infection which is as sensitive and reproducible as the in vivo model. We found that the viral envelope (Env) protein is crucial for target cell infection but not for presentation of Sag. Furthermore, we show that infection of purified B cells with MMTV induces entry of Sag-responsive T cells into the cell cycle, while other professional antigen-presenting cells, such as dendritic cells, are much less efficient in inducing a response.

Absence of p53 gene mutations in a tumor panel representative of pilocytic astrocytoma diversity using a p53 functional assay.

Although p53-gene mutations occur with significant frequency in diffuse low-grade and high-grade astrocytomas, and are postulated to play an important role in tumorigenesis in these cases, the role of the p53 gene in pilocytic astrocytomas remains unclear. Published data using DNA-based assays for p53-gene analysis in these tumors have shown contradictory results in mutation frequency (0-14%). It is not known whether these heterogeneous results stem from the biological diversity of this tumor group or from technical problems. To re-evaluate p53-gene status in pilocytic tumors, we analyzed 18 tumors chosen to represent the clinical and biological heterogeneity of this tumor type with respect to anatomical location, patient age, gender, ethnic origin (Caucasian or Japanese) and the concomitant occurrence of neurofibromatosis type 1 (NF1). All primary tumors were histologically diagnosed as pilocytic astrocytoma (WHO grade I), except for one anaplastic pilocytic astrocytoma (WHO grade III) which developed in an NF1 patient and recurred as glioblastoma multiforme (WHO grade IV). p53 mutations were detected using an assay in yeast which tests the transcriptional activity of p53 proteins synthesized from tumor mRNA-derived p53-cDNA templates. None of 18 tumors, including 3 NF1-related tumors, showed p53-gene mutations between and including exons 4 and 11. We conclude that p53-gene mutations are extremely rare findings in pilocytic astrocytomas, and are absent even in those exceptional cases in which malignant progression of such tumors has occurred.

Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants.

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.

QuantiFERON-TB Gold assay for the diagnosis of latent tuberculosis infection.

Tuberculin skin test (TST) has been used for 100 years for the diagnosis of latent tuberculosis (TB) infection (LTBI). In recent years, increasing interest in the diagnosis of TB has led to the development of new assays. QuantiFERON-TB Gold (QFT-G) is an IFN-gamma-release assay that measures the release of interferon after stimulation in vitro by Mycobacterium tuberculosis antigens. The main advantage of this assay with respect to TST is the lack of crossreaction with bacillus Calmette-Guérin and most nontuberculous mycobacteria. QFT-G also eliminates the need for the patient to return for test reading in 48-72 h. In the immunocompromised host and in pediatric populations, studies suggest that the QFT-G better correlates with the risk of TB than the TST, but data remain inconclusive. In contrast to TST, there are no prospective studies regarding the association of the QFT-G result and the risk for development of TB. Given its advantages, the QFT-G may become the standard test for the diagnosis of LTBI.

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment. OBJECTIVE: To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures. METHODS: Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples. RESULTS: PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp. grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis, Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture. CONCLUSIONS: Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.

Pattern-based sensing of nucleotides in aqueous solution with a multicomponent indicator displacement assay

A multicomponent indicator displacement assay ( MIDA) based on an organometallic receptor and three dyes can be used for the identification and quantification of nucleotides in aqueous solution at neutral pH.

The ager tarraconensis project (right side of river Francoli) (PAT): the application of geophysical survey to identify rural Roman settlement typologies

The methodology of the ager Tarraconensis project also included geophysical surveys aiming to distinguish different categories of rural settlements. Two geophysical techniques (resistivity and magnetometry) were combined to reveal traces of unearth structures from a selection of sites identified from the field survey. Results of geophysical surveys of these seven sites as well as conclusions obtained from this approach are discussed here.

Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease.

OBJECTIVES: Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. METHODS: Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). RESULTS: Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P = 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P = 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P = 0.044) compared to the IBD group. CONCLUSIONS: Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay.

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.

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

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. OBJECTIVE: To compare the TST and the T-SPOT.TB IGRA for identifying LTBI in patients with psoriasis before anti-TNF treatment. METHODS: A retrospective study was carried out over a 4-year period on patients with psoriasis requiring anti-TNF treatment. All were subjected to the TST, T-SPOT.TB and chest X-ray. Risk factors for LTBI and history of bacillus Calmette-Guérin (BCG) vaccination were recorded. The association of T-SPOT.TB and TST results with risk factors for LTBI was tested through univariate logistic regression models. Agreement between tests was quantified using kappa statistics. Treatment for LTBI was started 1 month before anti-TNF therapy when indicated. RESULTS: Fifty patients were included; 90% had prior BCG vaccination. A positive T-SPOT.TB was strongly associated with a presumptive diagnosis of LTBI (odds ratio 7.43; 95% confidence interval 1.38-39.9), which was not the case for the TST. Agreement between the T-SPOT.TB and TST was poor, kappa = 0.33 (SD 0.13). LTBI was detected and treated in 20% of the patients. In 20% of the cases, LTBI was not retained in spite of a positive TST but a negative T-SPOT.TB. All patients received an anti-TNF agent for a median of 56 weeks (range 20-188); among patients with a positive TST/negative T-SPOT.TB, no tuberculosis was detected with a median follow-up of 64 weeks (44-188). One case of disseminated tuberculosis occurred after 28 weeks of adalimumab treatment in a patient with LTBI in spite of treatment with rifampicin. CONCLUSION: This study is the first to underline the frequency of LTBI in patients with psoriasis (20%), and to support the use of IGRA instead of the TST for its detection. Nevertheless, there is still a risk of tuberculosis under anti-TNF therapy, even if LTBI is correctly diagnosed and treated.

Real-time quantitative PCR assay for measurement of bird telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba. This technique is based on the PCR amplification of telomeric (TTAGGG)(n) sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.