Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction.

In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.

State-of-the-art serological techniques for detection of antibodies against tick-borne encephalitis virus.

Indirect immunofluorescence, ELISA and immunoblot test systems for the detection of antibodies against tick-borne encephalitis (TBE) virus were developed and evaluated. Sera from 112 patients with clinically characterized TBE virus infections, 27 patients with antibodies against dengue or yellow fever virus and 100 healthy blood donors were investigated for anti-TBE virus antibodies. The assays yielded sensitivities of 91-95% and specificities of 91-94%. The test systems are valuable new tools for the diagnosis and monitoring of TBE virus infections.

Prognostic value of MCM2 immunoreactivity in stage T1 transitional cell carcinoma of the bladder.

OBJECTIVE: Due to the heterogeneous biologic behavior of stage T1 bladder carcinomas, there is a need for new markers allowing to assess the prognosis more accurately. To our knowledge, there are no reports on studies investigating minichromosome maintenance protein 2 (MCM2) expression in bladder carcinomas. Thus, we investigated the prognostic value of MCM2 immunoreactivity in stage T1 bladder tumors. METHODS: Fifty-four tumors were analyzed using Biochip microarrays. Also p53 and Ki67 antigen expression were examined. Immunohistochemical scores were compared with the clinical outcome. RESULTS: During a median follow-up of 43 months, tumor recurrence was registered in 43 and progression to stage T2 in 19 patients. Kaplan-Meier curves demonstrated that high-level MCM2 expression was significantly associated with early tumor recurrence when using a cutoff of 60% (p=0.0035 by log-rank test), and with early tumor progression when using a cutoff of 20% (p=0.0454). There was no relationship (p=0.604) between MCM2 and p53, but a tendentious relationship (p=0.082) between MCM2 and Ki67 antigen expression. MCM2 (p=0.006), Ki67 antigen (p=0.035) and p53 expression (p=0.049) as well as tumor grade (p=0.026) and age (p=0.025) were found significantly associated with recurrence-free survival by univariate Cox regression analysis, among which only Ki67 antigen expression (p=0.015) and age (p=0.019) proved to be of independent predictive value by multivariate analysis. Concerning tumor progression, MCM2 expression was identified as the only predictive parameter by log-rank test, but it was not of independent predictive value by multivariate analysis (p=0.101). CONCLUSION: Our data suggest that MCM2 expression may bear some prognostic relevance in stage T1 bladder carcinomas.