During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and beta-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4'-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and beta-actin DNA could be quantified in parallel in a wide linear range from 10(1) to 10(7) copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 10(4) copies/2x10(5) beta-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.

Human cytomegalovirus (hCMV) infection is still a cause of morbidity and mortality after solid organ and bone marrow transplantation and in other immunocompromised states. 'Preemptive therapy' strategies necessitate sensitive and specific methods for rapid diagnosis of symptomatic hCMV infection and monitoring of antiviral treatment. For analysis of the lytic stage of viral replication, the molecular determination of late transcripts is a useful approach for diagnosis of hCMV disease. In the present study we established an absolute quantitation of hCMV spliced late gene (SLG) RNA transcripts by real-time reverse transcription-polymerase chain reaction. Intron spanning primers were used for amplification to discriminate between viral DNA and cDNA. For standardization of the varying amounts of cDNA analyzed, cytoplasmic beta2-microglobulin (beta2-MG) cDNA was quantitated in parallel. cDNA copy numbers of both target sequences could be quantitated in a wide linear range from 10 to 10(7) copies. To investigate the applicability of the developed assay, diploid lung fibroblasts were infected with the virus strain AD169. SLG expression was measured during a 48-h period after inoculation. After an only low expression during the first 10 h (approximately 5 x 10(2) copies/sample or SLG/beta2-MG ratio <0.001), SLG transcription increased dramatically after 24 h, peaking at 5x104 copies/sample or SLG/beta2-MG ratio of 0.035 after 48 h. Intra- and interassay variability was less than 5% for calibrators and less than 10% samples. We conclude that quantitation of SLG transcripts by the presented method might be a powerful tool for differentiating between hCMV latency and active replication in vitro end in vivo, and thus may be a promising tool for diagnosis of symptomatic hCMV infection and monitoring of antiviral treatment.