beta-Herpesvirus (human cytomegalovirus and human herpesvirus 6) reactivation in at-risk lung transplant recipients and in human immunodeficiency virus-infected patients.

Human herpesvirus (HHV)-6 is a beta-herpesvirus-like human cytomegalovirus (HCMV) with the potential to reactivate in immunocompromised persons. HHV-6 and HCMV were assessed in the peripheral blood leukocytes of 26 lung transplant recipients and of 37 human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy, to determine the degree of concordance between HHV-6 and HCMV reactivation in different biologic settings. In the lung transplant recipients (145 samples), HHV-6 was not detected, even though 44 (30%) of 145 samples were from 9 HCMV DNA-positive patients (13 episodes of HCMV pneumonitis). Among the HIV-infected patients (172 samples), HCMV DNA was detected in 29 (17%) of 172 samples from 10 patients (4 episodes of HCMV disease). HHV-6 DNA was detected in 2 HIV-infected patients who did not have HCMV detected at that time. These findings suggest that the pathobiologic control mechanisms for these 2 beta-herpesviruses may be significantly different.

Improvement of non-radioactive in situ hybridization in human airway tissues: use of PCR-generated templates for synthesis of probes and an antibody sandwich technique for detection of hybridization.

We describe the use of non-traditional methods of probe synthesis and quantification and detection of hybridization that appreciably improved non-radioactive in situ hybridization (ISH) in human airway tissue. To avoid the problems of bacterial cloning, plasmid digestion, and probe hydrolysis, we synthesised complementary RNA probes (riboprobes) for ISH from PCR-generated DNA. DNA template was produced by nested PCR incorporation of T7 and SP6 RNA polymerase promoters. We then compared the efficiency of in vitro transcription from PCR-generated template with traditional plasmid template by quantifying the relative probe fluorescence in denaturing gels. Transcription with SP6 or T7 polymerase in either orientation produced TNF riboprobes from a single PCR-generated template more efficiently than from plasmid, providing there were no primer hairpin loops. Fluorescence quantification enabled equal amounts of probe label to be used in ISH, eliminating signals from the sense probe and demonstrating that probes transcribed from PCR templates were as sensitive as hydrolyzed probe transcribed from plasmid. Detection of ISH by a conventional anti-hapten, alkaline phosphatase-based technique was found to cause tissue damage due to extended substrate incubation at high pH. We therefore developed a four-layer, avidin-biotin-peroxidase technique that afforded greater sensitivity, allowing brief substrate incubation and resulting in structural preservation of tissue.